Patent Application
20200251186
The present invention generally relates to systems and methods for manufacturing biologically-produced products.
Biologically-produced pharmaceutical products, which are therapeutic drugs produced by biological organisms, have revolutionized the pharmaceutical industry. Biological organisms are an attractive source of therapeutic drugs because they are often capable of producing molecules that would be challenging, if not impossible, to synthesize chemically. For example, some biological organisms can be engineered to produce complex proteins, such as antibodies and signaling proteins, which can be used to treat or prevent diseases ranging from cancer to rheumatoid arthritis. There are already hundreds of approved biologically-produced pharmaceutical products on the market and thousands of new products in development, and demand for these products continues to grow. However, conventional systems and methods of manufacturing biologically-produced products, such as proteins, are not systematic, continuous, integrated, or modular, resulting in production systems and methods that are often slow, cumbersome, expensive, and/or not amenable to miniaturization. Accordingly, improved systems and methods that are systematic, more integrated, more continuous, and/or modular are needed to allow for more efficient protein production and purification with machines that are portable, amenable to miniaturization, and/or easier to use by operators with a variety of skill levels. Such improved systems and methods would be of significant interest to the biopharmaceutical industry, but would also have applications in bio-energy, medical research, pollution remediation, manufacturing, agriculture, and other fields.
The present invention generally relates to systems and methods for manufacturing biologically-produced products. The subject matter of the present invention involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.
In some embodiments, a method is provided, for generating and evaluating candidate sequences of partitioning steps to partition at least one biologically produced product from at least one impurity, the method comprising acts of: generating a plurality of candidate sequences of partitioning steps, wherein at least one candidate sequence of the plurality of candidate sequences comprises a plurality of partitioning steps in a specified order; and evaluating the plurality of candidate sequences, comprising, for at least one partitioning step in the at least one candidate sequence: accessing a data set associated with the at least one partitioning step, the data set comprising: first data indicative of a behavior of the at least one biologically produced product with respect to the at least one partitioning step; and second data indicative of a behavior of the at least one impurity with respect to the at least one partitioning step; and scoring the at least one candidate sequence based at least in part on the data set.
In some embodiments, a method is provided, for generating and evaluating candidate sequences of chromatography steps to partition at least one pharmaceutical product from at least one impurity, the at least one pharmaceutical product being biologically produced, the method comprising acts of: generating a plurality of candidate sequences of chromatography steps, wherein at least one candidate sequence of the plurality of candidate sequences comprises a plurality of chromatography steps in a specified order; and evaluating the plurality of candidate sequences, comprising, for at least one chromatography step in the at least one candidate sequence: accessing a data set associated with the at least one chromatography step, the data set comprising: first data indicative of a behavior of the at least one pharmaceutical product with respect to the at least one chromatography step; and second data indicative of a behavior of the at least one impurity with respect to the at least one chromatography step; and scoring the at least one candidate sequence based at least in part on the data set.
In some embodiments, a method is provided, for characterizing at least one behavior of at least one biologically produced product, the method comprising acts of: for each partitioning step of a plurality of partitioning steps: using the partitioning step to partition one or more mixtures comprising the at least one biologically produced product and/or the at least one impurity; generating a data set for the partitioning step, the data set comprising: first data indicative of a behavior of the at least one biologically produced product with respect to the partitioning step; and second data indicative of a behavior of the at least one impurity with respect to the partitioning step; and storing the data set in association with the partitioning step.
In some embodiments, a method is provided, comprising acts of: generating a plurality of first data sets for a first mixture from a biological expression system, comprising, for each chromatography step of a plurality of chromatography steps: using the chromatography step to partition the first mixture and thereby obtain at least one first fraction; analyzing the at least one first fraction to generate one or more first data patterns indicative of chemical composition of the at least one first fraction; and storing a first data set in association with the chromatography step and the at least one first fraction, wherein the first data set comprises the one or more first data patterns indicative of the chemical composition of the at least one first fraction; and generating a plurality of second data sets for a second mixture comprising a target protein, comprising, for each chromatography step of the plurality of chromatography steps: using the chromatography step to partition the second mixture and thereby obtain at least one second fraction; analyzing the at least one second fraction to generate one or more second data pattern indicative of chemical composition of the at least one second fraction; and storing a second data set in association with the chromatography step and the at least one second fraction, wherein the second data set comprises the one or more second data patterns indicative of the chemical composition of the at least one second fraction.
In some embodiments, a method is provided, for generating and evaluating candidate sequences of chromatography steps to partition at least one pharmaceutical product from at least one process-related impurity and/or at least one product-related impurity, the at least one pharmaceutical product being biologically produced, the method comprising acts of: generating a plurality of candidate sequences of chromatography steps, wherein at least one candidate sequence of the plurality of candidate sequences comprises a plurality of chromatography steps in a specified order; evaluating the plurality of candidate sequences, comprising, for at least one chromatography step in the at least one candidate sequence: accessing a data set comprising first data indicative of a behavior of the at least one process-related impurity with respect to the at least one chromatography step; accessing a data set comprising second data indicative of a behavior of the at least one product-related impurity with respect to the at least one chromatography step; assigning a combined score to each of the candidate sequences, wherein the combined score is a combination of a first score based on the at least one process-related impurity and a second score based on the at least one product-related impurity; and ranking the candidate sequences based on the combined scores.
In some embodiments, at least one computer-readable medium is provided, having stored thereon executable instructions that, when executed by at least one processor, perform one or more of the acts of any of the above methods.
In some embodiments, a system comprising: at least one processor; and at least one computer-readable medium having stored thereon executable instructions that, when executed by the at least one processor, perform one or more of the acts of any of the above methods.
Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.
Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures:
Aspects of the present disclosure relate to systems and methods for manufacturing biologically-produced products, which may include pharmaceutical and/or protein products. Some of the systems described herein comprise an upstream component comprising a bioreactor and at least one filter (e.g., a filter probe) integrated with a downstream component comprising a purification module comprising at least a first partitioning unit and a second partitioning unit. In some embodiments, these integrated biological manufacturing systems may be operated under continuous conditions and may be capable of efficiently producing pure, high-quality pharmaceutical and/or protein products. In some embodiments, these integrated biological systems may be operated as semi-continuous processes. A semi-continuous process is a process characterized by periods of continuous operation intentionally interrupted by periods of non-operation.
In some cases, the downstream component of the integrated system can be designed through a framework that involves generating a plurality of data sets and using the plurality of data sets to evaluate candidate sequences of partitioning units and/or partitioning conditions. This framework may, in some cases, allow for the efficient design of systems to capture and purify various biologically produced products, such as pharmaceutical and/or protein products. Accordingly, the systems and methods described herein may provide a flexible platform for efficiently manufacturing a wide array of biologically produced products, which may include pharmaceutical and/or protein products.
Typically, in conventional approaches, downstream processing systems are designed based on engineers' experience and intuition. For instance, a chromatography engineer may select a bind-elute step based on a target product to be recovered, identify a fraction in which the target product is eluted, and analyze the fraction to determine which impurities are present. The engineer may then select and experiment with one or more subsequent chromatography steps that the engineer believes will be effective in partitioning the target product from the identified impurities. The engineer may repeat this design process with different chromatography techniques (e.g., ion exchange chromatography, size exclusion chromatography, hydrophobic charge induction chromatography, etc.) and/or different sets of parameters (e.g., different chromatographic resins, pH gradient vs. salt gradient, etc.), and may select a sequence of chromatography steps that achieve one or more objectives (e.g., speed, cost, purity, yield, etc.).
The inventors have recognized and appreciated that such a highly subjective and sequential design process is typically costly and time consuming, and may result in suboptimal downstream processes. For instance, choosing chromatography techniques and/or parameters in a step-by-step fashion may be akin to a greedy algorithm that chooses a local optimum at each step. Such an approach may not always lead to a global optimum. For example, for a list of two candidate processes and a starting mixture of a product and 100 impurities, a first candidate process may remove impurities 1-90 and a second candidate process may remove impurities 1-100, so the second candidate process may be more desirable overall. However, the first candidate process may comprise a partitioning step F that is effective in removing impurities 1-80 followed by a partitioning step G that is effective in removing impurities 70-90, while the second candidate process may comprise a partitioning step H that is effective in removing impurities 40-100 followed by partitioning step J that is effective in removing impurities 1-50. A greedy algorithm may select the first candidate process because the partitioning step F is more effective than the partitioning step H, even though the second candidate process is more effective overall. In some embodiments, an improved framework is provided for downstream process (e.g. purification) design. The framework may include generating a plurality of data sets that may be used to generate and evaluate candidate processes, where each candidate process may include a sequence of partitioning steps. In some embodiments, a redundant sequence may be selected for assuring clearance (e.g., 1-80 and 60-100).
In some embodiments, a partitioning step may be represented based on a partitioning technique and/or a set of one or more parameters for the partitioning technique. Examples of partitioning techniques include, but are not limited to, chromatography, filtration, crystallization, density differential separations, extraction, applied force-based separations, or any of a wide variety of other separation or purification techniques known in the field of biologically-based production of pharmaceutical products. Non-limiting illustrative suitable categories of partitioning techniques, non-limiting examples of partitioning techniques falling under those categories, and parameters influencing the outcome of the partitioning techniques are described in Table 1.
In some embodiments, candidate processes may be generated by first grouping available partitioning steps into multiple clusters so that partitioning steps within each cluster are functionally similar. Two partitioning steps may be grouped into a same cluster based on functional similarity, even if the partitioning steps are based on different partitioning techniques. An ordering of clusters may then be selected (where a cluster may, although need not, appear multiple times), and a candidate process may be generated by selecting a partitioning step from each cluster while maintaining the ordering of the clusters. In some embodiments, different orderings of clusters may be selected. In this manner, more candidate processes may be considered, compared to where candidate processes are designed in a step-by-step fashion. As a result, a better process may be discovered. In some embodiments, a candidate process may be evaluated based on one or more criteria, such as a number of steps in the sequence, need for an adjustment of one or more conditions between two consecutive steps (e.g., pH, salt concentration, etc.), cost, product purity, product yield, product concentration, product activity, etc. For instance, a numerical score may be generated, which may, although need not, reflect a penalty for one or more undesirable aspects of a candidate sequence. As an example, a penalty may be imposed on any adjustment required between two consecutive steps, and an even higher penalty may be imposed if the adjustment is costly and/or difficult to implement (e.g., an adjustment of salt concentration). One or more best scoring processes may then be selected.
In some embodiments, one or more best scoring processes may be evaluated using one or more known experimental methods to refine one or more conditions for one or more partitioning step. For instance, for a chromatography step, column load conditions, wash conditions, elution conditions, etc. may be refined.
In some embodiments, a criterion may relate to a degree to which partitioning steps in a candidate process are orthogonal to each other. Orthogonality between two or more partitioning steps, as is understood by the skilled practitioner, is achieved when the partitioning steps have selectivity for different impurities or sets of impurities. For instance, a partitioning step A and a subsequent partitioning step B may be considered orthogonal if the partitioning step B is effective in removing impurities that co-elute from the partitioning step A with a target product. Thus, a higher degree of orthogonality may be desirable. For example, for a set of three candidate partitioning steps—partitioning step C, partitioning step D, and partitioning step E—and a starting mixture of a product and 100 impurities, the step C may be effective in removing impurities 1-80, the step D may be effective in removing impurities 40-90, and step E may be effective in removing impurities 81-100. A two-step process starting with the step C followed by the step D may recover the product with impurities 90-100 still present. A two-step process starting with the step C followed by the step E, by contrast, may recover the product with zero impurities. Although the step D on its own may be a better performing partitioning step than the step E, the step D may not be sufficiently orthogonal to the step C to result in a high performing process. On the other hand, the step E on its own may be a poor partitioning step, but together with the step C may provide a high performing process because all impurities are removed from the product by the end of the process.
In some embodiments, a criterion may relate to a degree to which partitioning steps in a candidate process are complementary to each other. Complementarity between two or more partitioning steps, as is understood by the skilled practitioner, is achieved when an outcome of a process incorporating the partitioning steps results in overall high impurity removal, even in the absence of orthogonality. For instance, a partitioning step A and a subsequent partitioning step B may be considered complementary if the partitioning step A is effective in increasing a surface charge on impurity M, and the partitioning step B is effective in binding the impurity M only when the impurity M's surface charge is above a certain threshold. Thus, passing the impurity M through the partitioning step A may improve effectiveness of the partitioning step B in removing the impurity M from the target product. Thus, a higher degree of complementarity may be desirable.
In some embodiments, a plurality of weights may be assigned, respectively, to a plurality of criteria. Such weights may reflect relative importance and abundance among the plurality of criteria. For instance, a numerical score may be generated for each criterion, and the plurality of weights may be used to combine the plurality of scores (e.g., via a weighted sum) into an overall score.
The inventors have recognized and appreciated that downstream process design may be informed by an understanding of how certain impurities behave with respect to a partitioning technique, relative to how a target product behaves with respect to the partitioning technique. Such an understanding may be used to facilitate in silico evaluation of the partitioning technique, even where physical properties of the impurities are not precisely characterized. For instance, an understanding of how much of certain impurities is co-eluted with the target product in a bind-elute chromatography step, and/or how much of the impurities is eluted after the target product in a subsequent flow-through chromatography step, may be sufficient for evaluating orthogonality of the chromatography steps, without having to precisely identify the impurities or their physical properties.
Accordingly, in some embodiments, a downstream process design framework may include, for each partitioning step of a plurality of partitioning steps, data indicative of how the target product behaves with respect to the partitioning step, as well as data indicative of how one or more impurities behave with respect to the partitioning step. For example, the target product may be a biologically produced product (which may be a pharmaceutical and/or protein product), and the one or more impurities may include one or more upstream process related impurities such as host cell proteins, host cell DNA, media components, etc., and/or one or more downstream process related impurities such as leachants, extractables, and residual proteins such as Protein A used in resins for chromatography. Additionally, or alternatively, the one or more impurities may include one or more product-related impurities such as product variants, product aggregates, etc. As illustrative, non-limiting examples, product-related impurities may comprise N-terminal additions, substitutions, and/or deletions; C-terminal additions, substitutions, and/or deletions; one or more misincorporated amino acids; acidic or basic species; one or more post-translational modifications, including but not limited to glycosylation, glycation, trisulfide bonds, oxidation, and deamidation; proteolytically-cleaved variants; charged variants; and/or product aggregates.
The inventors have recognized and appreciated that, by allowing different partitioning techniques to be used within a single process, more candidate processes may be considered, and as a result a better process may be found. For instance, a process may begin with centrifugation to remove some impurities by a density differential, followed by a filtration step and then a chromatography step.
Accordingly, in some embodiments, a downstream process design framework may include a plurality of data sets for each of a plurality of partitioning techniques. For each partitioning technique, the plurality of data sets may include target product data and impurity data (e.g., as described above) for each partitioning step of a plurality partitioning steps that use the partitioning technique. For example, the partitioning technique may be IEC, and the plurality partitioning steps may be a plurality of IEC steps, each with a different parameter set (e.g., different resins, buffers, etc.) The plurality of data sets for IEC may include target product data and impurity data for each parameter set.
In some embodiments, data sets may be stored in a manner that allows retrieval based on partitioning technique and/or one or more parameters.
As discussed above, the inventors have recognized and appreciated that precise characterizations of physical properties of impurities may not be necessary in evaluating candidate processes. In some embodiments, a data set for a partitioning step may be generated by conducting one or more experiments using the partitioning step. For instance, one or more experiments may be conducted with each parameter set of a plurality of parameter sets. Raw and/or processed data from the one or more experiments may be stored in the data set.
In some embodiments, one or more experiments may be designed to study how a target product, and/or one or more impurities, behave with respect to the partitioning technique and/or the parameter set. As an example, two IEC experiments may be conducted using the same resin, pH gradient, and operating conditions. The first experiment may be conducted on a cell culture fluid where a target protein is not expressed, while the second experiment may be conducted on a pure solution of the target protein. Thus, data from the first experiment may be indicative of how one or more process related impurities behave with respect to the partitioning technique and the parameter set, whereas data from the second experiment may be indicative of how the target protein behaves with respect to the partitioning technique and the parameter set.
As another example, two IEC experiments may be conducted using the same resin, pH gradient, and operating conditions, and the first experiment may be conducted on a cell culture fluid where a target protein is not expressed, but the second experiment may be conducted on a cell culture fluid where the target protein is expressed. The same organism may be used to produce both cell culture fluids. Thus, data from the first experiment may be indicative of how one or more process related impurities behave with respect to the partitioning technique and the parameter set, whereas data from the second experiment may be indicative of how the target protein and one or more product-related impurities (e.g., one or more variants and/or aggregates of the target protein) behave with respect to the partitioning technique and the parameter set.
As yet another example, two IEC experiments may be conducted using the same resin, pH gradient, and operating conditions, but the first experiment may be conducted on a cell culture fluid where a first target protein is expressed, while the second experiment may be conducted on a cell culture fluid where a second target protein is expressed. The same organism may be used to produce both cell culture fluids, and the two target proteins may differ in one or more properties (e.g., different molecular weights). The inventors have recognized and appreciated that the two cell culture fluids may have similar impurities and therefore similar impurity behavior. Thus, impurity behavior may be determined by comparing results from the two experiments.
Any suitable raw and/or processed data may be stored in a data set for a partitioning technique. For instance, any one or more suitable techniques may be used to analyze an output of a partitioning step (e.g., one or more fractions collected from a chromatography step), and an outcome of the analysis may be stored. Non-limiting examples of analysis techniques are listed in Table 2 below.
In some embodiments, an input of partitioning step 1 may include a cell culture fluid, which may be an output of an upstream process (e.g., a bioreactor process) using any suitable organism, such as yeast, Chinese hamster ovary (CHO), E. coli, etc. This cell culture fluid may include a target protein, as well as upstream process related impurities such as host cell proteins, host cell DNA, etc. The process 100 may be designed to remove these impurities and output a purified product at partitioning step P. Additionally, or alternatively, the process 100 may be designed to remove downstream process related impurities (e.g., leachants) and/or product-related impurities (e.g., product variants, product aggregates, etc.).
Although not shown in
In some embodiments, the process 100 may be selected from a set of candidate sequences of partitioning steps using one or more optimization techniques. For instance, a set of candidate sequences may be reduced by eliminating candidate sequences that do not satisfy one or more constraints. The remaining candidate sequences may be evaluated based on one or more criteria, and a selection may be made accordingly.
The number of partitioning steps P may be selected using a cost/benefit analysis. Typically, the greater the number of partitioning steps, the higher the purity of the product, which may stand as a benefit. However, a greater number of partitioning steps, typically, results in lower overall yields and higher overall costs. For pharmaceutical products, a minimum of two partitioning steps are generally required/recommended by the FDA. Generally, a maximum of ten partitioning steps are used in industrial separations, above which costs are prohibitive and/or product yields are too low. The design of the downstream process may involve minimizing the number of partitioning steps in the process while maintaining recovery of the target product, product purity, and/or product activity. However, aspects of the present disclosure are not limited to the use of ten or fewer, or any particular number of, partitioning steps. For instance, in some embodiments, four or more steps may be used, six or more steps may be used, ten or more steps may be used, etc.
As discussed above, the inventors have recognized and appreciated that downstream process design may be informed by an understanding of how certain impurities behave with respect to a partitioning technique, relative to how a target product behaves with respect to the partitioning technique. Accordingly, in some embodiments, one or more experiments may be designed to study how a target product and one or more impurities behave with respect to a partitioning technique and a parameter set.
In some embodiments, the target product may be a protein to be produced using a cell culture. An input of the experiment 210 may include a pure sample of the target product dissolved in a solution (e.g., an aqueous solution), while an input of the experiment 220 may include a cell culture fluid in which the target product is not expressed. However, this arrangement is not required, as in some embodiments, an input of the experiment 210 may include a cell culture fluid in which the target product is expressed, where the cell culture fluid is produced using the same organism as for the input of the experiment 220.
In some embodiments, one or more fractions collected from the experiment 210 may be analyzed using one or more analysis techniques, and resulting data 215 may be stored (and may subsequently be used to design downstream processes, e.g., as discussed below in connection with
The inventors have recognized and appreciated that, in some instances, the data 225 (behavior of one or more impurities) may be generated once for a host organism, and may be re-used when a different pharmaceutical and/or protein product is to be made using the same host organism. In this manner, only the data 215 (behavior of target product) may be re-generated when a different product is to be produced using the same host. Likewise, the data 215 (behavior of target product) may be generated once for a target product, and may be re-used when the target product is to be made using a different host organism. In this manner, only the data 225 (behavior of one or more impurities) may be re-generated when the same target product is to be produced using a different host. This may allow efficient design of downstream processes for different combinations of products and hosts.
In some embodiments, the data 215 and the data 225 may be stored in a data set 230, which may be associated with one or more tags indicating the partitioning step used in the experiments 210 and 220 and/or the one or more analysis techniques used to analyze the collected fractions. For instance, there may be a tag indicating a partitioning technique used in the partitioning step (e.g., IEC, SEC, HCIC, TFF, etc.), one or more tags indicating one or more parameters for the partitioning technique (e.g., resin, gradient, input pH, flow rate, etc. for a chromatography step), and a tag indicating an analysis technique (e.g., LC-MS, ELISA, MALDI, UV, SDS-PAGE, IEF, etc.). In this manner, the data set 230 may be retrieved by querying based on one or more tags.
Although various details of implementation are described herein, it should be appreciated that such details are illustrative of more general systems and methods. As one example, in some embodiments, the data 215 (behavior of target product) and the data 225 (behavior of one or more impurities) may be compared and one or more differences may be stored instead of, or in addition to, the data 215 and the data 225. As another example, aspects of the present disclosure are not limited to the use of tags, as other ways to organize and/or search for data may also be used.
In the example shown in
In some embodiments, a pH gradient may be used, in which case the two mobile phase materials 310 and 315 may have different pH values. The inflow may begin with a first ratio (e.g., 100% mobile phase material 310 and 0% mobile phase material 315) at a first time, and end with a second ratio (e.g., 0% mobile phase material 310 and 100% mobile phase material 315) at a second time. The ratio may vary in any suitable manner between the first and second times. For example, the ratio may vary continuously (e.g., linearly or according to some other continuous function), and/or there may be one or more discrete changes.
In some embodiments, a salt gradient may be used, instead of, or in addition to, a pH gradient. In this case, the two mobile phase materials 310 and 315 may have different concentrations of one or more salts, and a ratio of these materials may change over time in any suitable manner as described above.
In some embodiments, one or more fractions may be collected from an outflow of the structure. For instance, fraction 1 may be collected during a first time interval, fraction 2 may be collected during a second time interval, etc. These time intervals may, although need not, be non-overlapping consecutive intervals.
In the example shown in
In some embodiments, the partitioning step 300 may be a flow-through step. In a flow-through step, the target product flows through the column containing the stationary phase material, leaving one or more impurities of interest bound to the stationary phase material. However, aspects of the present disclosure are not limited to the use of a bind-elute step or a flow-through step, or any chromatography step at all.
In some embodiments, the partitioning step 300 may be used to study a behavior of a target product and/or one or more impurities of interest, and each fraction of the plurality of fractions may be analyzed to obtain data relating to chemical composition of the fraction. In some embodiments, the partitioning step 300 may be used to partition the target product from the one or more impurities of interest, and one or more fractions in which the target product is eluted (e.g., fraction RElute shown in
As another example, an input to the illustrative partitioning step 300 may include a pure sample of a target protein dissolved in an aqueous solution, so that the chromatograms 400-1, . . . , 400-N may represent behavior of the target protein. Thus, one or more of the chromatograms 400-1, . . . , 400-N may be stored as the illustrative data 215 shown in
In the example shown in
The inventors have recognized and appreciated that it may be efficient to use a rapid chromatography technique to analyze one or more fractions. A chromatography technique may be considered “rapid” if a fraction may be analyzed in no more than 15 minutes. However, aspects of the present disclosure are not limited to the use of a rapid chromatography technique.
It should also be appreciated that aspects of the present disclosure are not limited to the use of chromatography to analyze fractions collected from a partitioning step. For example, in some instances, product-related impurities (e.g., product variants, product aggregates, etc.) may not be expected to be present in significant quantities, and a collected fraction may be analyzed directly using any suitable analysis technique, without being further partitioned by a higher resolution chromatography technique.
In some embodiments, each of the chromatograms 400-1, . . . , 400-N may be generated by measuring UV absorbance at an outflow of an analytical chromatography step and plotting the measurement against time. UV absorbance may be measured at one or more wavelengths, and the chromatograms 400-1, . . . , 400-N shown in
It should be appreciated that aspects of the present disclosure are not limited to the use of chromatography or UV absorbance analysis to analyze fractions collected from a partitioning step. Other examples of analytical techniques include, but are not limited to, LC-MS, ELISA, MALDI, SDS-PAGE, IEF, etc. Any one or more of these and/or other analytical techniques may be used to generate behavior data for a target product and/or one or more impurities of interest.
In some embodiments, one or more of the chromatograms 400-1, . . . , 400-N may be stored in discretized form, for example, to reduce storage and/or to speed up one or more optimization techniques that use the chromatograms 400-1, . . . , 400-N to evaluate candidate downstream processes. For instance, a plurality of time intervals t1, . . . , tK may be selected. These intervals may, although need not, be non-overlapping consecutive intervals. For each i=1, . . . , K, and n=1, N, an integral ai (n) of the chromatogram 400-n may be calculated over the interval ti, as shown in
In the example shown in
Examples of partitioning techniques and associated parameters include, but are not limited to, those listed in Table 1 above. However, it should be appreciated that aspects of the present disclosure are not limited to the use of any particular partitioning technique or parameter.
In some embodiments, one or more output conditions may be stored in a data set for a partitioning step. Examples of output conditions include, but are not limited to, pH, temperature, flow rate, etc. In some embodiments, such an output condition may be used to determine whether an adjustment is needed to match an input condition of an immediately following partitioning step.
In some embodiments, each data set may include data indicative of how a target protein and/or one or more impurities of interest behave with respect to the partitioning step associated with the data set. Such data may be obtained using any one or more suitable analytical techniques. For instance, chromatography in combination with UV absorbance analysis may be used to generate a plurality of chromatograms (e.g., as shown in
Some examples of analytical techniques are provided below. However, it should be appreciated that aspects of the present disclosure are not limited to the use of any particular analytical technique. Non-limiting illustrative suitable categories of analytical techniques and non-limiting examples of analytical techniques falling under those categories, are described in Table 2.
As discussed above, the inventors have recognized and appreciated that data sets such as the illustrative data sets shown in
In some embodiments, the illustrative data sets shown in
At act 605, a plurality of available partitioning steps may be generated. As discussed above, a partitioning step may be represented based on a partitioning technique (e.g., IEC, SEC, HCIC, TFF, etc.) and/or one or more parameters for the partitioning technique. Accordingly, in some embodiments, a plurality of parameter sets may be generated for each available partitioning technique. As an example, for chromatography, different combinations of available resin, gradient, and/or one or more conditions (e.g., temperature, flow rate, etc.) may be generated. Each such parameter set may represent a different partitioning step.
In some embodiments, one or more partitioning techniques and/or one or more parameters may be determined based on user input. For instance, a user interface may be provided via which a user may indicate which one or more partitioning techniques and/or materials (e.g., chromatography resins and/or buffers) are available. Additionally, or alternatively, the user interface may allow a user to specify one or more conditions (e.g., based on available equipment, product to be made, amount of product desired, amount of time available, etc.).
At act 610, a plurality of candidate sequences may be generated. The inventors have recognized and appreciated that, in many instances, at least two partitioning steps may be needed to effectively partition a target product from one or more impurities, and/or at least two partitioning steps may be desired to meet expectations of regulatory approval agencies. The inventors have further recognized and appreciated that, in many instances, three partitioning steps may be sufficient to effectively partition a target product from one or more impurities.
Accordingly, in some embodiments, only two-step sequences and three-step sequences are included. In some other embodiments, only two-step sequences or only three-step sequences are included. However, neither is required, as in some embodiments, sequences with more than three steps (e.g., more than four steps, more than five steps, more than six steps, more than seven steps, more than eight steps, more than nine steps, more than ten steps, etc.), and/or single partitioning steps (i.e., sequences of length 1), may be included. For instance, the inventors have recognized and appreciated that one or more of the techniques described herein may be used to speed up evaluation of candidate sequences, so that a larger length (and hence a broader pool of candidate sequences) may be efficiently evaluated.
In some embodiments, every possible sequence of a desired length may be included. However, the inventors have recognized and appreciated that such an inclusive approach may result in too many candidate sequences, because it may take too much time and/or computing resource to evaluate all of the candidate sequences. Accordingly, in some embodiments, one or more constraints may be used during generation of candidate sequences.
In some embodiments, a sequence may be included only if the sequence recovers a target product. The sequence may be considered to recover the target product if, for each partitioning step in the sequence, no target product is retained, and/or all target product is available for a next step in the sequence. For example, in a case where a partitioning step in a candidate sequence is a chromatography step in a column format, whether or not the partitioning step recovers the product may be determined by performing a regeneration process for the chromatography column and analyzing an eluting solution by spectrophotometry to determine a presence or absence of the target product.
In some embodiments, one or more constraints may relate to an initial step in a sequence. As one example, resins may be categorized as multimodal cation (MMC), multimodal anion (MMA), HCIC, etc., and a constraint may be imposed based on resin category. For instance, a constraint may prohibit using a partitioning step with an HCIC resin as a capture step.
As another example, partitioning steps may be categorized as bind-elute or flow-through, and a constraint may be imposed accordingly. In some embodiments, categorization of partitioning steps may be performed based on behavior data of a target product. For instance, target product behavior data for a partitioning step may be accessed (e.g., from the illustrative data arrangement 500 shown in
In some embodiments, a constraint may be imposed according to how a partitioning step is categorized. For instance, only those partitioning steps categorized as bind-elute steps may be used for capture (e.g., to concentrate the product early in the process). In some embodiments, one or more constraints may relate to a degree of orthogonality between steps. As an example, a same resin may not be used in two different steps in a sequence, as using a resin a second time may provide minimal benefit.
In some embodiments, one or more constraints may relate to connectivity between steps. As an example, an output condition (e.g., pH, salt concentration, etc.) of a partitioning step must sufficiently match an input condition of an immediately following partitioning step in a sequence of partitioning steps. Otherwise, the sequence may not be included as a candidate sequence. In this manner, no adjustment may be needed between steps, which may facilitate integrated manufacturing.
Any suitable combination of these and/or other constraints may be used during generation of candidate sequences, as aspects of the present disclosure are not limited to the use of any particular constraint, or any constraint at all.
Returning to
At act 620, remaining candidate sequences may be scored, and one or more best scoring sequences may be output (e.g., stored in a computer-readable file, shown to a user on a computer screen, communicated to a computer program for automating a biological manufacturing plant, etc.). In some embodiments, all candidate sequences may be output (e.g., in ranked order).
The scoring may be done in any suitable manner. In some embodiments, a candidate sequence may be scored based on one or more criteria, such as a number of steps in the sequence, lack of need for an adjustment between two consecutive steps (e.g., of pH, conductivity, etc.), cost, product purity, product yield, product concentration, product activity, etc. For instance, a numerical score may be generated, which may, although need not, reflect a penalty for one or more undesirable aspects of a candidate sequence. As an example, a penalty may be imposed on any adjustment required between two consecutive steps, and an even higher penalty may be imposed if the adjustment is costly and/or difficult to implement (e.g., a salt concentration adjustment). However, it should be appreciated that aspects of the present disclosure are not limited to the use of a penalty score. In some embodiments, a score may be generated that reflects a reward, in addition to, or instead of, a penalty score.
In some embodiments, a score may be calculated that is indicative of a degree to which partitioning steps in a candidate sequence are orthogonal to each other. For instance, a partitioning step A and a subsequent partitioning step B may be considered orthogonal if the partitioning step B is effective in removing impurities that co-elute with a target product in the partitioning step A.
As an example, for an embodiment involving a sequence of chromatographic partitioning steps, a penalty score may be calculated as follows using behavior data of one or more impurities of interest.
In this example, there are P partitioning steps in each candidate sequence. For each i=1, . . . , K, j=1, . . . , P, and n=1, . . . , N, ai,j(n) is an integral, over the ith time interval, of a chromatogram obtained at a given wavelength from the nth fraction (e.g., as shown in
In the illustrative formula above, F denotes a set of flow-through steps, B denotes a set of bind-elute steps, and fraction RElute(j) is a fraction in which the target product is eluted during the jth partitioning step. The value RElute(j) may be determined based on target product behavior data for the jth partitioning step, which may be accessed (e.g., from the illustrative data arrangement 500 shown in
Thus, for a bind-elute step (j in the set B), the above formula penalizes impurities co-eluted with the target product (as reflected by the value ai,j (RElute(j))). By contrast, for a flow-through step (j in the set F), the above formula rewards impurities eluted after the target product (as reflected by the reciprocal of the sum of the values ai,j(n) for n greater than RElute(j)).
The inventors have recognized and appreciated that the above formula may functionally captures orthogonality. For example, if the term Ai,j of a flow-throw step is large, the product Πj=1pAi,j may be small even if the term the term Ai,j of a bind-elute step in the same sequence is large. However, it should be appreciated that aspects of the present disclosure are not limited to the use of the above formula, or any formula designed based on orthogonality. For instance, in some embodiments, one or more scores may be calculated to capture complementarity, instead of, or in addition to, orthogonality.
In some embodiments, one or more best scoring processes may be evaluated using one or more known experimental methods to refine one or more conditions for one or more partitioning step. For instance, for a chromatography step, column load conditions, wash conditions, elution conditions, etc. may be refined.
As discussed above, the inventors have recognized and appreciated that some partitioning steps may be functionally similar to each other, even if the partitioning steps use different partitioning techniques and/or different parameters (e.g., different resins, gradients, etc.). For example, two partitioning steps may produce similar results when used to partition a target product from one or more impurities.
Process 3600 may use data sets such as the illustrative data sets shown in
At act 3605, a plurality of available partitioning steps may be generated, and product retention data may be read for each available partitioning step. As discussed above, a partitioning step may be represented based on a partitioning technique (e.g., IEC, SEC, HCIC, TFF, etc.) and/or one or more parameters for the partitioning technique. Accordingly, in some embodiments, a plurality of parameter sets may be generated for each available partitioning technique. As an example, for chromatography, different combinations of available resin, gradient, and/or one or more conditions (e.g., temperature, flow rate, etc.) may be generated. Each such parameter set may represent a different partitioning step.
In some embodiments, one or more partitioning techniques for removal of process-related impurities (e.g., host-related impurities) and/or product-related impurities and/or one or more parameters may be determined based on user input. For instance, a user interface may be provided via which a user may indicate which one or more partitioning techniques and/or materials (e.g., chromatography resins and/or buffers) are available. Additionally, or alternatively, the user interface may allow a user to specify one or more conditions (e.g., based on available equipment, product to be made, amount of product desired, amount of time available, etc.).
At act 3610, a plurality of candidate sequences for removal of process-related impurities (e.g., host-related impurities) and/or product-related impurities may be generated. As described above, the inventors have recognized and appreciated that, in many instances, sequences with two and/or three partitioning steps may be desirable. However, in some embodiments, sequences with more than three steps (e.g., more than four steps, more than five steps, more than six steps, more than seven steps, more than eight steps, more than nine steps, more than ten steps, etc.), and/or single partitioning steps (i.e., sequences of length 1), may be included. For instance, the inventors have recognized and appreciated that one or more of the techniques described herein may be used to speed up evaluation of candidate sequences, so that a larger length (and hence a broader pool of candidate sequences) may be efficiently evaluated.
In some embodiments, every possible sequence of a desired length may be included. However, the inventors have recognized and appreciated that such an inclusive approach may result in too many candidate sequences for removal of process-related impurities (e.g., host-related impurities) and/or product-related impurities, because it may take too much time and/or computing resource to evaluate all of the candidate sequences. Accordingly, in some embodiments, one or more constraints may be used during generation of candidate sequences.
In some embodiments, a sequence for removal of process-related impurities (e.g., host-related impurities) and/or product-related impurities may be included only if the sequence recovers a target product. The sequence may be considered to recover the target product if, for each partitioning step in the sequence, no target product is retained, and/or all target product is available for a next step in the sequence. For example, in a case where a partitioning step in a candidate sequence is a chromatography step in a column format, whether or not the partitioning step recovers the product may be determined by performing a regeneration process for the chromatography column and analyzing an eluting solution by spectrophotometry to determine a presence or absence of the target product.
In some embodiments, one or more constraints may relate to an initial step in a sequence. As one example, resins may be categorized as multimodal cation (MMC), multimodal anion (MMA), HCIC, etc., and a constraint may be imposed based on resin category. For instance, a constraint may prohibit using a partitioning step with an HCIC resin as a capture step.
As another example, partitioning steps may be categorized as bind-elute or flow-through, and a constraint may be imposed accordingly. In some embodiments, categorization of partitioning steps may be performed based on behavior data of a target product. For instance, target product behavior data for a partitioning step may be accessed (e.g., from the illustrative data arrangement 500 shown in
In some embodiments, a constraint may be imposed according to how a partitioning step is categorized. For instance, only those partitioning steps categorized as bind-elute steps may be used for capture (e.g., to concentrate the product early in the process). In some embodiments, one or more constraints may relate to a degree of orthogonality between steps. As an example, a same resin may not be used in two different steps in a sequence, as using a resin a second time may provide minimal benefit.
In some embodiments, one or more constraints may relate to connectivity between steps. As an example, an output condition (e.g., pH, salt concentration, etc.) of a partitioning step must sufficiently match an input condition of an immediately following partitioning step in a sequence of partitioning steps. Otherwise, the sequence may not be included as a candidate sequence. In this manner, no adjustment may be needed between steps, which may facilitate integrated manufacturing, or improve volumetric productivity or efficiency of operation of a process by reducing total numbers of steps required.
Any suitable combination of these and/or other constraints may be used during generation of candidate sequences, as aspects of the present disclosure are not limited to the use of any particular constraint, or any constraint at all.
Returning to
At act 3615, the data collected for each partitioning step in a sequence on process-related impurities (e.g., host-related impurities) may be mapped to the remaining candidate sequences (e.g., processes). Following this, at act 3625, the remaining candidate sequences may be scored or ranked for expected removal of process-related impurities (e.g., host-related impurities), and one or more best scoring sequences may be output (e.g., stored in a computer-readable file, shown to a user on a computer screen, communicated to a computer program for automating a biological manufacturing plant, etc.). In some embodiments, all candidate sequences for removal of process-related impurities (e.g., host-related impurities) may be output (e.g., in ranked order).
The scoring may be done in any suitable manner. In some embodiments, a candidate sequence for removal of process-related impurities (e.g., host-related impurities) may be scored based on one or more criteria, such as a number of steps in the sequence, lack of need for an adjustment between two consecutive steps (e.g., of pH, conductivity, etc.), cost, product purity, product yield, product concentration, product activity, etc. For instance, a numerical score may be generated, which may, although need not, reflect a penalty for one or more undesirable aspects of a candidate sequence. As an example, a penalty may be imposed on any adjustment required between two consecutive steps, and an even higher penalty may be imposed if the adjustment is costly and/or difficult to implement (e.g., a salt concentration adjustment). However, it should be appreciated that aspects of the present disclosure are not limited to the use of a penalty score. In some embodiments, a score may be generated that reflects a reward, in addition to, or instead of, a penalty score. As described above, in some embodiments, a score may be calculated that is indicative of a degree to which partitioning steps in a candidate sequence are orthogonal to each other. For instance, a partitioning step A and a subsequent partitioning step B may be considered orthogonal if the partitioning step B is effective in removing impurities that co-elute with a target product in the partitioning step A. However, it should be appreciated that aspects of the present disclosure are not limited to the use of the above formula, or any formula designed based on orthogonality. For instance, in some embodiments, one or more scores may be calculated to capture complementarity, instead of, or in addition to, orthogonality.
At act 3620, the data collected for each partitioning step in a sequence on product-related impurities may be mapped to the candidate sequences (e.g., processes) remaining from act 3610. Following this, at act 3630, the remaining candidate sequences may be scored or ranked for expected removal of product-related impurities, and one or more best scoring sequences may be output (e.g., stored in a computer-readable file, shown to a user on a computer screen, communicated to a computer program for automating a biological manufacturing plant, etc.). In some embodiments, all candidate sequences for removal of product-related impurities may be output (e.g., in ranked order).
The scoring may be done in any suitable manner. In some embodiments, a candidate sequence for removal of product-related impurities may be scored in a manner described herein as being used for the scoring of candidate sequences for removal of process-related impurities (e.g., host-related impurities). In some embodiments, a candidate sequence for removal of product-related impurities may be scored based on a metric of selectivity. In a non-limiting illustrative embodiment, for each partitioning step, all chromatographic curves of the product and the product-related impurities may be normalized based on area under the curve. From there, in the same non-limiting illustrative embodiment, the percent overlap between the product and each product-related impurity may be considered for a given partitioning step in a given candidate sequence generated from act 3610. This selectivity metric may be used to score processes for removal of product-related impurities.
Following the performance of acts 3625 and 3630, a selection step may be performed at act 3635, wherein sequences (e.g., processes) are selected that perform well based on the performance rankings resulting from acts 3625 and 3630. Act 3635 selects sequences that perform well in removal of both process-related impurities (e.g., host-related impurities) and product-related impurities. As an illustrative example, each candidate sequence may be assigned a combined score comprising a combination (e.g., a weighted or non-weighted sum) of a first score based on removal of process-related impurities and a second score based on removal of product-related impurities. The candidate sequences may be ranked based on the combined scores, and act 3635 may select one or more top-ranking sequences.
In some embodiments, one or more top-ranking sequences may be evaluated using one or more known experimental methods to refine one or more conditions for one or more partitioning step. For instance, for a chromatography step, column load conditions, wash conditions, elution conditions, etc. may be refined.
As discussed above, the inventors have recognized and appreciated that some partitioning steps may be functionally similar to each other, even if the partitioning steps use different partitioning techniques and/or different parameters (e.g., different resins, gradients, etc.). For example, two partitioning steps may produce similar results when used to partition a target product from one or more impurities.
The inventors have recognized and appreciated that generation of candidate processes (e.g., at act 610 of the illustrative process 600 shown in
In some embodiments, an ordering of clusters may be selected (where a cluster may, although need not, appear multiple times), and a candidate process may be generated by selecting a partitioning step from each cluster while maintaining the ordering of the clusters. For instance, with reference to
In some embodiments, functional similarity may be defined explicitly based on existing knowledge. For instance, as discussed above in connection with
The inventors have further recognized and appreciated that functional similarity may depend on an analysis technique used to analyze fractions obtained using the partitioning steps. For instance, a first analytical technique (e.g., RPLC) may provide information regarding purity, while a second analytical technique (e.g., ELISA) may provide information regarding product concentration. As a result, two partitioning steps may be functionally similar with respect to the first analytical technique (e.g., similar purities) but not with respective to the second analytical technique (e.g., different concentrations), or vice versa.
In some embodiments, clustering may be performed based on multiple analytical techniques. For instance, two partitioning steps may be considered functionally similar if the steps are functionally similar with respect to each of the analytical techniques (e.g., similar concentration and similar purity). However, it should be appreciated that aspects of the present disclosure are not limited to the use of multiple analytical techniques to perform clustering, or any clustering at all. Thus, the inventors have recognized and appreciated that fractions obtained from partitioning experiments (e.g., chromatography screening experiments) may be analyzed, and data sets may be stored based on results of analyzing the fractions, and that such data sets may be used to efficiently develop downstream processes. For example, such data sets may be used to select downstream processes, where orthogonality is used as a criterion.
In some embodiments, one or more automated techniques may be used to perform the partitioning experiments rapidly. Examples of automated techniques include, but are not limited to, a robotic fluid handling workstation, pre-packed chromatography columns and/or filter plates, etc.). Such techniques may allow a large number of experiments to be conducted, and hence a large amount of data to be collected, in a small amount of time.
In some embodiments, the cell culture fluid may include multiple host cell proteins. Samples collected from the experiments may be analyzed using one or more suitable techniques. In a non-limiting example, a liquid chromatography mass spectrometry (LC/MS) technique may be used, and results may be compared to LC/MS data of known host cell proteins (e.g., those routinely detected in Pichia cell culture supernatant) to identify which host cell proteins are retained. Additionally, or alternatively, a retention percentage may be determined for each host cell protein. Thus, the data arrangement 705 may indicate which partitioning techniques and/or parameters are effective in removing which host cell proteins.
In the example shown in
In some embodiments, each data set may include data indicative of how one or more host cell proteins (e.g., a target protein and/or one or more impurities of interest) behave with respect to the partitioning step associated with the data set. For instance, each data set may include a retention percentage for each host cell protein. As discussed herein, the inventors have recognized and appreciated that data sets such as the illustrative data sets shown in
In some embodiments, the illustrative data sets shown in
At act 715-1, data may be collected and/or analyzed to map identities of host cell proteins to behaviors with respect to different partitioning techniques and/or parameters. For instance, data sets such as those shown in the illustrative data arrangement 705 of
At act 715-2, one or more biophysical characteristics of proteins may be considered. Examples of biophysical characteristics include, but are not limited to, isoelectric point (pI), secondary structure, tertiary structure, hydrophobicity, size, number of certain amino acids (e.g., number of lysines), etc. For each value (or range of values) for a biophysical characteristic, one or more host cell proteins having that value (or falling into that range of values) may be identified. At act 715-3, behaviors of such host cell proteins with respect to different partitioning techniques and/or parameters (e.g., as determined at act 715-1) may be analyzed to identify potential correlations. For instance, host cell proteins may be grouped based on a biophysical characteristic (e.g., pI), and one or more partitioning techniques and/or parameters suitable for separating host cell proteins with different values of that biophysical characteristic may be identified.
At act 715-4, one or more amino acid sequences may be mapped to one or more corresponding biophysical characteristics. For instance, given an amino acid sequence, a lookup may be performed in one or more databases to determine one or more biophysical characteristics of that protein.
At act 715-5, a predictive model may be built to predict, based on amino acid sequence, how different proteins may behave with respect to different partitioning techniques and/or parameters. For instance, the one or more biophysical characteristics identified at act 715-4 for each amino acid sequence may be used to retrieve behavior data with respect to different partitioning techniques and/or parameters, and the retrieved behavior data may be used to train a machine learning model.
In some embodiments, partitioning experiments may be performed to obtain data relating to how host-related impurities behave with respect to a partitioning technique (e.g., a chromatography technique).
In some embodiments, partitioning experiments may be performed to obtain data relating to how a target product and/or one or more product-related impurities behave with respect to a partitioning technique (e.g., a chromatography technique).
In some embodiments, chromatography experiments may be performed using various chromatography materials, including multimodal, ion exchange, hydrophobic charge induction, and salt tolerant chromatographic materials.
In some embodiments, chromatography experiments may be performed using various linear salt and pH gradients in a column format.
In some embodiments, chromatography experiments may be performed using batch adsorption at various salt and pH conditions.
In some embodiments, fractions obtained from partitioning experiments (e.g., chromatography screening experiments) may be analyzed using any suitable analytical technique, and analysis results may be used to quantify orthogonality.
In some embodiments, fractions obtained from partitioning experiments (e.g., chromatography screening experiments) may be analyzed using a high resolution chromatographic technique.
In some embodiments, fractions obtained from partitioning experiments (e.g., chromatography screening experiments) may be analyzed using a reversed phase based high resolution chromatographic technique.
In some embodiments, fractions obtained from partitioning experiments (e.g., chromatography screening experiments) may be analyzed using a size exclusion phase based high resolution chromatographic technique.
In some embodiments, fractions obtained from partitioning experiments (e.g., chromatography screening experiments) may be analyzed using a mass spectrometry technique.
In some embodiments, fractions obtained from partitioning experiments (e.g., chromatography screening experiments) may be analyzed using a chip based (e.g., microfluidic chip, assay chip) technique.
In some embodiments, results from analyzing fractions obtained from partitioning experiments (e.g., chromatography screening experiments) may be organized to facilitate retrieval and/or querying. For instance, a data set may be stored in association with one or more identifiers indicating partitioning technique used (e.g., IEC, SEC, HCIC, etc.), materials used (e.g., resin, gradient, etc.), fraction number, one or more detection parameters (e.g. wavelength of UV absorbance), etc.
In some embodiments, data from analyzing fractions obtained from partitioning experiments (e.g., chromatography screening experiments) may be discretized. For instance, a chromatogram resulting from analyzing the fractions using high resolution chromatography may be integrated over discrete time intervals to obtain discrete values, which may be stored as a vector.
In some embodiments, data relating to how a target product and/or one or more product-related impurities behave with respect to a partitioning technique (e.g., a chromatography technique) may be interrogated to identify sequences of partitioning steps which will recover the product while satisfying one or more constraints.
In some embodiments, one or more constraints may relate to a number of partitioning steps in a sequence, connectivity between steps, a mode of operation of a step, an ordering of steps, identities of steps, a material used (e.g., prohibition against use of a single resin in more than one step), etc.
In some embodiments, modes of operation may include flow-through, weak partitioning, bind-elute, isocratic, and/or gradient.
In some embodiments, data relating to how a target product and/or one or more product-related impurities behave with respect to various partitioning techniques, and/or data relating to how one or more host-related impurities behave with respect to various partitioning techniques, may be used to rank candidate sequences of portioning steps based on likelihoods of removing host-related impurities and product-related impurities, with appropriate weightings.
In some embodiments, one or more top ranking processes may be selected and process refinement may be performed to accommodate for effects of column load, column washes, and elution conditions, for example, to improve product purity and product recovery. For instance, multiple high ranking processes may be refined, and results may be compared.
In some embodiments, process refinement may include using column modeling to facilitate identification of process conditions which satisfy product specific purity and recovery requirements.
In some embodiments, one or more of the above described techniques may be used to design a downstream process for hGH from yeast (e.g., Pichia pastoris) cell culture supernatant.
In some embodiments, one or more of the above described techniques may be used to design a downstream process for GCSF from yeast (e.g., Pichia pastoris) cell culture supernatant.
In some embodiments, one or more of the above described techniques may be used to design a downstream process for interferon-alpha 2B from yeast (e.g., Pichia pastoris) cell culture supernatant.
The computer 800 may have one or more input devices and/or output devices, such as devices 806 and 807 illustrated in
As shown in
In some embodiments, one or more aspects of the present disclosure may be implemented on a server. As an example, one or more data sets such as the illustrative data sets 510 and 520 shown in
Any suitable computer may be used as a server. In some embodiments, a server may include a single computer, or multiple computers configured to perform parallel processing. Any suitable types of computers may be used, such as a desktops, laptops, tablets, smartphones, etc. Additionally, or alternatively, a server may include computing resources from one or more public and/or private clouds.
In some embodiments, one or more computers may be used to present a user interface (e.g., to receive user input and/or display results as discussed in connection with the illustrative process 600 shown in
Certain systems and methods described herein are systems and methods of manufacturing biologically-produced products, which may include pharmaceutical and/or protein products. In some cases, these systems and methods incorporate innovations in the upstream and/or downstream processes. In certain embodiments, the above-described framework involving generating a plurality of data sets and using the plurality of data sets to evaluate candidate sequences of partitioning units and/or partitioning conditions is used to design downstream processes for purifying at least one biologically-produced product. Some embodiments described herein are inventive systems and methods of manufacturing exemplary biologically-produced products, including G-CSF, a single-domain antibody (e.g., nanobody), hGH, and IFN-α2b. In certain of these embodiments, at least one step was designed using the above-described design framework.
As shown in
In some embodiments, biomanufacturing system 900 further comprises purification module 906. According to some embodiments, purification module 906 is fluidically connected (e.g., directly fluidically connected) to filter 904 and/or bioreactor 902. Purification module 906 may, in some embodiments, be configured to remove at least a first type of impurity and a second type of impurity from an output of filter 904 and/or bioreactor 902. In certain cases, purification module 906 comprises a first partitioning unit configured to remove at least the first type of impurity and a second partitioning unit configured to remove at least the second type of impurity. In certain embodiments, the first partitioning unit is fluidically connected (e.g., directly fluidically connected) to the second partitioning unit. In some embodiments, the sequence of partitioning units and/or partitioning conditions may be designed using the above-described framework.
It should be understood that direct fluidic connection between the first partitioning unit and the second partitioning unit may allow for compact design and efficient space utilization in the biomanufacturing system. In some conventional systems, direct fluidic connection between the first partitioning unit and the second partitioning unit would not be possible due to the need for additional processing steps (e.g., hold steps, modifications step) between the first partitioning unit and the second partitioning unit.
In operation, bioreactor 902 receives a first type of biological cells configured to express at least one biologically-produced product, according to some embodiments. In some embodiments, bioreactor 902 further receives feed stream 908 comprising a cell culture medium. The cell culture medium may, for example, be a growth cell culture medium configured to promote growth of the first type of biological cells and/or a production cell culture medium configured to promote expression of the at least one biologically-produced product. In some embodiments, the first type of biological cells are suspended in the cell culture medium, such that the reactor chamber of bioreactor 902 contains a suspension comprising the first type of biological cells and the cell culture medium. According to some embodiments, the first type of biological cells in the suspension proliferate and/or express the at least one biologically-produced product. In certain embodiments, the first type of biological cells secrete the at least one biologically-produced product into the cell culture medium of the suspension.
In some embodiments, at least a portion of the suspension is directed to flow through filter 904 as cell suspension stream 910 to produce first filtrate 912. According to some embodiments, first filtrate 912 comprises the at least one biologically-produced product and is lean in the first type of biological cells relative to cell suspension stream 910.
In some embodiments, first filtrate 912 is directed to flow to purification module 906 to produce purified filtrate 914. In some cases, purified filtrate 914 comprises the at least one biologically-produced product and is lean in at least a first type of impurity and a second type of impurity relative to first filtrate 912. According to certain embodiments, first filtrate 912 is directed to flow to a first partitioning unit of purification module 906 to remove at least the first type of impurity to produce a first partitioned filtrate that comprises the at least one biologically-produced product and is lean in the first type of impurity relative to first filtrate 912. In certain embodiments, the first partitioned filtrate is subsequently directed to flow to a second partitioning unit of purification module 906 to remove at least the second type of impurity to produce a second partitioned filtrate that comprises the at least one biologically-produced product and is lean in the second type of impurity relative to the first partitioned filtrate. In certain embodiments, the second partitioned filtrate is directed to flow to additional partitioning units within purification module 906. In certain embodiments, the second partitioned filtrate is collected as purified filtrate 914. As discussed in further detail below, in some embodiments, purified filtrate 914 is directed to flow to additional modules of biomanufacturing system 900 for further processing. In some embodiments, purified filtrate 914 is discharged from biomanufacturing system 900 as a biologically-produced product stream.
As shown in
In some embodiments, biomanufacturing system 900 further comprises optional formulation module 920. For example,
In some embodiments, biomanufacturing system 900 (as illustrated in any one of
In some embodiments, biomanufacturing system 900 (as illustrated in any one of
In some embodiments, biomanufacturing system 900 comprises at least two modules that are housed under a single isolator. For example, biomanufacturing system 900 may further comprise at least one isolator housing any two of the following modules: bioreactor 902, filter 904, purification module 906, adjustment module 916, and formulation module 920. In certain embodiments, at least one isolator houses one or more of the following combinations of modules: bioreactor 902, filter 904, and purification module 906; bioreactor 902, filter 904, and adjustment module 916; bioreactor 902, filter 904, and formulation module 920; adjustment module 916 and purification module 906; adjustment module 916 and formulation module 920; or purification module 906 and adjustment module 916. In certain embodiments, a single isolator houses all modules of biomanufacturing system 900 and aseptically isolates all components from the surrounding environment. For example, a single isolator may house bioreactor 902, filter 904, adjustment module 916, and purification module 906. As another example, a single isolator may house bioreactor 902, filter 904, adjustment module 916, purification module 906, and formulation module 920. In some embodiments, biomanufacturing system 900 comprises two or more isolators. In certain embodiments, each of the two or more isolators house at least two modules of biomanufacturing system 900.
As used herein, a direct fluid connection exists between a first component and a second component (and the two components are said to be “directly fluidically connected” to each other) when they are fluidically connected to each other such that the composition of a connecting fluid stream does not substantially change (i.e., no phase change occurs and no fluid component changes in relative abundance by more than 5%) as it flows from the first component to the second component. As an illustrative example, a first component and a second component are “directly fluidically connected” if a connecting fluid stream undergoes changes in pressure and/or temperature during passage from the first component to the second component, but not if the connecting fluid stream undergoes a separation step or a chemical reaction that substantially alters the chemical composition of the connecting fluid stream during passage from the first component to the second component. In some embodiments, one or more fluidic connections (e.g., direct fluidic connections) between one or more modules are “functionally closed” (e.g., assembled so as to maintain aseptic conditions within the one or more modules).
In some embodiments, biomanufacturing system 900 (as illustrated in any one of
According to certain embodiments, a biomanufacturing system (e.g., system 900) comprises a bioreactor (e.g., bioreactor 902) comprising a reactor chamber having an internal volume in the range of about 50 mL to about 1 L. In some cases, a feed stream (e.g., stream 908) and at least one of a purified filtrate stream (e.g., stream 914) and a formulated product stream (e.g., stream 922) of the biomanufacturing system each have a flow rate of at least about 0.01 mL/min, at least about 0.05 mL/min, at least about 0.1 mL/min, at least about 0.15 mL/min, at least about 0.2 mL/min, at least about 0.3 mL/min, at least about 0.4 mL/min, at least about 0.5 mL/min, at least about 0.6 mL/min, at least about 0.7 mL/min, at least about 0.8 mL/min, at least about 0.9 mL/min, at least about 1 mL/min, at least about 1.5 mL/min, or at least about 2 mL/min over a specified time period. In some embodiments, the feed stream (e.g., stream 908) and at least one of the purified filtrate stream (e.g., stream 914) and the formulated product stream (e.g., stream 922) of the biomanufacturing system each have a flow rate in the range of about 0.01 mL/min to about 0.1 mL/min, 0.01 mL/min to about 0.5 mL/min, about 0.01 mL/min to about 1 mL/min, about 0.01 mL/min to about 2 mL/min, about 0.03 mL/min to about 0.1 mL/min, 0.05 mL/min to about 0.1 mL/min, about 0.05 mL/min to about 0.5 mL/min, about 0.05 mL/min to about 1 mL/min, about 0.05 mL/min to about 2 mL/min, about 0.07 mL/min to about 0.2 mL/min, about 0.1 mL/min to about 0.4 mL/min, about 0.1 mL/min to about 1 mL/min, about 0.3 mL/min to about 1 mL/min, about 0.5 mL/min to about 1 mL/min, about 0.5 mL/min to about 2 mL/min, or about 1 mL/min to about 2 mL/min over a specified time period. The flow rate of any fluid stream within a biomanufacturing system may be measured using any suitable flow rate measurement device known in the art. Non-limiting examples of suitable flow rate measurement devices include ultrasonic flow meters, paddle wheel flow meters, rotameters, vortex flow meters, magnetic flow meters, turbine flow meters, and optical flow sensors (e.g., microparticle or bubble detection devices).
In some cases, each fluid stream of a biomanufacturing system comprising a reactor chamber having an internal volume in the range of about 50 mL to about 1 L (e.g., feed stream 908, cell suspension stream 910, first filtrate stream 912, adjusted filtrate stream 918, purified filtrate stream 914, formulated product stream 922) has a flow rate of at least about 0.01 mL/min, at least about 0.05 mL/min, at least about 0.1 mL/min, at least about 0.15 mL/min, at least about 0.2 mL/min, at least about 0.3 mL/min, at least about 0.4 mL/min, at least about 0.5 mL/min, at least about 0.6 mL/min, at least about 0.7 mL/min, at least about 0.8 mL/min, at least about 0.9 mL/min, at least about 1 mL/min, at least about 1.5 mL/min, or at least about 2 mL/min over a specified time period. In some embodiments, each fluid stream of this biomanufacturing system has a flow rate in the range of about 0.01 mL/min to about 0.1 mL/min, 0.01 mL/min to about 0.5 mL/min, about 0.01 mL/min to about 1 mL/min, about 0.01 mL/min to about 2 mL/min, about 0.03 mL/min to about 0.1 mL/min, 0.05 mL/min to about 0.1 mL/min, about 0.05 mL/min to about 0.5 mL/min, about 0.05 mL/min to about 1 mL/min, about 0.05 mL/min to about 2 mL/min, about 0.07 mL/min to about 0.2 mL/min, about 0.1 mL/min to about 0.4 mL/min, about 0.1 mL/min to about 1 mL/min, about 0.3 mL/min to about 1 mL/min, about 0.5 mL/min to about 1 mL/min, about 0.5 mL/min to about 2 mL/min, or about 1 mL/min to about 2 mL/min over a specified time period.
In some embodiments, the specified time period over which the flow rate is measured is at least about 1 hour, at least about 2 hours, at least about 5 hours, at least about 10 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 5 weeks, or at least about 10 weeks.
In some embodiments, a biomanufacturing system comprising a reactor chamber having an internal volume in the range of about 50 mL to about 1 L produces at least about 10 μg, at least about 50 μg, at least about 100 μg, at least about 500 μg, at least about 1 mg, at least about 5 mg, at least about 10 mg, at least about 20 mg, at least about 50 mg, at least about 100 mg, at least about 200 mg, at least about 500 mg, at least about 1 g, at least about 2 g, at least about 5 g, at least about 10 g, at least about 20 g, or at least about 50 g of the at least one biologically-produced product per day. In some embodiments, the system is configured to produce an amount of the at least one biologically-produced product in the range of about 10 μg to about 1 mg, about 10 μg to about 10 mg, about 10 μg to about 50 mg, about 10 μg to about 100 mg, about 10 μg to about 500 mg, about 10 μg to about 1 g, about 10 μg to about 5 g, about 10 μg to about 10 g, about 10 μg to about 50 g, about 100 μg to about 1 mg, about 100 μg to about 10 mg, about 100 μg to about 50 mg, about 100 μg to about 100 mg, about 100 μg to about 500 mg, about 100 μg to about 1 g, about 100 μg to about 5 g, about 100 μg to about 10 g, about 100 μg to about 50 g, about 1 mg to about 10 mg, about 1 mg to about 50 mg, about 1 mg to about 100 mg, about 1 mg to about 500 mg, about 1 mg to about 1 g, about 1 mg to about 5 g, about 1 mg to about 10 g, about 1 g to about 50 g, about 10 mg to about 50 mg, about 10 mg to about 100 mg, about 10 mg to about 500 mg, about 10 mg to about 1 g, about 10 mg to about 5 g, about 10 mg to about 10 g, about 10 mg to about 50 g, about 50 mg to about 100 mg, about 50 mg to about 200 mg, about 50 mg to about 500 mg, about 50 mg to about 1 g, about 50 mg to about 2 g, about 50 mg to about 5 g, about 50 mg to about 10 g, about 50 mg to about 20 g, about 50 mg to about 50 g, about 100 mg to about 500 mg, about 100 mg to about 1 g, about 100 mg to about 2 g, about 100 mg to about 5 g, about 100 mg to about 10 g, about 100 mg to about 20 g, about 100 mg to about 50 g, about 500 mg to about 1 g, about 500 mg to about 2 g, about 500 mg to about 5 g, about 500 mg to about 10 g, about 500 mg to about 50 g, about 1 g to about 5 g, about 1 g to about 10 g, or about 1 g to about 50 g per day.
According to certain embodiments, a biomanufacturing system (e.g., system 900) comprises a bioreactor (e.g., bioreactor 902) comprising a reactor chamber having an internal volume in the range of about 1 L to about 10 L. In some cases, a feed stream (e.g., stream 908) and at least one of a purified filtrate stream (e.g., stream 914) and a formulated product stream (e.g., stream 922) of the biomanufacturing system each have a flow rate of at least about 0.5 mL/min, at least about 1 mL/min, at least about 1.5 mL/min, at least about 2 mL/min, at least about 5 mL/min, at least about 10 mL/min, at least about 15 mL/min, or at least about 20 mL/min over a specified time period. In some embodiments, the feed stream (e.g., stream 908) and at least one of the purified filtrate stream (e.g., stream 914) and the formulated product stream (e.g., stream 922) of the biomanufacturing system each have a flow rate in the range of about 0.5 mL/min to about 2 mL/min, about 0.5 mL/min to about 5 mL/min, about 0.5 mL/min to about 10 mL/min, about 0.5 mL/min to about 15 mL/min, about 0.5 mL/min to about 20 mL/min, about 3 mL/min to about 10 mL/min, about 5 mL/min to about 10 mL/min, about 5 mL/min to about 15 mL/min, about 5 mL/min to about 20 mL/min, about 7 mL/min to about 20 mL/min, about 10 mL/min to about 20 mL/min, or about 15 mL/min to about 20 mL/min over a specified time period.
In some cases, each fluid stream of a biomanufacturing system comprising a reactor chamber having an internal volume in the range of about 1 L to about 10 L (e.g., feed stream 908, cell suspension stream 910, first filtrate stream 912, adjusted filtrate stream 918, purified filtrate stream 914, formulated product stream 922) has a flow rate of at least about 0.5 mL/min, at least about 1 mL/min, at least about 1.5 mL/min, at least about 2 mL/min, at least about 5 mL/min, at least about 10 mL/min, at least about 15 mL/min, or at least about 20 mL/min over a specified time period. In some embodiments, each fluid stream of this biomanufacturing system has a flow rate in the range of about 0.5 mL/min to about 2 mL/min, about 0.5 mL/min to about 5 mL/min, about 0.5 mL/min to about 10 mL/min, about 0.5 mL/min to about 15 mL/min, about 0.5 mL/min to about 20 mL/min, about 3 mL/min to about 10 mL/min, about 5 mL/min to about 10 mL/min, about 5 mL/min to about 15 mL/min, about 5 mL/min to about 20 mL/min, about 7 mL/min to about 20 mL/min, about 10 mL/min to about 20 mL/min, or about 15 mL/min to about 20 mL/min over a specified time period.
In some embodiments, the specified time period over which the flow rate is measured is at least about 1 hour, at least about 2 hours, at least about 5 hours, at least about 10 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 5 weeks, or at least about 10 weeks.
In some embodiments, a biomanufacturing system comprising a reactor chamber having an internal volume in the range of about 1 L to about 10 L produces at least about 100 μg, at least about 1 mg, at least about 5 mg, at least about 10 mg, at least about 50 mg, at least about 100 mg, at least about 500 mg, at least about 1 g, at least about 5 g, at least about 10 g, at least about 50 g, at least about 100 g, at least about 200 g, or at least about 500 g of the at least one biologically-produced product per day. In some embodiments, the system is configured to produce an amount of the at least one biologically-produced product in the range of about 100 μg to about 1 mg, about 100 μg to about 10 mg, about 100 μg to about 50 mg, about 100 μg to about 100 mg, about 100 μg to about 500 mg, about 100 μg to about 1 g, about 100 μg to about 5 g, about 100 μg to about 10 g, about 100 μg to about 50 g, about 100 μg to about 100 g, about 100 μg to about 500 g, about 1 mg to about 10 mg, about 1 mg to about 50 mg, about 1 mg to about 100 mg, about 1 mg to about 500 mg, about 1 mg to about 1 g, about 1 mg to about 5 g, about 1 mg to about 10 g, about 1 mg to about 50 g, about 1 mg to about 100 g, about 1 mg to about 500 g, about 100 mg to about 500 mg, about 100 mg to about 1 g, about 100 mg to about 5 g, about 100 mg to about 10 g, about 100 mg to about 50 g, about 100 mg to about 100 g, about 100 mg to about 500 g, about 1 g to about 5 g, about 1 g to about 10 g, about 1 g to about 50 g, about 1 g to about 100 g, about 1 g to about 500 g, about 10 g to about 50 g, about 10 g to about 100 g, about 10 g to about 500 g, or about 100 g to about 500 g per day. According to certain embodiments, a biomanufacturing system (e.g., system 900) comprises a bioreactor (e.g., bioreactor 902) comprising a reactor chamber having an internal volume in the range of about 10 L to about 100 L. In some cases, a feed stream (e.g., stream 908) and at least one of a purified filtrate stream (e.g., stream 914) and a formulated product stream (e.g., stream 922) of the biomanufacturing system each have a flow rate of at least about 5 mL/min, at least about 10 mL/min, at least about 15 mL/min, at least about 20 mL/min, at least about 50 mL/min, at least about 100 mL/min, at least about 150 mL/min, or at least about 200 mL/min over a specified time period. In some embodiments, the feed stream (e.g., stream 908) and at least one of the purified filtrate stream (e.g., stream 914) and the formulated product stream (e.g., stream 922) of the biomanufacturing system each have a flow rate in the range of about 5 mL/min to about 20 mL/min, about 5 mL/min to about 100 mL/min, about 5 mL/min to about 150 mL/min, about 5 mL/min to about 200 mL/min, about 10 mL/min to about 50 mL/min, about 10 mL/min to about 100 mL/min, about 10 mL/min to about 150 mL/min, about 10 mL/min to about 200 mL/min, about 35 mL/min to about 100 mL/min, about 35 mL/min to about 150 mL/min, about 35 mL/min to about 200 mL/min, about 50 mL/min to about 100 mL/min, about 50 mL/min to about 150 mL/min, about 50 mL/min to about 200 mL/min, or about 100 mL/min to about 200 mL/min over a specified time period.
In some cases, each fluid stream of a biomanufacturing system comprising a reactor chamber having an internal volume in the range of about 10 L to about 100 L (e.g., feed stream 908, cell suspension stream 910, first filtrate stream 912, adjusted filtrate stream 918, purified filtrate stream 914, formulated product stream 922) has a flow rate of at least about 5 mL/min, at least about 10 mL/min, at least about 15 mL/min, at least about 20 mL/min, at least about 50 mL/min, at least about 100 mL/min, at least about 150 mL/min, or at least about 200 mL/min over a specified time period. In some embodiments, each fluid stream of this biomanufacturing system has a flow rate in the range of about 5 mL/min to about 20 mL/min, about 5 mL/min to about 100 mL/min, about 5 mL/min to about 150 mL/min, about 5 mL/min to about 200 mL/min, about 10 mL/min to about 50 mL/min, about 10 mL/min to about 100 mL/min, about 10 mL/min to about 150 mL/min, about 10 mL/min to about 200 mL/min, about 35 mL/min to about 100 mL/min, about 35 mL/min to about 150 mL/min, about 35 mL/min to about 200 mL/min, about 50 mL/min to about 100 mL/min, about 50 mL/min to about 150 mL/min, about 50 mL/min to about 200 mL/min, or about 100 mL/min to about 200 mL/min over a specified time period.
In some embodiments, the specified time period over which the flow rate is measured is at least about 1 hour, at least about 2 hours, at least about 5 hours, at least about 10 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 5 weeks, or at least about 10 weeks.
In some embodiments, a biomanufacturing system comprising a reactor chamber having an internal volume in the range of about 10 L to about 100 L produces at least about 1 mg, at least about 5 mg, at least about 10 mg, at least about 50 mg, at least about 100 mg, at least about 200 mg, at least about 500 mg, at least about 1 g, at least about 2 g, at least about 5 g, at least about 10 g, at least about 50 g, at least about 100 g, at least about 200 g, at least about 500 g, at least about 1 kg, or at least about 5 kg of the at least one biologically-produced product per day. In some embodiments, the system is configured to produce an amount of the at least one biologically-produced product in the range of about 1 mg to about 10 mg, about 1 mg to about 50 mg, about 1 mg to about 100 mg, about 1 mg to about 500 mg, about 1 mg to about 1 g, about 1 mg to about 5 g, about 1 mg to about 10 g, about 1 mg to about 50 g, about 1 mg to about 100 g, about 1 mg to about 500 g, about 1 mg to about 1 kg, about 1 mg to about 5 kg, about 10 mg to about 50 mg, about 10 mg to about 100 mg, about 10 mg to about 500 mg, about 10 mg to about 1 g, about 10 mg to about 5 g, about 10 mg to about 10 g, about 10 mg to about 50 g, about 10 mg to about 100 g, about 10 mg to about 500 g, about 10 mg to about 1 kg, about 10 mg to about 5 kg, about 100 mg to about 500 mg, about 100 mg to about 1 g, about 100 mg to about 5 g, about 100 mg to about 10 g, about 100 mg to about 50 g, about 100 mg to about 100 g, about 100 mg to about 500 g, about 100 mg to about 1 kg, about 100 mg to about 5 kg, about 500 mg to about 1 g, about 1 g to about 5 g, about 1 g to about 10 g, about 1 g to about 50 g, about 1 g to about 100 g, about 1 g to about 500 g, about 1 g to about 1 kg, about 1 g to about 5 kg, about 10 g to about 50 g, about 10 g to about 100 g, about 10 g to about 500 g, about 10 g to about 1 kg, about 10 g to about 5 kg, about 100 g to about 500 g, about 100 g to about 1 kg, about 100 g to about 5 kg, about 500 g to about 1 kg, about 500 g to about 5 kg, or about 1 kg to about 5 kg per day.
Bioreactor
In some embodiments, the bioreactor (e.g., bioreactor 902 in
In some embodiments, the bioreactor is a chemostat. A chemostat generally refers to a bioreactor that is continuously operated such that an input stream comprising a cell culture medium is continuously supplied and an output stream comprising at least a portion of the cell culture medium and the biological cells is continuously removed such that the wet cell weight of the biological cells is maintained at a substantially constant value. In some embodiments, the bioreactor is a continuous stirred tank reactor (CSTR).
In some embodiments, the bioreactor comprises a reactor chamber. In certain embodiments, the reactor chamber has an internal volume (i.e., a volume capable of containing a fluid such as a cell suspension) of at least about 50 mL, at least about 100 mL, at least about 200 mL, at least about 500 mL, at least about 1 L, at least about 2 L, at least about 5 L, at least about 10 L, at least about 50 L, or at least about 100 L. In some embodiments, the reactor chamber has an internal volume of about 100 L or less, about 50 L or less, about 10 L or less, about 5 L or less, about 2 L or less, about 1 L or less, about 500 mL or less, about 200 mL or less, about 100 mL or less, or about 50 mL or less. In some embodiments, the reactor chamber has an internal volume in the range of about 50 mL to about 100 mL, about 50 mL to about 500 mL, about 50 mL to about 1 L, about 50 mL to about 5 L, about 50 mL to about 10 L, about 50 mL to about 50 L, about 50 mL to about 100 L, about 100 mL to about 500 mL, about 100 mL to about 1 L, about 100 mL to about 5 L, about 100 mL to about 10 L, about 100 mL to about 50 L, about 100 mL to about 100 L, about 500 mL to about 1 L, about 500 mL to about 5 L, about 500 mL to about 10 L, about 500 mL to about 50 L, about 500 mL to about 100 L, about 1 L to about 10 L, about 1 L to about 50 L, about 1 L to about 100 L, about 10 L to about 50 mL, about 10 L to about 100 L, or about 50 L to about 100 L.
The reactor chamber of the bioreactor may have any suitable shape. According to certain embodiments, for example, the reactor chamber may be substantially cylindrical. The reactor chamber also may be formed of any suitable material. Non-limiting examples of a suitable material include stainless steel, glass, and plastic. In some embodiments, the reactor chamber comprises one or more internal components, such as an agitator and/or impeller. An agitator and/or impeller may, for example, promote suspension of the cells within the cell culture medium and/or may maintain a sufficiently high level of dissolved oxygen within a cell culture. The impeller may be any suitable type of impeller. Non-limiting examples of suitable types of impellers include Rushton impellers, Marine impellers, and angled-Rushton impellers. In certain embodiments, the reaction chamber of a bioreactor comprises 1, 2, 3, or more impellers. In some instances, 2 or more impellers may be arranged on a single shaft. In those instances, the 2 or more impellers may be Rushton impellers, Marine impellers, and/or angled-Rushton impellers. In some embodiments, the bioreactor is fluidically connected (e.g., directly fluidically connected) to a gas concentration device or a gas source (e.g., gas tank). In certain cases, the gas concentration device is an oxygen concentrator.
In some embodiments, the bioreactor is operated in at least two phases: a cell growth phase and a biologically-produced product production phase. According to certain embodiments, in the cell growth phase, the bioreactor receives a first type of biological cells configured to express at least one biologically-produced product (i.e., the bioreactor is “inoculated” with the first type of biological cells) and receives a feed stream comprising a growth cell culture medium configured to promote the growth of the first type of biological cells. In some embodiments, the first type of biological cells are incubated in the growth cell culture medium for a period of at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 32 hours, at least about 36 hours, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, or at least about 14 days. In some embodiments, the first type of biological cells are incubated in the growth cell culture medium for a period in the range of about 1 hour to about 12 hours, about 1 hour to about 24 hours, about 1 hour to about 32 hours, about 1 hour to about 36 hours, about 1 hour to about 48 hours, about 1 hour to about 72 hours, about 1 hour to about 4 days, about 1 hour to about 5 days, about 1 hour to about 6 days, about 1 hour to about 7 days, about 1 hour to about 14 days, about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 12 hours to about 48 hours, about 12 hours to about 72 hours, about 12 hours to about 4 days, about 12 hours to about 5 days, about 12 hours to about 6 days, about 12 hours to about 7 days, about 12 hours to about 14 days, about 24 hours to about 36 hours, about 24 hours to about 48 hours, about 24 hours to about 72 hours, about 24 hours to about 4 days, about 24 hours to about 5 days, about 24 hours to about 6 days, about 24 hours to about 7 days, about 24 hours to about 14 days, about 36 hours to about 72 hours, about 3 days to about 4 days, about 3 days to about 5 days, about 3 days to about 6 days, about 3 days to about 7 days, about 3 days to about 14 days, about 4 days to about 7 days, about 4 days to about 14 days, or about 5 days to about 7 days, about 5 days to about 14 days, about 7 days to about 14 days, or about 10 days to about 14 days.
In some embodiments, the first type of biological cells are incubated in the growth cell culture medium until they reach a wet cell weight of at least about 150 g/L, at least about 200 g/L, at least about 250 g/L, at least about 300 g/L, at least about 350 g/L, at least about 400 g/L, at least about 450 g/L, or at least about 500 g/L. In some embodiments, the first type of biological cells are incubated in the growth cell culture medium until they reach a wet cell weight in the range of about 150 g/L to about 200 g/L, about 150 g/L to about 300 g/L, about 150 g/L to about 400 g/L, about 150 g/L to about 500 g/L, about 200 g/L to about 300 g/L, about 200 g/L to about 400 g/L, about 200 g/L to about 500 g/L, about 300 g/L to about 500 g/L, or about 400 g/L to about 500 g/L. The wet cell weight may be measured by a mass balance.
According to some embodiments, the cell growth phase is ended by removing the growth cell culture medium from the reaction chamber of the bioreactor. In some embodiments, the biologically-produced product production phase is initiated by introducing a production cell culture medium configured to promote expression of the at least one biologically-produced product into the reaction chamber. According to some embodiments, in the biologically-produced product production phase, the bioreactor receives a feed stream comprising the production cell culture medium.
In some embodiments, the first type of biological cells suspended in the production cell culture medium produce at least one biologically-produced product for a period of at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 4 weeks, at least about 6 weeks, or at least about 10 weeks. In some embodiments, the first type of biological cells suspended in the production cell culture medium produce at least one biologically-produced product for a period in the range of about 1 day to about 7 days, about 1 day to about 2 weeks, about 1 day to about 4 weeks, about 1 day to about 6 weeks, about 1 day to about 10 weeks, about 7 days to about 2 weeks, about 7 days to about 4 weeks, about 7 days to about 6 weeks, about 7 days to about 10 weeks, about 4 weeks to about 6 weeks, or about 4 weeks to about 10 weeks.
In some embodiments, the first type of biological cells suspended in the production cell culture medium have a wet cell weight of at least about 150 g/L, at least about 200 g/L, at least about 250 g/L, at least about 300 g/L, at least about 350 g/L, at least about 400 g/L, at least about 450 g/L, or at least about 500 g/L. In some embodiments, the first type of biological cells suspended in the production cell culture medium have a wet cell weight in the range of about 150 g/L to about 200 g/L, about 150 g/L to about 300 g/L, about 150 g/L to about 400 g/L, about 150 g/L to about 500 g/L, about 200 g/L to about 300 g/L, about 200 g/L to about 400 g/L, about 200 g/L to about 500 g/L, about 300 g/L to about 500 g/L, or about 400 g/L to about 500 g/L.
In some embodiments, the first type of biological cells are configured to express at least one biologically-produced product. For example, the first type of biological cells may be genetically engineered to express at least one biologically-produced product (e.g., via site-directed mutagenesis, gene insertion, viral vectors, microinjection, plasmids, recombinant DNA, metal nanoparticles, electroporation, chemical poration). In some embodiments, the biologically-produced product is a protein product and/or a pharmaceutical product. Non-limiting examples of a suitable biologically-produced product include a cytokine, an antibody, an antibody fragment, a single-domain antibody (e.g., a nanobody), a hormone, an enzyme, a growth factor, a blood factor, a recombinant immunogen, a recombinant vaccine or subunit, and a fusion protein. In some embodiments, the antibody is a single-chain antibody, a bispecific antibody, and/or a monoclonal antibody. In some embodiments, the cytokine is an interferon. According to certain embodiments, the at least one biologically-produced product comprises human growth hormone (hGH), granulocyte-colony stimulating factor (G-CSF), a single-domain antibody (e.g., nanobody), and/or interferon-α2b (IFN-α2b).
In some embodiments, the first type of biological cells is a prokaryotic cell. Non-limiting examples of prokaryotic cells include cyanobacteria algae and bacteria. The bacterium may be a gram-negative bacterium, including, but not limited to, including Escherichia, Salmonella, Shigella, Pseudomonas, Neisseria, Chlamydia, Yersinia, Moraxella, Haemophilus, Helicobacter, Acinetobacter, Stenotrophomonas, Bdellovibrio, Legionella, and acetic acid bacteria. In other embodiments, the bacterium may be a gram-positive bacterium, including, but not limited to, Streptococcus, Staphylococcus, Corynebacterium, Listeria, Bacillus, Clostridium, Lactobacillus, and Mycobacterium.
In some embodiments, the first type of biological cells is a lower eukaryote. Lower eukaryotes include yeast, fungi, collar-flagellates, microsporidia, alveolates (e.g., dinoflagellates), stramenopiles (e.g, brown algae, protozoa), rhodophyta (e.g., red algae), plants (e.g., green algae, plant cells, moss) and other protists. In some embodiments, the first type of biological cells are microalgae cells. A non-limiting example of microalgae cells is Chlamydomonas reinhardtii cells. In some embodiments, the first type of biological cells are diatom cells. A non-limiting example of diatom cells is Phaeodactylum tricornutum cells.
In some embodiments, the first type of biological cells is a yeast cell. Examples of yeast cells include, but are not limited to, Arxula adeninivorans, Aureobasidium pullulans, Aureobasidium melanogenum, Aureobasidium namibiae, Aureobasidium subglaciale, Brettanomyces bruxellensis, Brettanomyces claussenii, Candida albicans, Candida auris, Candida bracarensis, Candida bromeliacearum, Candida dubliniensis, Candida glabrata, Candida humilis, Candida keroseneae, Candida krusei, Candida lusitaniae, Candida oleophila, Candida parapsilosis, Candida rhizophoriensis, Candida sharkiensis, Candida stellate, Candida theae, Candida tolerans, Candida tropicalis, Candida ubatubensis, Candida viswanathii, Candida zemplinina, Cryptococcus gattii, Cryptococcus neoformans, Debaryomyces hansenii, Hansenula polymorpha, Hanseniaspora guilliermondii, Kluyveromyces lactis and like kinds, Kluyveromyces marxianus, Leucosporidium frigidum, Macrorhabdus ornithogaster, Malassezia caprae, Malassezia dermatis, Malassezia equine, Malassezia japonica, Malassezia nana, Malassezia sympodialis, Ogataea methanolica, Ogataea polymorpha, Pachysolen tannophilus, Pichia anomala, Pichia guilliermondii, Pichia pastoris, Pichia stipites, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Rhodotorula cladiensis, Rhodotorula evergladiensis, Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomyces paradoxus, Schizosaccharomyces pombe, Yarrowia lipolytica, and Zygosaccharomyces bailii. In one embodiment, the yeast is Pichia pastoris.
In some embodiments, the first type of biological cells is a filamentous fungi. Non-limiting examples of filamentous fungi include Trichoderma, for example from Trichoderma reesei; Neurospora, for example from Neurospora crassa; Sordaria, for example from Sordaria macrospora; Aspergillus, for example from Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, or from Aspergillus sojae; Fonsecaea, for example from Fonsecaea pedrosoi; Cladosporium, for example from Cladosporium carrionii; Chrysosporium luchiowense; Fusarium sp. (for example, Fusarium gramineum, Fusarium venenatum); Physcomitrella patens; or Phialophora, for example from Phialophora verrucosa.
The smaller secretome of organisms such as Pichia pastoris and similar cells are surprisingly advantageous in the context of designing and operating the integrated systems of certain embodiments. For example, Pichia pastoris, which is a methylotrophic yeast, contains the necessary cellular machinery for protein folding, glycosylation, and secretion, so it can be used to produce complex heterologous proteins used as therapeutics. However, its smaller secretome (e.g., the smaller number of proteins expected to contain a secretion signal peptide for entry into the secretory system and eventual secretion into the cell culture medium) allows for more streamlined downstream (e.g., purification) processes than higher eukaryotic cells.
In some embodiments, the reaction chamber contains a growth cell culture medium configured to promote growth of the first type of biological cells. Non-limiting examples of a suitable growth cell culture medium include chemically defined media comprising a carbon source, buffered glycerol-complex medium (BMGY), basal salt media, FM22, and d′Anjou media. Non-limiting examples of suitable additives include methanol, glycerol, sorbitol, glucose, arabinose, corn syrup, corn steep liquor, mannose, galactose, lactose trehaolse, maltitol, xylose, ribose, melibiose, maltose, raffinose, inulin, inositol, sorbose, arabitol, xylitol, ribitol, myo-inositol, glucono-1,5-lactone, lactate, quinic acid, gluconate, and trehalose. In some embodiments, the pH of the growth cell culture medium is at least about 4.0, at least about 5.0, at least about 5.5, at least about 6.0, at least about 6.5, at least about 7.0, at least about 7.5, at least about 8.0, or at least about 8.5. In some embodiments, the pH of the growth cell culture medium is about 8.5 or less, about 8.0 or less, about 7.5 or less, about 7.0 or less, about 6.5 or less, about 6.0 or less, about 5.5 or less, about 5.0 or less, or about 4.0 or less. In some embodiments, the pH of the growth cell culture medium is in the range of about 4.0 to about 6.0, about 4.0 to about 7.0, about 4.0 to about 8.0, about 4.0 to about 8.5, about 5.0 to about 7.0, about 5.0 to about 8.0, about 5.0 to about 8.5, about 6.0 to about 7.0, about 6.0 to about 8.0, about 6.0 to about 8.5, about 7.0 to about 8.0, or about 7.0 to about 8.5. The pH of the growth cell culture medium may be measured according to any method known in the art. For example, the pH may be measured using a digital pH meter.
In some embodiments, the reaction chamber contains a production cell culture medium configured to promote expression of at least one biologically-produced product by the first type of biological cells. Non-limiting examples of a suitable production cell culture medium include chemically defined media comprising a carbon source and/or other additive for induction of protein expression, buffered methanol-complex medium (BMMY), basal salt media with methanol, FM22 with methanol, and d′Anjou media with methanol. Non-limiting examples of suitable additives include methanol, glycerol, sorbitol, glucose, arabinose, corn syrup, corn steep liquor, mannose, galactose, lactose trehaolse, maltitol, xylose, ribose, melibiose, maltose, raffinose, inulin, inositol, sorbose, arabitol, xylitol, ribitol, myo-inositol, glucono-1,5-lactone, lactate, quinic acid, gluconate, and trehalose. In some embodiments, the pH of the production cell culture medium is at least about 4.0, at least about 5.0, at least about 5.5, at least about 6.0, at least about 6.5, at least about 7.0, at least about 7.5, at least about 8.0, or at least about 8.5. In some embodiments, the pH of the production cell culture medium is about 8.5 or less, about 8.0 or less, about 7.5 or less, about 7.0 or less, about 6.5 or less, about 6.0 or less, about 5.5 or less, about 5.0 or less, or about 4.0 or less. In some embodiments, the pH of the production cell culture medium is in the range of about 4.0 to about 6.0, about 4.0 to about 7.0, about 4.0 to about 8.0, about 4.0 to about 8.5, about 5.0 to about 7.0, about 5.0 to about 8.0, about 5.0 to about 8.5, about 6.0 to about 7.0, about 6.0 to about 8.0, about 6.0 to about 8.5, about 7.0 to about 8.0, or about 7.0 to about 8.5.
In some embodiments, the biomanufacturing system (e.g., system 900 in
According to some embodiments, the level sensing system is a non-invasive level sensing system. In some cases, non-invasive level sensing systems are associated with certain advantages, such as reduced risk of contamination, reduced geometric complexity, and cost savings. In single-use bioreactors, non-invasive level sensing systems may be particular advantageous because they may allow for reuse of the level sensing system without requiring sterilization. However, conventional non-invasive level sensing systems that have been used in other industries are generally unsuitable for use in bioreactors. For example, external capacitive level sensors, which measure change in dielectric constant through a vessel wall, are generally unsuitable for measuring liquid levels in a bioreactor because the ionic conductivity of the bioreactor liquid generally changes during the course of a run and interferes with dielectric constant measurements. Methods employing load cells and pressure transducers are similarly unsuitable for measuring liquid levels in a bioreactor since vibrational noise and variations in the density of the bioreactor liquid may interfere with measurements. Time-of-flight methods, such as ultrasonic or laser-based methods, are also generally unsuitable for measuring liquid levels in a bioreactor because such methods often require very high resolution and are susceptible to change due to a wide range of parameters.
In some embodiments, the level sensing system may be a magnetic level sensing system and/or an optical level sensing system. In some cases, a magnetic and/or optical level sensing system may advantageously provide accurate readings of a liquid level in a bioreactor since the magnetic and/or optical level sensing system may not rely upon properties (e.g., dielectric constant, density) of the bioreactor liquid to measure the liquid level. In addition, a magnetic and/or optical level sensing system may be associated with other advantages compared to other types of level sensing system, including reduced vessel and head plate complexity, reduced risk of contamination, reduced in-vessel wiring requirements, and increased cost savings. In some cases, the magnetic and/or optical level sensing system may allow continuous real-time monitoring.
According to some embodiments, the level sensing system is an in-vessel level sensing system. In certain embodiments, the level sensing system comprises a capacitance-based probe (e.g., an in-vessel capacitance probe).
In certain embodiments, the level sensing system is a magnetic level sensing system. In some embodiments, the magnetic level sensing system comprises a magnetic float, a non-magnetic shaft, and one or more magnetically-activated switches (e.g., reed switches). A schematic illustration of an exemplary magnetic level sensing system is shown in
In operation, magnetic float 2740 may float on the surface of bioreactor liquid 2730. As the height of bioreactor liquid 2730 increases or decreases, magnetic float 2740 may move vertically along non-magnetic shaft 2750. As magnetic float 2740 moves vertically along non-magnetic shaft 2750, magnetic float 2740 may become sufficiently close to a magnetically-activated switch of array 2760 to activate the switch, and an LED may be illuminated. Based on the color of the LED, an observer may be able to determine the level of bioreactor liquid 2730 within reaction chamber 2720.
In some embodiments, the magnetic float of the magnetic level sensing system comprises a flotation ring. The flotation ring may comprise any material suitable for use in a bioreactor. In certain embodiments, the material of the flotation ring is biocompatible.
In some instances, exposure of cells (e.g., the first type of biological cells) to a biocompatible material (e.g., the material of the flotation ring) results in the death of a relatively low percentage of cells (or substantially no cells). In certain embodiments, exposure of cells to a biocompatible material results in a cell death percentage of about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2% or less, about 1% or less, about 0.5% or less, about 0.2% or less, or about 0.1% or less. In some cases, exposure of cells to a biocompatible material results in the death of substantially no cells. The cell death percentage may be calculated as the number of dead cells divided by the number of live cells, multiplied by 100. The numbers of dead and live cells may be measured according to any method known in the art, such as flow cytometry or a Trypan blue exclusion assay. In some cases, a biocompatible material (e.g., the material of the flotation ring) may be inserted or injected into a living subject (e.g., a human or non-human animal) without inducing an adverse response (e.g., inflammation or other immune response). In some embodiments, a biocompatible material meets USP Class VI requirements, complies with the ISO 10993 standard, and/or has been designated as Generally Recognized as Safe. The biocompatible material may be a plastic, metal, ceramic, or other suitable material. Non-limiting examples of suitable biocompatible materials include titanium, titanium alloys, stainless steel, cobalt-chromium alloys, glass, alumina, polyethylene, polypropylene, polyethylene terephthalate, polymethyl methacrylate, polyvinyl alcohol, polyvinyl chloride, polystyrenes, polyamides, polyesters, polyurethanes, and silicones.
In some embodiments, the material of the flotation ring can withstand sterilization. A material that can withstand sterilization generally refers to a material that can withstand exposure to radiation (e.g., gamma radiation), steam, dry heat, sterilizing chemicals (e.g., ethylene oxide, nitrogen dioxide, ozone, hydrogen peroxide).
In certain embodiments, the material of the flotation ring has a specific gravity less than the liquid within the bioreactor. A non-limiting example of a material having a suitable specific gravity is polystyrene foam. In some embodiments, the flotation ring comprises a material (e.g., a biocompatible material) encompassing a hollow region comprising a gas (e.g., air). The material encompassing the hollow region may be any suitable material.
In some embodiments, the magnetic float of the magnetic level sensing system comprises one or more magnets. According to some embodiments, the one or more magnets are substantially equidistantly spaced around the flotation ring. In some such embodiments, magnetic material may be substantially uniformly distributed around the flotation ring, which may advantageously result in a rotation-independent magnetic field. In some embodiments, the one or more magnets comprise at least 1 magnet, at least 2 magnets, at least 3 magnets, at least 4 magnets, at least 5 magnets, at least 6 magnets, at least 7 magnets, at least 8 magnets, at least 9 magnets, or at least 10 magnets. In certain cases, the one or more magnets comprise between about 1 and 2 magnets, between 1 and 5 magnets, between 1 and 10 magnets, between 2 and 5 magnets, between about 2 and 10 magnets, or between about 5 and 10 magnets.
In some embodiments, the one or more magnets of the magnetic float are sufficiently strong to generate a magnetic field that can penetrate a wall of a bioreactor reaction chamber. In some embodiments, at least one of the one or more magnets has a pull force of at least about 1 pound, at least about 2 pounds, at least about 3 pounds, at least about 4 pounds, at least about 5 pounds, at least about 5.5 pounds, at least about 6 pounds, at least about 6.5 pounds, at least about 7 pounds, at least about 8 pounds, at least about 9 pounds, at least about 10 pounds, at least about 20 pounds, at least about 30 pounds, at least about 40 pounds, or at least about 50 pounds. In certain embodiments, at least one of the one or more magnets has a pull force between about 1 pound and about 5 pounds, between about 1 pound and about 10 pounds, between about 1 pound and about 20 pounds, between about 1 pound and about 30 pounds, between about 1 pound and about 40 pounds, between about 1 pound and about 50 pounds, between about 5 pounds and about 10 pounds, between about 5 pounds and about 20 pounds, between about 5 pounds and about 30 pounds, between about 5 pounds and about 40 pounds, between about 5 pounds and about 50 pounds, between about 10 pounds and about 20 pounds, between about 10 pounds and about 30 pounds, between about 10 pounds and about 40 pounds, between about 10 pounds and about 50 pounds, between about 20 pounds and about 50 pounds, between 30 pounds and about 50 pounds, or between about 40 pounds and about 50 pounds.
The one or more magnets of the magnetic float may comprise any suitable magnetic material. Non-limiting examples of suitable magnetic materials include neodymium iron boron (NdFeB), samarium cobalt (SmCo), and aluminum nickel cobalt (Alnico). In certain embodiments, the magnetic material is coated (e.g., to promote biocompatibility). Examples of suitable coating materials include, but are not limited to, parylene, silicone, nickel, titanium, and titanium nitride.
The one or more magnets may have any suitable size. In some embodiments, at least one of the one or more magnets has a diameter and/or thickness of at least about 0.05 inches (in), at least about 0.1 in, at least about 0.2 in, at least about 0.5 in, at least about 1 in, at least about 1.5 in, at least about 2 in, at least about 3 in, at least about 4 in, or at least about 5 in. In some embodiments, at least one of the one or more magnets has a diameter and/or thickness of about 5 in or less, about 4 in or less, about 3 in or less, about 2 in or less, about 1.5 in or less, about 1 in or less, about 0.5 in or less, about 0.2 in or less, about 0.1 in or less, or about 0.05 in or less. In some embodiments, at least one of the one or more magnets has a diameter and/or thickness between about 0.05 in and about 0.1 in, between about 0.05 in and about 0.5 in, between about 0.05 in and about 1 in, between about 0.05 in and about 2 in, between about 0.05 in and about 5 in, between about 0.1 in and about 0.5 in, between about 0.1 in and about 1 in, between about 0.1 in and about 2 in, between about 0.1 in and about 5 in, between about 0.5 in and about 1 in, between about 0.5 in and about 2 in, between about 0.5 in and about 5 in, between about 1 in and 5 in, or between about 2 in and about 5 in.
In some embodiments, the magnetic level sensing system comprises a non-magnetic shaft configured such that a magnetic float can move vertically along the non-magnetic shaft. In certain instances, the non-magnetic shaft comprises a probe (e.g., a probe used for process control) or a baffle. In some cases, the non-magnetic shaft is positioned sufficiently closely to a wall of a reactor chamber of a bioreactor such that at least a portion of the magnetic field of the magnetic float can penetrate the reactor chamber wall. The non-magnetic shaft may be formed of any non-magnetic material suitable for use in a bioreactor. In some embodiments, the material of the non-magnetic shaft is biocompatible. In some instances, the material of the non-magnetic shaft can withstand sterilization. Non-limiting examples of suitable materials for the non-magnetic shaft include metals (e.g., titanium, titanium alloys, stainless steel, cobalt-chromium alloys), glass, plastics (e.g., polyethylene, polypropylene, polyethylene terephthalate, polymethyl methacrylate, polyvinyl alcohol, polyvinyl chloride, polystyrenes, polyamides, polyesters, polyurethanes, silicones), and ceramics (e.g., alumina).
In some embodiments, the magnetic level sensing system further comprises one or more magnetically-activated switches. In certain instances, the one or more magnetically-activated switches comprise one or more reed switches. A reed switch generally refers to an electric switch that turns on in the presence of a magnetic field. In certain instances, the one or more magnetically-activated switches comprise one or more Hall Effect sensors. A Hall Effect sensor generally refers to a transducer that varies output in response to a magnetic field. In certain instances, a Hall Effect sensor acts as a substantially continuous sensor due to its ability to linearly increase output voltage (until saturation) with increasing magnetic field density.
The magnetic level sensing system may comprise any number of magnetically-activated switches. In certain embodiments, the magnetic level sensing system comprises at least 1 switch, at least 2 switches, at least 5 switches, at least 10 switches, at least 20 switches, at least 50 switches, or at least 100 switches. In certain embodiments, the magnetic level sensing system comprises 100 switches or less, 50 switches or less, 20 switches or less, 10 switches or less, 5 switches or less, 2 switches or less, or 1 switch. In some embodiments, the magnetic level sensing system comprises between 1 and 5 switches, between 1 and 10 switches, between 1 and 20 switches, between 1 and 50 switches, between 1 and 100 switches, between 5 and 10 switches, between 5 and 20 switches, between 5 and 50 switches, between 5 and 100 switches, between 10 and 20 switches, between 10 and 50 switches, between 10 and 100 switches, between 20 and 100 switches, or between 50 and 100 switches. The switches may be any suitable type of switch. Examples of suitable types of switches include, but are not limited to, single pole-single throw switches, single pole-double throw switches, normal-open switches, and normal-close switches.
In some embodiments, the one or more magnetically-activated switches are positioned external to a bioreactor. In some cases, the one or more magnetically-activated switches are positioned equidistant to each other. The one or more magnetically-activated switches may, in certain instances, be attached to an adhesive strip that can be externally mounted to a bioreactor.
In some embodiments, at least one switch of the one or more magnetically-activated switches is placed in series with a light-emitting diode (LED), a current sensor, a switch state detector, a processor, or another electronic component. In certain embodiments, each switch of the one or more magnetically-activated switches is placed in series with an LED, a current sensor, a switch state detector, a processor, or another electronic component. In some instances, each switch of the one or more magnetically-activated switches is placed in series with an LED of a different color. In some such instances, the LEDs provide visual information regarding a liquid level within a bioreactor.
Certain embodiments are directed to kits comprising one or more components of the magnetic level sensing system. In some instances, the kit comprises a magnetic float and one or more magnetically-activated switches. In some embodiments, the kit comprises a magnetic float and a strip (e.g., an adhesive strip) comprising one or more magnetically-activated switches. In certain cases, the kit may further comprise a non-magnetic shaft. In certain instances, the kit further comprises one or more containers.
Some embodiments are directed to bioreactors (e.g., single-use bioreactors) comprising one or more components of the magnetic level sensing system. In certain instances, for example, a magnetic float and/or a non-magnetic shaft may be manufactured as part of the bioreactor.
In certain embodiments, the level sensing system is an optical level sensing system (e.g., a system in which a liquid level is measured based on one or more visual cues). According to some embodiments, the optical level sensing system may be a discrete optical level sensing system, a continuous coordinate-based optical level sensing system, and/or a continuous area-based optical level sensing system. The optical level sensing system may function at varying cultivation densities. In some cases, the optical level sensing system may accurately measure bioreactor liquid level at a cultivation density corresponding to an optical density of at least about 0.01 OD600, at least about 0.02 OD600, at least about 0.05 OD600, at least about 0.1 OD600, at least about 0.15 OD600, at least about 0.2 OD600, at least about 0.25 OD600, at least about 0.3 OD600, at least about 0.4 OD600, at least about 0.5 OD600, at least about 1 OD600, or at least about 5 OD600. In some embodiments, the optical level sensing system may accurate measure bioreactor liquid level at a cultivation density corresponding to an optical density of about 5 OD600 or less, about 1 OD600 or less, about 0.5 OD600 or less, about 0.4 OD600 or less, about 0.3 OD600 or less, about 0.25 OD600 or less, about 0.2 OD600 or less, about 0.15 OD600 or less, about 0.1 OD600 or less, about 0.05 OD600 or less, about 0.02 OD600 or less, or about 0.01 OD600 or less, or less. In some embodiments, the optical level sensing system may accurate measure bioreactor liquid level at a cultivation density corresponding to an optical density between about 0.01 OD600 and about 0.05 OD600, between about 0.01 OD600 and about 0.1 OD600, between about 0.01 OD600 and about 0.15 OD600, between about 0.01 OD600 and about 0.2 OD600, between about 0.01 OD600 and about 0.25 OD600, between about 0.01 OD600 and about 0.3 OD600, between about 0.01 OD600 and about 0.5 OD600, between about 0.01 OD600 and about 1 OD600, between about 0.01 OD600 and about 5 OD600, between about 0.05 OD600 and about 0.1 OD600, between about 0.05 OD600 and about 0.15 OD600, between about 0.05 OD600 and about 0.2 OD600, between about 0.05 OD600 and about 0.25 OD600, between about 0.05 OD600 and about 0.3 OD600, between about 0.05 OD600 and about 0.5 OD600, between about 0.05 OD600 and about 1 OD600, between about 0.05 OD600 and about 5 OD600, between about 0.1 OD600 and about 0.15 OD600, between about 0.1 OD600 and about 0.2 OD600, between about 0.1 OD600 and about 0.25 OD600, between about 0.1 OD600 and about 0.3 OD600, between about 0.1 OD600 and about 0.5 OD600, between about 0.1 OD600 and about 1 OD600, between about 0.1 OD600 and about 5 OD600, between about 0.15 OD600 and about 0.2 OD600, between about 0.15 OD600 and about 0.25 OD600, between about 0.15 OD600 and about 0.3 OD600, between about 0.15 OD600 and about 0.5 OD600, between about 0.15 OD600 and about 1 OD600, between about 0.15 OD600 and about 5 OD600, between about 0.2 OD600 and about 0.25 OD600, between about 0.2 OD600 and about 0.3 OD600, between about 0.2 OD600 and about 0.5 OD600, between about 0.2 OD600 and about 1 OD600, between about 0.2 OD600 and about 5 OD600, between about 0.25 OD600 and about 0.3 OD600, between about 0.25 OD600 and about 0.5 OD600, between about 0.25 OD600 and about 1 OD600, between about 0.25 OD600 and about 5 OD600, between about 0.3 OD600 and about 0.5 OD600, between about 0.3 OD600 and about 1 OD600, between about 0.3 OD600 and about 5 OD600, between about 0.5 OD600 and about 1 OD600, between about 0.5 OD600 and about 5 OD600, or between about 1 OD600 and about 5 OD600. In some cases, the optical level sensing system may accurately measure the level of a bioreactor liquid that is substantially opaque corresponding to an optical density of 100 OD600 units or more.
In some embodiments, an optical level sensing system may permit monitoring of additional aspects of a bioreactor with a single system. As a non-limiting, illustrative example, image processing may detect foaming of a bioreactor liquid, which can result in fluids entering regions or sensors at the top of the reactor (e.g., off-gassing). In certain instances, an image that is used for level sensing may also be used to monitor foam levels, which may advantageously allow closed-loop control of anti-foam additives. As another illustrative example, an optical level sensing system may permit estimation of the optical opacity of a bioreactor liquid (e.g., relative to the reference color of an optical float or a probe), which may provide a method of determining cell culture density.
In some embodiments, the optical level sensing system is a discrete optical level sensing system (e.g., an optical level sensing system comprising a plurality of discrete visual markings). In some embodiments, the discrete optical level sensing system comprises a probe having two or more discrete visual markings. In some embodiments, at least one discrete visual marking comprises a band having a color distinct from the color of a bioreactor fluid (e.g., a cell suspension). In certain instances, a probe comprises at least 2 colored bands, at least 3 colored bands, at least 4 colored bands, at least 5 colored bands, at least 10 colored bands, at least 20 colored bands, or at least 50 colored bands. In some embodiments, each colored band of a probe has a distinct color (e.g., the colors are sufficiently different that they can be visually distinguished from each other and from the bioreactor liquid). In some instances, each colored band of the probe is associated with a pre-defined liquid level. It should be understood that though certain examples and embodiments of optical level sensing are described with respect to color, any suitable visual marking may be used. In some embodiments, visual markings may comprise a high-contrast region with differential light scattering properties, a different material of distinct color or contrast, a geometric form or pattern, or any other fiduciary marking to indicate a region of contrast.
The probe of the discrete optical level sensing system may or may not be an existing component of a bioreactor. In certain instances, for example, the probe comprises a shaft of an agitator or impeller of a bioreactor. In certain other instances, the probe comprises a separate component (e.g., an otherwise non-functional baffle). The probe may comprise any suitable material. In some embodiments, the material of the probe is biocompatible. In some embodiments, the material of the probe can withstand sterilization. Non-limiting examples of suitable materials for the probe include metals (e.g., titanium, titanium alloys, stainless steel, cobalt-chromium alloys), glass, plastics (e.g., polyethylene, polypropylene, polyethylene terephthalate, polymethyl methacrylate, polyvinyl alcohol, polyvinyl chloride, polystyrenes, polyamides, polyesters, polyurethanes, silicones), and ceramics (e.g., alumina).
In some embodiments, the probe is coated or otherwise surrounded by a biocompatible material. In certain instances, the probe is positioned inside an optically transparent sleeve within a bioreactor. In embodiments in which the probe is coated or surrounded by a biocompatible material, or otherwise not in direct contact with biological cells (e.g., the first type of biological cells), the probe itself may or may not comprise a biocompatible material.
A schematic illustration of an exemplary discrete optical level sensor is shown in
In operation, the level of bioreactor liquid 2830 may rise and/or fall, rendering different colored bands visible to camera 2850. Camera 2850 may be used to acquire images of agitator shaft 2840, and the images may be processed by one or more algorithms.
In some embodiments, a color image acquired by an image acquisition device (e.g., camera 2850) may be transmitted (e.g., electronically transmitted) to a computer (e.g., computer 2860) configured to run one or more image processing algorithms. In some embodiments, the one or more image processing algorithms comprise a chrominance-based binarization (CBB) algorithm. In some instances, the CBB algorithm may convert a color image to a binary (e.g., black and white) image by selecting for colors of interest. In certain embodiments, the acquired image may be converted from the RGB (red, green, blue) space to the HSV (hue, saturation, value) space. The HSV image may then be filtered using thresholding of the different hue, saturation, and value data against the known ranges of the colors of interest. To remove optical and physical noise, the holes in the binary image (e.g., a couple black pixels among many white ones) may be filled (e.g., by using the “imfill” function in Matlab) in order to ensure objects remain together and are not compromised.
In some embodiments, a colored object detection (COD) algorithm may be used to identify discrete colored objects in a binary image generated by the CBB algorithm. In some cases, the COD algorithm may apply a Gaussian blur and filter to the binary image. In certain instances, this may smooth erroneous pixels, physical and optical imperfections, and may reduce vibrational noise. In some cases, the COD algorithm is performed by clustering binary data and creating “blobs” that represent objects of a specific color in the original image. To avoid flecks of the specific color in the image, and other minor objects of the same color, an area-based filter may be applied to retain objects within a certain pixel area range. The pixel area range may vary based on the colored object being searched for. As an illustrative example, the COD algorithm may filter out objects smaller than 40 pixels when searching for a colored float or painted bands, and may filter out objects smaller than 500 pixels when searching for a colored shaft. The COD algorithm may then count the retained objects of the specific color. Only one object should remain for each color of interest.
In some embodiments, a Painted Bands (PB) algorithm may use information from the COD algorithm to determine a level of liquid within the bioreactor. In some instances, the number of objects for each band color may be counted in the COD algorithm and fed to the PB algorithm. The PB algorithm may use those counts to detect the presence or absence of a specific painted band in the image and correlate that to a point level being above or below certain values associated with the bands at those levels.
In some embodiments, the optical level sensing system is a continuous coordinate-based optical level sensing system. In certain embodiments, the continuous coordinate-based optical level sensing system comprises a probe and an optical float configured to move vertically along the probe. In some instances, a level of liquid within a bioreactor may be determined from coordinates of the optical float. In certain embodiments, the optical float has a color distinct from the color of a liquid within the bioreactor (e.g., a cell suspension). Examples of suitable colors for the optical float include, but are not limited to, red, green, blue, orange, and purple.
In some embodiments, the continuous coordinate-based optical level sensing system comprises an optical float comprising a flotation ring. The flotation ring may comprise any material suitable for use in a bioreactor. In certain embodiments, the material of the flotation ring is biocompatible. In certain embodiments, the material of the flotation ring can withstand sterilization. Non-limiting examples of suitable materials for the flotation ring include metals (e.g., titanium, titanium alloys, stainless steel, cobalt-chromium alloys), glass, plastics (e.g., polyethylene, polypropylene, polyethylene terephthalate, polymethyl methacrylate, polyvinyl alcohol, polyvinyl chloride, polystyrenes, polyamides, polyesters, polyurethanes, silicones), and ceramics (e.g., alumina).
The flotation ring may comprise any suitable material having a specific gravity less than the liquid within the bioreactor. Examples of materials having a suitable specific gravity include, but are not limited to, polypropylene and polystyrene foam. In some embodiments, the flotation ring comprises a material (e.g., a biocompatible material) encompassing a hollow region comprising a gas (e.g., air). The material encompassing the hollow region may be any suitable material.
In certain embodiments, the flotation ring may comprise a colored material (e.g., a colored plastic). In some cases, one or more suitable colors may be imparted to the flotation ring according to any method known in the art. In certain instances, the flotation ring may be painted a color and/or wrapped in a colored tape.
In some embodiments, the continuous coordinate-based optical level sensing system comprises a probe. The probe may or may not be an existing component of a bioreactor. In certain instances, for example, the probe comprises a shaft of an agitator or impeller of a bioreactor. In certain other instances, the probe comprises a separate component (e.g., an otherwise non-functional baffle). The probe may be formed of any material suitable for use in a bioreactor. In some embodiments, the material of the probe is biocompatible. In some embodiments, the material of the probe can withstand sterilization. Non-limiting examples of suitable materials for the probe include metals (e.g., titanium, titanium alloys, stainless steel, cobalt-chromium alloys), glass, plastics (e.g., polyethylene, polypropylene, polyethylene terephthalate, polymethyl methacrylate, polyvinyl alcohol, polyvinyl chloride, polystyrenes, polyamides, polyesters, polyurethanes, silicones), and ceramics (e.g., alumina).
A schematic illustration of an exemplary continuous coordinate-based optical level sensing system is shown in
In operation, optical float 2870 may move vertically along probe 2880 as the level of bioreactor liquid 2830 rises and falls. Camera 2850 may obtain images of optical float 2870, and the acquired images may be transmitted to computer 2860, which may run one or more image processing algorithms.
In some embodiments, a chrominance-based binarization (CBB) algorithm (e.g., the CBB algorithm described above) may be employed to convert a color image acquired by a camera (e.g., camera 2850) to a binary image. In certain embodiments, a colored object detection (COD) algorithm (e.g., the COD algorithm described above) may be used to identify discrete colored objects in a binary image generated by the CBB algorithm.
In some embodiments, a colored float (CF) algorithm may use information from the COD algorithm to determine a level of liquid within the bioreactor. In certain embodiments, the CF algorithm may detect the location of the colored float (e.g., a red float) and determine its centroid. In some instances, by pre-determined geometric calculations and knowledge of camera-acquired image specifications, the centroid of the colored float that was detected may be used to correlate to liquid level.
It should be understood that though certain examples and embodiments of optical level sensing are described with respect to color, any suitable visual marking may be used. In some embodiments, visual markings may comprise a high-contrast region with differential light scattering properties, a different material of distinct color or contrast, a geometric form or pattern, or any other fiduciary marking to indicate a region of contrast.
Continuous Area-Based Optical Level Sensing System
In some embodiments, the optical level sensing system is a continuous area-based optical level sensing system. In certain embodiments, the continuous area-based optical level sensing system comprises a probe having a color distinct from the color of a liquid within the bioreactor. In some instances, a level of liquid within a bioreactor may be determined from the amount (e.g., area) of the colored probe that is visible (e.g., the portion of the probe that is not submerged in the bioreactor liquid).
The probe may or may not be an existing component of a bioreactor. In certain instances, for example, the probe comprises a shaft of an agitator or impeller of a bioreactor. In certain other instances, the probe comprises a separate component (e.g., an otherwise non-functional baffle). The probe may be formed of any material suitable for use in a bioreactor. In some embodiments, the material of the probe is biocompatible. In some embodiments, the material of the probe can withstand sterilization. Non-limiting examples of suitable materials for the probe include metals (e.g., titanium, titanium alloys, stainless steel, cobalt-chromium alloys), glass, plastics (e.g., polyethylene, polypropylene, polyethylene terephthalate, polymethyl methacrylate, polyvinyl alcohol, polyvinyl chloride, polystyrenes, polyamides, polyesters, polyurethanes, silicones), and ceramics (e.g., alumina).
The probe may have any suitable color. Examples of suitable colors for the probe include, but are not limited to, red, green, blue, orange, and purple. In certain embodiments, the probe may comprise a colored material (e.g., a colored plastic). In some cases, the color may be imparted to the probe according to any method known in the art. In certain embodiments, at least a portion of the probe may be painted and/or wrapped in colored tape.
A schematic illustration of an exemplary continuous area-based optical level sensing system is shown in
In operation, the level of bioreactor liquid 2830 may rise and/or fall, which may change the amount of colored agitator shaft 2840 that is visible to camera 2850. Camera 2850 may be used to acquire images of agitator shaft 2840, and the images may be processed by one or more algorithms.
In some embodiments, a chrominance-based binarization (CBB) algorithm (e.g., the CBB algorithm described above) may be employed to convert a color image acquired by a camera (e.g., camera 2850) to a binary image. In certain embodiments, a colored object detection (COD) algorithm (e.g., the COD algorithm described above) may be used to identify discrete colored objects in a binary image generated by the CBB algorithm.
In some embodiments, a colored shaft (CS) algorithm may use information from the COD algorithm to determine a level of liquid within the bioreactor. In certain embodiments, the CS algorithm may detect the residual size of the colored probe. By pre-determined geometrical calculations and knowledge of camera-acquired image specifications, the area of the colored probe may then be used to determine the liquid level.
Certain embodiments are directed to kits comprising one or more components of the optical level sensing system. In some instances, the kit comprises an optical float, a colored probe, and/or a probe having two or more discrete visual markings. In some embodiments, the kit further comprises a camera. In some embodiments, the kit further comprises one or more containers.
Some embodiments are directed to bioreactors (e.g., single-use bioreactors) comprising one or more components of the optical level sensing system. In certain instances, for example, a colored probe, a probe having two or more discrete visual markings, and/or an optical float may be manufactured as part of the bioreactor.
It should be understood that though certain examples and embodiments of optical level sensing are described with respect to color, any suitable visual marking may be used. In some embodiments, visual markings may comprise a high-contrast region with differential light scattering properties, a different material of distinct color or contrast, a geometric form or pattern, or any other fiduciary marking to indicate a region of contrast.
In some embodiments, the biomanufacturing system (e.g., system 900 in
In some embodiments, the filter is a filter probe. Suitable filter probes include those described in a to U.S. Provisional Patent Application Ser. No. 62/553,104, filed Aug. 31, 2017, and entitled “Filtration Systems and Methods for Manufacturing Biologically-Produced Products, which is incorporated herein by reference in its entirety for all purposes.
The filter probe may have any suitable size or shape. In certain embodiments, for example, the filter probe is substantially cylindrical. In certain embodiments, the filter probe comprises a plurality of fibers. In some embodiments, the filter probe is constructed of materials that are chemically stable upon exposure to the cell culture medium (e.g., growth cell culture medium, production cell culture medium). In some embodiments, the filter probe is constructed of materials that are chemically stable upon exposure to methanol and/or glycerol. In some embodiments, the filter comprises a ceramic filter and/or a filtration membrane. In some embodiments, the at least one filter has a pore size that is sufficiently large to allow the at least one biologically-produced product to pass through the filter but sufficiently small to prevent the passage of the first type of biological cells. In certain embodiments, the filter has a pore size of at least about 0.01 microns (μm), at least about 0.02 μm, at least about 0.05 μm, at least about 0.08 μm, at least about 0.1 μm, at least about 0.2 μm, at least about 0.3 μm, at least about 0.4 μm, at least about 0.5 μm, at least about 0.8 μm, at least about 1 μm, at least about 2 μm, or at least about 3 μm. In some embodiments, the filter has a pore size of about 4 μm or less, about 3 μm or less, about 2 μm or less, about 1 μm or less, about 0.8 μm or less, about 0.5 μm or less, about 0.4 μm or less, about 0.3 μm or less, about 0.2 μm or less, about 0.1 μm or less, about 0.08 μm or less, about 0.05 μm or less, about 0.02 μm or less, or about 0.01 μm or less. In some embodiments, the filter has a pore size in the range of about 0.01 μm to about 0.05 μm, about 0.01 μm to about 0.1 μm, about 0.01 μm to about 0.5 μm, about 0.01 μm to about 1 μm, about 0.01 μm to about 2 μm, about 0.01 μm to about 3 μm, about 0.01 μm to about 4 μm, about 0.05 μm to about 0.1 μm, about 0.05 μm to about 0.5 μm, about 0.05 μm to about 1 μm, about 0.05 μm to about 2 μm, about 0.05 μm to about 3 μm, about 0.05 μm to about 4 μm, about 0.1 μm to about 0.5 μm, about 0.1 μm to about 1 μm, about 0.1 μm to about 2 μm, about 0.1 μm to about 3 μm, about 0.1 μm to about 4 μm, about 0.2 μm to about 0.5 μm, about 0.2 μm to about 0.8 μm, about 0.2 μm to about 1 μm, about 0.2 μm to about 2 μm, about 0.2 μm to about 3 μm, about 0.2 μm to about 4 μm, or about 0.5 μm to about 1 μm, about 0.5 μm to about 2 μm, about 0.5 μm to about 3 μm, about 0.5 μm to about 4 μm. In some embodiments, the filter has a pore size in the range of about about 0.2 μm to about 0.8 μm.
In some embodiments, the at least one filter has a sufficiently large surface area exposed to the cell suspension in the reactor chamber of the bioreactor that at least a portion of the cell suspension (e.g., the first filtrate stream) flows through the at least one filter at a relatively high flow rate.
In certain embodiments in which the bioreactor comprises a reactor chamber having an internal volume of about 50 mL to about 1 L, the first filtrate stream has a flow rate of at least about 0.01 mL/min, at least about 0.05 mL/min, at least about 0.1 mL/min, at least about 0.15 mL/min, at least about 0.2 mL/min, at least about 0.3 mL/min, at least about 0.4 mL/min, at least about 0.5 mL/min, at least about 0.6 mL/min, at least about 0.7 mL/min, at least about 0.8 mL/min, at least about 0.9 mL/min, at least about 1 mL/min, at least about 1.5 mL/min, or at least about 2 mL/min over a specified time period. In some embodiments, the first filtrate stream has a flow rate in the range of about 0.01 mL/min to about 0.1 mL/min, 0.01 mL/min to about 0.5 mL/min, about 0.01 mL/min to about 1 mL/min, about 0.01 mL/min to about 2 mL/min, about 0.03 mL/min to about 0.1 mL/min, 0.05 mL/min to about 0.1 mL/min, about 0.05 mL/min to about 0.5 mL/min, about 0.05 mL/min to about 1 mL/min, about 0.05 mL/min to about 2 mL/min, about 0.07 mL/min to about 0.2 mL/min, about 0.1 mL/min to about 0.4 mL/min, about 0.1 mL/min to about 1 mL/min, about 0.3 mL/min to about 1 mL/min, about 0.5 mL/min to about 1 mL/min, about 0.5 mL/min to about 2 mL/min, or about 1 mL/min to about 2 mL/min over a specified time period.
In certain embodiments in which the bioreactor comprises a reactor chamber having an internal volume of about 1 L to about 10 L, the first filtrate stream has a flow rate of at least about 0.5 mL/min, at least about 1 mL/min, at least about 1.5 mL/min, at least about 2 mL/min, at least about 5 mL/min, at least about 10 mL/min, at least about 15 mL/min, or at least about 20 mL/min over a specified time period. In some embodiments, the first filtrate stream has a flow rate in the range of about 0.5 mL/min to about 2 mL/min, about 0.5 mL/min to about 5 mL/min, about 0.5 mL/min to about 10 mL/min, about 0.5 mL/min to about 15 mL/min, about 0.5 mL/min to about 20 mL/min, about 3 mL/min to about 10 mL/min, about 5 mL/min to about 10 mL/min, about 5 mL/min to about 15 mL/min, about 5 mL/min to about 20 mL/min, about 7 mL/min to about 20 mL/min, about 10 mL/min to about 20 mL/min, or about 15 mL/min to about 20 mL/min over a specified time period.
In certain embodiments in which the bioreactor comprises a reactor chamber having an internal volume of about 10 L to about 100 L, the first filtrate stream has a flow rate of at least about 5 mL/min, at least about 10 mL/min, at least about 15 mL/min, at least about 20 mL/min, at least about 50 mL/min, at least about 100 mL/min, at least about 150 mL/min, or at least about 200 mL/min over a specified time period. In some embodiments, the first filtrate stream has a flow rate in the range of about 5 mL/min to about 20 mL/min, about 5 mL/min to about 100 mL/min, about 5 mL/min to about 150 mL/min, about 5 mL/min to about 200 mL/min, about 10 mL/min to about 50 mL/min, about 10 mL/min to about 100 mL/min, about 10 mL/min to about 150 mL/min, about 10 mL/min to about 200 mL/min, about 35 mL/min to about 100 mL/min, about 35 mL/min to about 150 mL/min, about 35 mL/min to about 200 mL/min, about 50 mL/min to about 100 mL/min, about 50 mL/min to about 150 mL/min, about 50 mL/min to about 200 mL/min, or about 100 mL/min to about 200 mL/min over a specified time period.
In some embodiments, the specified time period is at least about 1 hour, at least about 2 hours, at least about 5 hours, at least about 10 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 5 weeks, or at least about 10 weeks.
In some embodiments, the at least one filter comprises a plurality of filters. In some embodiments, the at least one filter comprises at least 2 filters, at least 3 filters, at least 4 filters, at least 5 filters, at least 10 filters, or at least 11 filters. In some embodiments, the at least one filter comprises 1 to 2 filters, 1 to 5 filters, 1 to 10 filters, 1 to 11 filters, 2 to 5 filters, 2 to 10 filters, 2 to 11 filters, 5 to 10 filters, 5 to 11 filters, or 10 to 11 filters.
In some embodiments, the first filtrate (e.g., stream 912 in
In some embodiments, the biomanufacturing system (e.g., system 900 in
According to some embodiments, the one or more properties comprise pH, and the adjustment module is configured to increase or decrease the pH of a fluid stream. As an illustrative example, the adjustment module may receive the first filtrate from the filter and adjust the pH of the first filtrate to produce an adjusted filtrate. In some embodiments, the adjusted filtrate has a pH that is compatible with a first partitioning unit of the purification module. For example, according to certain embodiments, the first partitioning unit of the purification module may comprise a chromatographic combination comprising a first stationary phase material and a first mobile phase material, where the first mobile phase material has a pH. In some embodiments, the difference between the pH of the adjusted filtrate and the pH of the first mobile phase material of the first partitioning unit of the purification module is about 4 or less, about 3 or less, about 2 or less, about 1 or less, about 0.5 or less, about 0.4 or less, about 0.3 or less, about 0.2 or less, about 0.1 or less, about 0.05 or less, or about 0.0. In some embodiments, the difference between the pH of the adjusted filtrate and the pH of the first mobile phase material of the first partitioning unit of the purification module is in the range of about 0.0 to about 0.1, about 0.0 to about 0.2, about 0.0 to about 0.3, about 0.0 to about 0.4, or about 0.0 to about 0.5. In some embodiments, the difference between the pH of the adjusted filtrate and the pH of the first mobile phase material of the first partitioning unit of the purification module is in the range of about 0.1 to about 1, about 0.1 to about 2, about 0.1 to about 3, or about 0.1 to about 4.
In some embodiments, the pH of a fluid stream may be adjusted by adding a pH-adjusting composition (e.g., an acid, a base) to the fluid stream. In certain embodiments, for example, an acid may be added to the fluid stream to decrease the pH of the stream. Non-limiting examples of suitable acids include citric acid, acetic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), hydrochloric acid, sulfuric acid, phosphoric acid, and nitric acid. In certain embodiments, a base may be added to the fluid stream to increase the pH of the stream. Examples of suitable bases include, but are not limited to, ammonium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, phosphate monobasic, phosphate dibasic, and tris(hydroxymethyl)aminomethane (Tris).
According to some embodiments, the one or more properties comprise conductivity, and the adjustment module is configured to increase or decrease the conductivity of a fluid stream. As an illustrative example, the adjustment module may receive the first filtrate from the filter and adjust the conductivity of the first filtrate to produce an adjusted filtrate. In some embodiments, the adjusted filtrate has a conductivity that is compatible with a first partitioning unit of the purification module. For example, according to certain embodiments, the first partitioning unit of the purification module comprises a chromatographic combination comprising a first stationary phase material and a first mobile phase material, where the first mobile phase material has a conductivity. In some embodiments, the difference between the conductivity of the adjusted filtrate and the conductivity of the first mobile phase material of the first partitioning unit of the purification module is 50 mS/cm or less, about 40 mS/cm or less, about 30 mS/cm or less, about 20 mS/cm or less, about 10 mS/cm or less, about 5 mS/cm or less, about 4 mS/cm or less, about 3 mS/cm or less, about 2 mS/cm or less, about 1 mS/cm or less, about 0.5 mS/cm or less, about 0.4 mS/cm or less, about 0.3 mS/cm or less, about 0.2 mS/cm or less, about 0.1 mS/cm or less, about 0.05 mS/cm or less, or about 0.0 mS/cm. In some embodiments, the difference between the conductivity of the adjusted filtrate and the first mobile phase material of the first partitioning unit of the purification module is in the range of about 0.0 mS/cm to about 0.1 mS/cm, about 0.0 mS/cm to about 0.2 mS/cm, about 0.0 mS/cm to about 0.3 mS/cm, about 0.0 mS/cm to about 0.4 mS/cm, about 0.0 mS/cm to about 0.5 mS/cm, about 0.0 mS/cm to about 1 mS/cm, about 0.0 mS/cm to about 5 mS/cm, about 0.0 mS/cm to about 10 mS/cm about 0.0 mS/cm to about 20 mS/cm, or about 0.0 mS/cm to about 50 mS/cm.
In some embodiments, the conductivity of a fluid stream may be adjusted by adding a conductivity-adjusting composition (e.g., a salt, a diluent) to the fluid stream. In some embodiments, for example, one or more salts may be added to the fluid stream to increase the conductivity of the stream. A non-limiting example of a suitable salt is sodium chloride. In some embodiments, a diluent may be added to the fluid stream to decrease the conductivity of the stream. A non-limiting example of a suitable diluent is water.
According to some embodiments, the one or more properties comprise biologically-produced product stability, and the adjustment module is configured to increase the stability of the biologically-produced product in a fluid stream. As an illustrative example, the adjustment module may receive the first filtrate from the filter and adjust the stability of the biologically-produced product in the first filtrate to produce an adjusted filtrate. In some embodiments, the stability of the biologically-produced product in a fluid stream may be adjusted by adding a stability-adjusting composition to the fluid stream. For example, the biologically-produced product present in the fluid stream may have one or more hydrophobic portions, while the remainder of the fluid stream (e.g., a cell culture medium) may be substantially hydrophilic. In certain embodiments, addition of one or more stability-adjusting compositions may enhance the stability of the biologically-produced product within the fluid stream. In some cases, the one or more stability-adjusting compositions comprise a surfactant, a lyoprotectant, a shear protectant, and/or an organic solvent. Non-limiting examples of suitable surfactants, lyoprotectants, shear protectants, and/or organic solvents include polysorbate 80, polysorbate 20, tween 20, triton-X 100, CHAPS, Breox, trehalose, sucrose, sorbitol, maltitol, and hexylene glycol.
According to some embodiments, the one or more properties comprise flow rate and/or pressure. As an illustrative example, the adjustment module may allow matching of desired flow rates between two or more components (e.g., units, modules). In some such cases, the adjustment module may serve as a flow rate and/or pressure converter between a first component (e.g., unit, module, bioreactor) and a second component (e.g., unit, module, purification module)
In some embodiments, the adjustment module is configured to minimize hold time after adjustment. In some cases, minimizing hold time after adjustment may advantageously maximize product quality. In some embodiments, the hold time of the adjusted filtrate is about 24 hours or less, about 18 hours or less, about 12 hours or less, about 6 hours or less, about 1 hour or less, about 30 minutes or less, or about 10 minutes or less. In some embodiments, the hold time of the adjusted filtrate is in the range of about 10 minutes to about 30 minutes, about 10 minutes to about 1 hour, about 10 minutes to about 6 hours, about 10 minutes to about 12 hours, about 10 minutes to about 18 hours, about 10 minutes to about 24 hours, about 30 minutes to about 1 hour, about 30 minutes to about 6 hours, about 30 minutes to about 12 hours, about 30 minutes to about 18 hours, about 30 minutes to about 24 hours, about 1 hour to about 6 hours, about 1 hour to about 12 hours, about 1 hour to about 18 hours, about 1 hour to about 24 hours, or about 6 hour to about 12 hours, about 6 hour to about 18 hours, about 6 hours to about 24 hours.
In some embodiments, the adjustment module comprises a surge tank. In certain cases, the surge tank has a volume of about 50 mL to about 2 L, about 2 L to about 10 L, or about 10 L to about 100 L. In some cases, the surge tank may advantageously facilitate connection of an upstream process associated with a first flow rate and a downstream process associated with a second, different flow rate. For instance, in some embodiments, a surge tank may help to release pressure from the filter.
In some embodiments, the adjustment module comprises a level sensing system. In certain instances, the level sensing system is configured to sense a level of a fluid in a fluid-containing vessel (e.g., a surge tank) of the adjustment module. In some embodiments, the level sensing system of the adjustment module comprises a capacitance-based probe (e.g., an in-vessel capacitance-based probe). In some embodiments, the level sensing system of the adjustment module comprises a magnetic level sensing system and/or an optical level sensing system.
In some embodiments, the biomanufacturing system (e.g., system 100) comprises a purification module (e.g., purification module 106) configured to remove at least a first type of impurity and a second type of impurity from a fluid (e.g., a cell suspension stream from the bioreactor, a first filtrate from the at least one filter, an adjusted filtrate from the adjustment module) to produce a purified filtrate. The purification module may comprise any number of partitioning units configured to remove at least one type of impurity from a fluid stream. In certain embodiments, the purification module comprises at least 2 partitioning units, at least 3 partitioning units, at least 4 partitioning units, at least 5 partitioning units, or at least 10 partitioning units. In some embodiments, the purification module comprises 2 to 5 partitioning units, 2 to 10 partitioning units, or 5 to 10 partitioning units.
According to some embodiments, the purification module comprises a first partitioning unit configured to remove at least the first type of impurity from the fluid stream to produce a first partitioned filtrate. In some embodiments, the purification module further comprises a second partitioning unit configured to remove at least the second type of impurity from the first partitioned filtrate to produce a second partitioned filtrate. In certain cases, the second partitioning unit is fluidically connected (e.g., directly fluidically connected) to the first partitioning unit.
In some embodiments, the purification module further comprises a third partitioning unit configured to remove at least a third type of impurity from the second partitioned filtrate to produce a third partitioned filtrate. In certain cases, the third partitioning unit is fluidically connected (e.g., directly fluidically connected) to the second partitioning unit. For example,
Any of the partitioning units (e.g., first partitioning unit, second partitioning unit, third partitioning unit) of the purification module may independently apply any partitioning technique. In some embodiments, the partitioning technique comprises chromatography, filtration, precipitation, crystallization, and/or extraction. The partitioning technique applied by one partitioning unit of the purification module may be the same or different from the partitioning technique applied by any other partitioning unit of the purification module.
In some embodiments, the partitioning technique applied by at least one partitioning unit of the purification module comprises chromatography. In certain embodiments, for example, the at least one partitioning unit comprises a column comprising a first stationary phase material. In some embodiments, the first stationary phase material is a cation exchange resin, a multimodal cation exchange resin, an anion exchange resin (e.g., a salt-tolerant anion exchange resin), a multimodal anion exchange resin, a hydrophobic charge induction chromatography (HCIC) resin, or an affinity chromatography resin. Non-limiting examples of suitable cation exchange resins include SP Sepharose HP. Non-limiting examples of suitable multi-modal cation exchange resins include Capto MMC, Capto MMC ImpRes, Nuvia cPrime, Toyopearl MX-Trp-650M, CMM HyperCel, and Eshmuno HCX. Non-limiting examples of anion exchange resins (e.g., salt-tolerant anion exchange resins) include HyperCel STAR AX, Toyopearl NH2-750F, and Q Sepharose HP. Non-limiting examples of suitable multi-modal anion exchange resins include Capto Adhere, PPA HyperCel, and HEA HyperCel. Non-limiting examples of suitable HCIC resins include MEP HyperCel, PPA HyperCel, and HEA HyperCel. Non-limiting examples of suitable affinity chromatography resins include MabSelect SuRe, KappaSelect, Eshmuno A, ProSep A, and immobilized antibody resins.
According to some embodiments, the column is associated with one or more mobile phase materials (i.e., one or more fluids that flow through the stationary phase material of the column). Non-limiting examples of suitable mobile phase materials include sodium citrate, sodium phosphate, sodium chloride, sodium acetate, Tris-HCl, glycine, and histidine. In some embodiments, the mobile phase material has a pH of at least about 3.0, at least about 3.5, at least about 4.0, at least about 4.5, at least about 5.0, at least about 5.5, at least about 6.0, at least about 6.5, at least about 7.0, at least about 7.5, at least about 8.0, at least about 8.5, or at least about 9.0. In some embodiments, the mobile phase material has a pH of about 9.0 or less, about 8.5 or less, about 8.0 or less, about 7.5 or less, about 7.0 or less, about 6.5 or less, about 6.0 or less, about 5.5 or less, about 5.0 or less, about 4.5 or less, about 4.0 or less, about 3.5 or less, or about 3.0 or less. In some embodiments, the mobile phase material has a pH in the range of about 3.0 to about 5.0, about 3.0 to about 6.0, about 3.0 to about 7.0, about 3.0 to about 8.0, about 3.0 to about 9.0, about 4.0 to about 6.0, about 4.0 to about 7.0, about 4.0 to about 8.0, about 4.0 to about 9.0, about 5.0 to about 7.0, about 5.0 to about 8.0, about 5.0 to about 9.0, about 6.0 to about 7.0, about 6.0 to about 8.0, about 6.0 to about 9.0, about 7.0 to about 8.0, or about 7.0 to about 9.0.
In some embodiments, the mobile phase material comprises a salt (e.g., sodium chloride). In some embodiments, the mobile phase material has a salt (e.g., sodium chloride) concentration of at least about 10 mM, at least about 20 mM, at least about 50 mM, at least about 100 mM, at least about 150 mM, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, at least about 450 mM, at least about 500 mM, at least about 1 M, at least about 1.5 M, or at least about 2 M. In some embodiments, the mobile phase material has a salt concentration of about 2 M or less, about 1.5 M or less, about 1 M or less, about 500 mM or less, about 450 mM or less, about 400 mM or less, about 350 mM or less, about 300 mM or less, about 250 mM or less, about 200 mM or less, about 150 mM or less, about 100 mM or less, about 50 mM or less, about 20 mM or less, or about 10 mM or less. In some embodiments, the mobile phase material has a salt concentration in the range of about 10 mM to about 150 mM, about 10 mM to about 250 mM, about 10 mM to about 500 mM, about 10 mM to about 1 M, about 10 mM to about 1.5 M, about 10 mM to about 2 M, about 50 mM to about 150 mM, about 50 mM to about 250 mM, about 50 mM to about 500 mM, about 50 mM to about 1 M, about 50 mM to about 1.5 M, about 50 mM to about 2 M, about 100 mM to about 250 mM, about 100 mM to about 500 mM, about 100 mM to about 1 M, about 100 mM to about 1.5 M, about 100 mM to about 2 M, about 250 mM to about 500 mM, about 250 mM to about 1 M, about 250 mM to about 1.5 M, about 250 mM to about 2 M, about 500 mM to about 1 M, about 500 mM to about 1.5 M, about 500 mM to about 2 M, or about 1 M to about 2 M.
The column may be operated in bind-elute mode, flow-through mode, or any other suitable mode. In bind-elute mode, two or more mobile phase materials may be directed to flow through the first stationary phase material of the column. In some embodiments, a first mobile phase material that is directed to flow through the column is configured to promote the binding of the at least one biologically-produced product to the first stationary phase material. Non-limiting examples of a suitable first mobile phase material (e.g., a bind buffer) include phosphate buffer, citrate buffer, formate buffer, acetate buffer, and tris buffer. In some embodiments, a second mobile phase material that is directed to flow through the column is configured to wash one or more materials other than the at least one biologically-produced product from the first stationary phase material. Non-limiting examples of a suitable second mobile phase material (e.g., a wash buffer) include, but are not limited to, phosphate buffer, citrate buffer, formate buffer, acetate buffer, and tris buffer. In some embodiments, the second mobile phase material is substantially similar to the first mobile phase material in terms of the types of components in the mobile phase (e.g. first phosphate buffer and second phosphate buffer), but substantially differs in at least one property (e.g. pH, ionic strength, etc.). In some embodiments, a third mobile phase material that is directed to flow through the column is configured to elute the at least one biologically-produced product from the first stationary phase material. Non-limiting examples of a suitable third mobile phase material (e.g., an elute buffer) include, but are not limited to, phosphate buffer, citrate buffer, formate buffer, acetate buffer, and tris buffer. In some embodiments, one or more fractions comprising the at least one biologically-produced product may be collected after the third mobile phase material is directed to flow through the column.
In flow-through mode, one or more mobile phase materials may be directed to flow through the first stationary phase material of the column. In some embodiments, a first mobile phase material that is directed to flow through the column is configured to promote the binding of one or more types of impurities to the stationary phase material. In some embodiments, the at least one biologically-produced product may “flow through” the stationary phase. In some embodiments, one or more fractions comprising the at least one biologically-produced product may be collected after the first mobile phase material is directed to flow through the column. Non-limiting examples of a suitable first mobile phase material include phosphate buffer, citrate buffer, formate buffer, acetate buffer, and tris buffer.
If a purification module comprises more than one partitioning unit applying chromatography as a partitioning technique, the first stationary phase material of the column of each partitioning unit may be the same or different. The one or more mobile phase materials associated with the column each partitioning unit may similarly be the same or different.
In some embodiments, the partitioning technique applied by at least one partitioning unit of the purification module comprises filtration. According to certain embodiments, the filtration technique comprises tangential flow filtration (also referred to as cross-flow filtration). A person of ordinary skill in the art would understand tangential flow filtration to refer to a type of filtration in which a fluid stream travels tangentially across the surface of a filter (e.g., a filtration membrane, a monolith). According to certain embodiments, the filtration technique comprises dead-end filtration. A person of ordinary skill in the art would understand dead-end filtration to refer to a type of filtration in which a fluid stream travels perpendicularly across the surface of a filter.
In some embodiments, a filter of the at least one partitioning unit (e.g., a filter in either a tangential flow filtration device or a dead-end filtration device) is a filtration membrane. In some cases, the filtration membrane comprises a plurality of pores having a pore size. In some embodiments, components of the fluid stream having a size smaller than the pore size of the filtration membrane may travel through the filtration membrane as part of a filtrate. In some embodiments, components of the fluid stream having a size larger than the pore size of the filtration membrane may be prevented from traveling through the filtration membrane and may be retained as part of a retentate. In certain embodiments, the filtration membrane has a pore size that permits passage of the at least one biologically-produced product and prohibits passage of one or more types of impurity in a fluid stream. In certain embodiments, the filtration membrane has a pore size that permits passage of one or more types of impurity and prohibits passage of the at least one biologically-produced product. In some embodiments, the filtration membrane has a pore size of at least about 0.01 μm, at least about 0.02 μm, at least about 0.05 μm, at least about 0.08 μm, at least about 0.1 μm, at least about 0.2 μm, at least about 0.3 μm, at least about 0.4 μm, at least about 0.5 μm, or at least about 1 μm. In some embodiments, the filtration membrane has a pore size of about 1 μm or less, about 0.5 μm or less, about 0.4 μm or less, about 0.3 μm or less, about 0.2 μm or less, about 0.1 μm or less, about 0.08 μm or less, about 0.05 μm or less, about 0.02 μm or less, or about 0.01 μm or less. In some embodiments, the filtration membrane has a pore size in the range of about 0.01 μm to about 0.05 μm, about 0.01 μm to about 0.1 μm, about 0.01 μm to about 0.5 μm, about 0.01 μm to about 1 μm, about 0.1 μm to about 0.5 μm, about 0.1 μm to about 1 μm, about 0.2 μm to about 0.5 μm, about 0.2 μm to about 1 μm, or about 0.5 μm to about 1 μm.
In some embodiments, the filter of the at least one partitioning unit is a monolith. A monolith generally refers to a filter formed from a porous solid material (e.g., a ceramic material). In some embodiments, components of the fluid stream having a size smaller than the average pore size of the monolith may travel through the monolith as part of a filtrate. In some embodiments, components of the fluid stream having a size larger than the pore size of the monolith may be prevented from traveling through the filtration membrane and may be retained as part of a retentate. In certain embodiments, the monolith has an average pore size that permits passage of the at least one biologically-produced product and prohibits passage of one or more types of impurity in a fluid stream. In certain embodiments, the monolith has an average pore size that permits passage of one or more types of impurity and prohibits passage of the at least one biologically-produced product. In some embodiments, the monolith has an average pore size of at least about 0.1 microns (μm), at least about 0.2 μm, at least about 0.3 μm, at least about 0.4 μm, at least about 0.5 μm, or at least about 1 μm. In some embodiments, the monolith has an average pore size of about 1 μm or less, about 0.5 μm or less, about 0.4 μm or less, about 0.3 μm or less, about 0.2 μm or less, or about 0.1 μm or less. In some embodiments, the monolith has an average pore size in the range of about 0.1 μm to about 0.5 μm, about 0.1 μm to about 1 μm, about 0.2 μm to about 0.5 μm, about 0.2 μm to about 1 μm, or about 0.5 μm to about 1 μm.
In some embodiments, the partitioning technique applied by at least one partitioning unit of the purification module comprises precipitation. In certain embodiments, the at least one partitioning unit comprises a precipitation apparatus. In some embodiments, the precipitation apparatus comprises a static mixer and/or a T-mixer. The precipitation apparatus may comprise a vessel (e.g., a settling tank), according to some embodiments. In some cases, the vessel may be sized to provide a fluid stream with sufficient residence time within the vessel for one or more types of impurity to precipitate from the fluid stream. In some embodiments, the residence time of a fluid stream flowing through the precipitation apparatus is at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 30 minutes, at least about 60 minutes, at least about 2 hours, at least about 5 hours, or at least about 10 hours. In some embodiments, the residence time is in the range of about 5 minutes to about 10 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 5 hours, about 5 minutes to about 10 hours, about 30 minutes to about 60 minutes, about 30 minutes to about 5 hours, about 30 minutes to about 10 hours, about 60 minutes to about 5 hours, about 60 minutes to about 10 hours, or about 5 hours to about 10 hours. The residence time may be calculated by dividing the volume of the vessel by the volumetric flow rate of the fluid stream flowing through the vessel.
In some embodiments, the partitioning technique applied by at least one partitioning unit of the purification module comprises crystallization. In certain embodiments, the at least one partitioning unit comprises a crystallization apparatus. The crystallization apparatus may comprise a vessel (e.g., a crystallization tank), according to some embodiments. In some cases, the vessel may be sized to provide a fluid stream with sufficient residence time within the vessel for one or more types of impurity to crystallize. In some embodiments, the residence time of a fluid stream flowing through the crystallization apparatus is at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 30 minutes, at least about 60 minutes, at least about 2 hours, at least about 5 hours, or at least about 10 hours. In some embodiments, the residence time is in the range of about 5 minutes to about 10 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 5 hours, about 5 minutes to about 10 hours, about 30 minutes to about 60 minutes, about 30 minutes to about 5 hours, about 30 minutes to about 10 hours, about 60 minutes to about 5 hours, about 60 minutes to about 10 hours, or about 5 hours to about 10 hours.
In some embodiments, the partitioning technique applied by at least one partitioning unit of the purification module comprises extraction. In certain embodiments, for example, the at least one partitioning unit comprises an extraction apparatus (e.g., a liquid-liquid extraction apparatus). In some embodiments, an extraction apparatus may be configured to receive a first solvent and a second solvent immiscible in the first solvent. In some embodiments, the extraction apparatus may be configured to further receive a fluid stream. In some embodiments, at least one component of the fluid stream (e.g., the at least one biologically-produced product) is miscible in a first solvent and at least one type of impurity in the fluid stream is miscible in a second, different solvent. In some embodiments, the solvent comprising the at least one biologically-produced product may be collected.
In some embodiments, the partitioning module further comprises at least one buffer delivery module configured to deliver at least one buffer to at least one partitioning unit. The at least one buffer delivery module may comprise one or more reservoirs containing one or more buffers. In some embodiments, the at least one buffer delivery module is in fluidic communication (e.g., direct fluidic communication) with at least one partitioning unit, at least two partitioning units, or at least three partitioning units. In some embodiments, the partitioning module contains at least one, at least two, or at least three buffer delivery modules.
In some embodiments, the purification module is configured to remove a relatively large percentage of at least a first type of impurity and at least a second type of impurity. In some embodiments, the purification module is configured to remove at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 99% of the first type of impurity and/or the second type of impurity from an input stream received by the purification module.
In some embodiments, the biomanufacturing system further comprises a formulation module. In some embodiments, the optional formulation module is configured to further process an output of the purification module to produce a formulated product. According to certain embodiments, for example, the formulation module may comprise a filtration unit configured to concentrate and/or further purify the at least one biologically-produced product, a viral filtration unit configured to remove and/or inactivate one or more viruses, and/or a product packaging unit configured to package doses of the at least one biologically-produced product into one or more sterile containers. In operation, the formulation module may receive a fluid stream (e.g., a purified filtrate stream) from the purification module and produce a formulated product stream.
In some embodiments, the formulation module comprises a filtration unit. According to some embodiments, the filtration unit is configured to increase the concentration of the at least one biologically-produced product in a fluid stream and/or further remove one or more types of impurity from the fluid stream. In some cases, the filtration unit comprises a tangential flow filtration (TFF) device. In certain embodiments, the TFF device comprises an ultrafiltration membrane. In some embodiments, the ultrafiltration membrane has a pore size of at least about 0.005 μm, at least about 0.01 μm, at least about 0.02 μm, at least about 0.03 μm, at least about 0.04 μm, at least about 0.05 μm, at least about 0.06 μm, at least about 0.07 μm, at least about 0.08 μm, at least about 0.09 μm, at least about 0.1 μm, at least about 0.2 μm, at least about 0.3 μm, about least about 0.4 μm, or at least about 0.5 μm. In some embodiments, the ultrafiltration membrane has a pore size of about 0.5 μm or less, about 0.4 μm or less, about 0.3 μm or less, about 0.2 μm or less, about 0.1 μm or less, about 0.09 μm or less, about 0.08 μm or less, about 0.07 μm or less, about 0.06 μm or less, about 0.05 μm or less, about 0.04 μm or less, about 0.03 μm or less, about 0.02 μm or less, about 0.01 μm or less, or about 0.005 μm or less. In some embodiments, the ultrafiltration membrane has a pore size in the range of about 0.005 μm to about 0.01 μm, about 0.005 μm to about 0.05 μm, about 0.005 μm to about 0.1 μm, about 0.005 μm to about 0.2 μm, about 0.005 μm to about 0.3 μm, about 0.005 μm to about 0.4 μm, about 0.005 μm to about 0.5 μm, about 0.01 μm to about 0.05 μm, about 0.01 μm to about 0.1 μm, about 0.01 μm to about 0.2 μm, about 0.01 μm to about 0.3 μm, about 0.01 μm to about 0.4 μm, about 0.01 μm to about 0.5 μm, about 0.05 μm to about 0.1 μm, about 0.05 μm to about 0.2 μm, about 0.05 μm to about 0.3 μm, about 0.05 μm to about 0.4 μm, about 0.05 μm to about 0.5 μm, or about 0.1 μm to about 0.5.
In some embodiments, the formulation module comprises a viral filtration unit. The viral filtration unit may be configured to receive a fluid stream and remove and/or inactivate one or more viruses from the fluid stream. In some embodiments, the viral filtration unit comprises a nanofiltration membrane. In some embodiments, the nanofiltration membrane has a pore size of about 500 nm or less, about 200 nm or less, about 100 nm or less, about 50 nm or less, about 20 nm or less, or about 10 nm or less. In some embodiments, the nanofiltration membrane has a pore size in the range of about 10 nm to about 20 nm, about 10 nm to about 50 nm, about 10 nm to about 100 nm, about 10 nm to about 200 nm, about 10 nm to about 500 nm, about 50 nm to about 100 nm, about 50 nm to about 200 nm, about 50 nm to about 500 nm, or about 100 nm to about 500 nm.
According to certain embodiments, the formulation module comprises a dilution adjustment unit. In some embodiments, the dilution adjustment unit is configured to add a diluent to an output of the purification module (e.g., the purified filtrate). Non-limiting examples of suitable diluents include polar protic solvents (e.g., water, aqueous solutions, buffers, methanol, ethanol, acetic acid), polar aprotic solvents (e.g. dimethylsulfoxide, acetonitrile, dimethylformamide, acetone), and nonpolar solvents (e.g., pentane, hexane, cyclohexane, benzene). In some embodiments, the diluent may include agents to stabilize the formulated purified filtrate to improve stability. Non-limiting examples include antioxidants (e.g., sodium bisulfite, sodium metabisulfite, ascorbate, sodium sulfite, thioglycerol), bulking agents (e.g., mannitol, dextran, glycine), viscosity enhancers/reducers or surfactants (e.g., polysorbate, 20, polysorbate 80), chelating agents (e.g., EDTA), preservatives (e.g., thimersol, sorbic acid), cryoprotectants (e.g., sucrose, trehalose, sorbitol), lyoprotectants, and adjuvants. (e.g., TLR agonists, CpG DNA, alum).
According to certain embodiments, the formulation module comprises a product packaging unit. In some embodiments, the product packaging unit is configured to package one or more doses of the at least one biologically-produced product into one or more containers. Non-limiting examples of a suitable containers include bags (e.g., bags configured to store intravenous liquid), vials, syringes, and bottles. The one or more containers may have any shape and may be formed of any suitable material (e.g., plastic, glass). In some embodiments, the one or more containers are airtight containers. In some embodiments, the one or more containers are sterile containers. In operation, the product packaging unit may receive a fluid stream comprising at least one biologically-produced product (e.g., a purified filtrate from a purification module) and package one or more portions of the fluid stream in one or more containers.
In other embodiments, the formulation module may not comprise a product packaging unit. In some embodiments, the product packaging unit may be a separate module that is distinct from the formulation module. In general, a product packaging module has the features described above with respect to the product packaging unit.
In some embodiments, the biomanufacturing system (e.g., system 900 in
As used herein, a unit that is “operatively associated with” one or more other components indicates that such components are directly connected to each other, in direct physical contact with each other without being connected or attached to each other, or are not directly connected to each other or in contact with each other, but are mechanically, electrically (including via electromagnetic signals transmitted through space), or fluidically interconnected (e.g., via channels) so as to cause or enable the components so associated to perform their intended functionality. For instance, in some embodiments, the controller may be electronically coupled to a component via a wireless or wired electronic connection. For example, the controller may be electronically coupled via a wireless or wired electronic connection to one or more processors for controlling flow rate, temperature, selection of reagent type, selection of reagent concentration, reaction time, selection of the ratio of reagents, the addition of an additive, or combinations thereof. In certain embodiments, the controller may be electronically coupled via a wireless or wired electronic connection to a temperature regulator for one or more fluid streams and/or the reactor. In some embodiments, the controller may be electronically coupled via a wireless or wired electronic connection to a fluid flow source (e.g., pump) for one or more fluid streams and/or the reactor. In certain embodiments, the controller may be electronically coupled via a wireless or wired electronic connection to one or more processors for controlling one or more selection of reagents (e.g., type, concentration, ratio).
In general, a unit (e.g., controller) may be used to conduct process monitoring and/or control by the use of feedback from one or more processes taking place in the biomanufacturing system. In some embodiments, a unit (e.g., controller) may be used to partially or fully automate the system. For instance, a controller may be configured to receive signal(s) (e.g., images) from the one or more sensors, to quantitatively analyze one or more signals or a pattern of signals (e.g., images), to compare one or more signals or a pattern of signals with other signals (e.g., reference signal) or values pre-programmed into the controller, and/or to modulate one or more parameters to control operation of the biomanufacturing system. For example, based at least in part on information derived from the signal, one or more parameters of the system may be modulated during a process and/or prior to and/or during a subsequent process in the system. In certain embodiments, the process operations are partially automated and may require minimal human intervention. This may allow a user or computer to partially operate the system to manufacture biologically-produced products without having any expertise in the biomanufacturing processes, equipment, or their operations. In some embodiments, the process operations are fully automated without any human intervention. This may allow a user or computer to operate the system to manufacture biologically-produced products without having any expertise in the biomanufacturing processes, equipment, or their operations.
In some embodiments, a user analyze the signal(s) from the sensor and identify the presence, absence, and/or source of a problem. In some such embodiments, a unit within the process monitoring and control system and or a user may determine appropriate corrective action to be implemented during the process and/or prior to and/or during a subsequent process. In certain embodiments, the unit may automatically identify a problem, determine the source of the problem, and/or implement appropriate corrective action. In some cases, a user may be involved in identifying a problem, determining the source of the problem, and/or implementing appropriate corrective action.
In some embodiments, implementation of the corrective action may be performed via a controller. For instance, a signal or pattern of signals (e.g., images) produced by the sensor(s) (e.g., cameras) can be transmitted to a controller. In some cases, the controller compares the signal or pattern of signals to a second set of signal(s). The second signal or pattern of signals may be, for example, signal(s) determined previously in the biomanufacturing system, or reference signal(s). In some cases, a reference signal or pattern of signals includes one or more threshold values or a range of threshold values. The controller may compare a first signal or pattern of signals with a second signal or pattern of signals (e.g., reference signals), and determine whether to modulate one or more parameters in the system. For instance, the controller can determine problems that have occurred or are occurring in the biomanufacturing system, and the controller may send one or more signal(s) to one or more components to cause modulation of a parameter in all or portions of the system. That is, the measured signal or pattern of signals (e.g., image or pattern of images) can be used by the controller to generate a drive signal and provide feedback control to the system. For example, based (at least in part) on the signal(s) received by the controller, this feedback can be used to modulate a parameter of the system by controlling, e.g., one or more of a pump, vacuum, valve, etc. The modulation may be performed, in certain embodiments, by the controller sending one or more drive signals to an appropriate component of the biomanufacturing system to actuate that or another component. Any suitable valve drive electronics circuit may be used to receive a drive signal and convert the drive signal to a voltage, current, or other signal capable of actuating the component. Alternatively, when corrective action cannot be taken, the controller may send one or more signal(s) to one or more components to cause the system or a component thereof to shut down. In some embodiments, one or more feedback control methods such as proportional control, integral control, proportional-integral control, derivative control, proportional-derivative control, integral-derivative control, nonlinear control, adaptive control, model-based control, and proportional-integral-derivative control can be used by a controller to modulate a parameter or cause the system or a component thereof to shut down.
Regardless of whether corrective action is implemented by a user or a controller, corrective action may be implemented prior to formation of the formulated product stream. In embodiments in which corrective action cannot be taken, the manufacturing of the biologically-produced product may be stopped. In general, the corrective actions may be implemented during the current and/or future biomanufacturing processes.
In certain embodiments, one or more measured signals (e.g., images) is processed or manipulated (e.g., before or after transmission, and/or before being compared to a signal). It should be appreciated, therefore, that when a signal is transmitted (e.g., to a controller, user), compared (e.g., with a reference signal or another signal), or otherwise used in a feedback process, that the raw signal may be used or a processed/manipulated signal based (at least in part) on the raw signal may be used. For example, in some cases, one or more derivative signals of a measured signal can be calculated (e.g., using a differentiator, or any other suitable method) and used to provide feedback. In other cases, signals are normalized (e.g., subtracting a measured signal from a background signal). In one set of embodiments, a signal comprises an optical image.
In some embodiments, the controller may be computer implemented. In general, any suitable calculation methods, steps, simulations, algorithms, systems, and system elements described herein may be implemented and/or controlled using one or more computer implemented controller(s). The methods, steps, controllers, and controller elements described herein are not limited in their implementation to any specific computer system described herein, as many other different machines may be used.
The computer implemented controller(s) can be part of or coupled in operative association with an image analysis system and/or other automated system components, and, in some embodiments, is configured and/or programmed to control and adjust operational parameters, as well as analyze and calculate values. In some embodiments, the computer implemented controller(s) can send and receive reference signals to set and/or control operating parameters of system apparatus. In other embodiments, the computer implemented system(s) can be separate from and/or remotely located with respect to the other system components and may be configured to receive data from one or more remote systems via indirect and/or portable means, such as via portable electronic data storage devices, such as magnetic disks, or via communication over a computer network, such as the Internet or a local intranet.
The computer implemented controller(s) may include several known components and circuitry, including a processing unit (i.e., processor), a memory system, input and output devices and interfaces (e.g., an interconnection mechanism), as well as other components, such as transport circuitry (e.g., one or more busses), a video and audio data input/output (UO) subsystem, special-purpose hardware, as well as other components and circuitry, as described below in more detail. Further, the computer system(s) may be a multi-processor computer system or may include multiple computers connected over a computer network.
In some embodiments, the process monitoring and control system is a non-invasive process monitoring and control system. In some cases, non-invasive process monitoring and control systems are associated with certain advantages, such as reduced risk of contamination, reduced geometric complexity, and cost savings as described herein with respect to the leveling sensor system. In some embodiments, the process monitoring and control system may comprise one or more optical sensor system and/or magnetic sensor system. Regardless of the sensor (e.g., camera) used, in some embodiments, the process monitoring and control system may allow continuous real-time monitoring. In some embodiments, the process monitoring and control system may allow for a relatively high amount of automation. For instance, in some embodiments, at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%, 100%) of the modules and/or processes in the biomaufacturing system are automated.
In some embodiments, one or more processes occurring in an upstream component may be monitored by one or more optical sensors (e.g., cameras). For example, optical sensors may be used to monitor fluid volume level, agitator or impeller speed, perfusion probe integrity, and gas sparging. In some embodiments, an upstream process monitoring and control sensor may monitor the level of fluid volume in the bioreactor. In some such cases, the upstream process monitoring and control sensor may operate as described herein with respect to the discrete level sensing system. In some embodiments, the process monitoring and control level sensing sensor may be configured to monitor the accuracy of the in-vessel level control system. In some embodiments, an upstream process monitoring and control sensor may monitor the vortex above the rotating agitator or impeller in the bioreactor. In some such embodiments, the process monitoring and control vortex sensor may be used to monitor the accuracy of the agitator or impeller speed control. In some embodiments, an upstream process monitoring and control sensor may be configured to monitor the filter probe. For example, an upstream process monitoring and control sensor may monitor fouling of the filter probe. In some such embodiments, the process monitoring and control filter probe sensor may be used to monitor indicia of fouling, such as formation of a cellular cake on the filter probe. In certain embodiments, indication of fouling may result in a user or a controller performing corrective action, such as washing the filter probe. As another example, an upstream process monitoring and control sensor may monitor the filtrate stream exiting the filter probe. In some such embodiments, the process monitoring and control filter probe sensor may be used to monitor indicia of filter probe failure (e.g., increased optical opacity of filtrate), such as the presence of cells in the filtrate stream. In certain embodiments, indication of filter probe failure may result in a user or a controller performing corrective action, such as washing the filter probe. In some embodiments, an upstream process monitoring and control sensor may monitor foam levels in the bioreactor. In some such embodiments, the process monitoring and control sensor may be configured to monitor the accuracy of the aeration system. In certain embodiments, over aeration, as indicated by foaming, may result in a user or a controller performing corrective action, such as administering anti-foaming additives or adjusting the aeration rate.
A schematic illustration of an exemplary upstream optical process monitoring and control sensor is shown in
As described herein, one or more modules (e.g., bioreactor, purification module, adjustment module) in the biomanufacturing system handle one or more fluids (e.g., liquids). In some embodiments, an optical process monitoring and control sensor may be used to prevent and/or correct problems associated with fluid delivery to a unit and/or a module. For example, the optical sensor may be configured to monitor the level of a fluid. Low levels of fluid may result in a user being alerted and/or a controller implementing a corrective action, such as connection to a new liquid supply source. As another example, an optical process monitoring and control sensor may be configured to validate that the correct fluid is entering the biomanufacturing system and/or component thereof (e.g., a module, a unit) by reading an identification element (e.g., visible registration mark, barcode) associated with (e.g., on the surface of) the container housing the fluid. In some embodiments, an optical process monitoring and control sensor may be configured to validate that a fluid exiting the biomanufacturing system and/or a component thereof (e.g., a module, a unit) is housed within the correct container by reading an identification element (e.g., visible registration mark, barcode) associated with (e.g., on the surface of) the container. In certain embodiments, the optical sensor may be configured to monitor the level of a fluid, which has exited the biomanufacturing system and/or a component thereof (e.g., a module, a unit), in a container. The information derived from the monitoring may be used to determine whether the biomanufacturing system and/or a component thereof (e.g., a module, a unit) produced the expected volume of the fluid.
A schematic illustration of an exemplary optical process monitoring and control sensor configured to monitor fluid handling is shown in
In some embodiments, one or more valves in the biomanufacturing system may be monitored by one or more optical sensors. In general, precise control of valve operation allows for optimal delivery of material (e.g., fluids) to the biomanufacturing system and/or component thereof (e.g., module, unit) at the optimal time. In some embodiments, certain processes in the biomanufacturing system may require the opening or closing of a certain sequence of valves, e.g., at certain times. In some embodiments, an optical process monitoring and control sensor may be configured to monitor the position of the valve(s) (e.g., opened, closed, on, off) prior to, during, and/or after a process in the biomanufacturing system and/or component thereof (e.g., module, unit). The optical process monitoring and control sensor may be configured to monitor any suitable valve. For example, an optical process monitoring and control sensor may be configured to monitor the valve position (on or off; opened or closed) of a pinch valve from a position above the pinch valve. As another example, an optical process monitoring and control sensor may be configured to monitor the internal actuator or gate position for a transparent multi-port valve. In one example, an optical process monitoring and control sensor may be configured to monitor the fluid flow before and after valve actuation to determine the direction of fluid flow for an opaque multi-port valve. As another example, an optical process monitoring and control sensor may be configured to monitor the presence or absence of fluid in a fluidic path (e.g., channel) to determine the valve position for valves for fluid input heads. Regardless of the type of valve, the process monitoring and control system may be configured to compare the actual valve position with the programmed valve position to determine if an error has occurred.
A schematic illustration of an exemplary optical process monitoring and control sensor for monitoring valve position is shown in
In some embodiments, one or more pumps in the biomanufacturing system may be monitored by one or more optical sensors. In certain embodiments, pumps may be used to induce fluid flow in the biomanufacturing system and/or components thereof (e.g., module, unit). In some instances, the revolutions per minute (also referred to as rpm) of a pump may be used to control the fluid flow rate. In some embodiments, an optical process monitoring and control sensor may be configured to monitor the rpm of a pump. For example, in a pump with a transparent housing, the optical process monitoring and control sensor may be configured to monitor the rotations of a rotating, colored object inside the pump that is indicative of or otherwise correlates with the revolutions per minute. As another example, for a pump with an opaque housing, a magnetic material may be positioned on or within a rotating portion of the pump. The rotation of the magnetic material may trigger an external reed switch (or hall effect sensor) that counts the number of instances and relates it to pump rpm. Regardless of the type of or the transparency of the pump housing, the process monitoring and control system may be configured to compare the actual rpm with the programmed rpm to determine if an error has occurred.
A schematic illustration of an exemplary optical process monitoring and control sensor for monitoring the rpm of a pump is shown in
In some embodiments, one or more flow rates in the biomanufacturing system may be monitored by one or more optical sensors. In some embodiments, the one or more flow rates may be monitored to confirm the accuracy of one or more flow rate measurement devices. For example, in a flow rate measurement device with a transparent housing, the optical process monitoring and control sensor may be configured to monitor the movement of a colored object inside the flow rate measurement device that is indicative of or otherwise correlates with the flow rate. As another example, for a flow rate measurement device with an opaque housing, a magnetic material may be positioned on or within a portion of the flow rate measurement device that moves due to fluid flow. The movement of the magnetic material may trigger an external reed switch (or hall effect sensor) that counts the number of instances of a certain movement and relates it to flow rate. Regardless of the type of or the transparency of the flow rate measurement device housing, the process monitoring and control system may be configured to compare the actual flow rate with the programmed flow rate and/or the flow rate determined by the flow rate measurement device to determine if an error has occurred.
A schematic illustration of an exemplary optical process monitoring and control sensor for monitoring the flow rate is shown in
In some embodiments, one or more optical sensors may be configured to monitor the facility in which the biomanufacturing system is located. In certain embodiments, the process monitoring and control system may be configured to monitor users and/or the environment (e.g., equipment, doors) around the biomanufacturing system. A schematic illustration of an exemplary optical process monitoring and control system for monitoring users and/or the environment around the biomanufacturing system is shown in
In general, the process monitoring and control system may include sensors to monitor any aspect of the biomanufacturing system and/or the facility in which the system is housed that would result in an adverse event (e.g., sub-optimal formulated product being recovered from the system, breach in good manufacturing practice, reduced system efficiency, system damage). For example, sensors may be used to determine leaks, blockages, breach of sterile barriers, user error, etc.
It should be understood that optical sensors as well as the process monitoring and control system are not limited to monitoring and/or deriving information from the biomanufacturing system or a component thereof based on color. It should be understood that though certain examples and embodiments of the process monitoring and control system and associated optical sensors are described with respect to color, any suitable visual marking may be used. In some embodiments, visual markings may comprise a high-contrast region with differential light scattering properties, a different material of distinct color or contrast, a geometric form or pattern, or any other fiduciary marking to indicate a region of contrast.
In some embodiments, a product stream exiting the biomanufacturing system (e.g., a purified filtrate stream, a formulated product stream) has a relatively high concentration of at least one biologically-produced product. In some embodiments, the product stream has a concentration of the at least one biologically-produced product of at least about 1 μg/mL, at least about 2 μg/mL, at least about 5 μg/mL, at least about 10 μg/mL, at least about 20 μg/mL, at least about 50 μg/mL, at least about 100 μg/mL, at least about 200 μg/mL, at least about 500 μg/mL, at least about 1 mg/mL, at least about 2 mg/mL, at least about 5 mg/mL, at least about 10 mg/mL, at least about 20 mg/mL, at least about 30 mg/mL, at least about 40 mg/mL, at least about 50 mg/mL, at least about 60 mg/mL, at least about 70 mg/mL, at least about 80 mg/mL, or at least about 90 mg/mL. In some embodiments, the product stream has a concentration of the at least one biologically-produced product in the range of about 1 μg/mL to about 10 μg/mL, about 1 μg/mL to about 50 μg/mL, about 1 μg/mL to about 100 μg/mL, about 1 μg/mL to about 200 μg/mL, about 1 μg/mL to about 500 μg/mL, about 10 μg/mL to about 50 μg/mL, about 10 μg/mL to about 100 μg/mL, about 10 μg/mL to about 200 μg/mL, about 10 μg/mL to about 500 μg/mL, about 50 μg/mL to about 100 μg/mL, about 50 μg/mL to about 200 μg/mL, about 50 μg/mL to about 500 μg/mL, about 100 μg/mL to about 500 μg/mL, or about 200 μg/mL to about 500 μg/mL. In certain embodiments, the product stream has a concentration of the at least one biologically-produced product in the range of about 1 μg/mL to about 100 mg/mL, about 10 μg/mL to about 100 mg/mL, about 50 μg/mL to about 100 mg/mL, about 100 μg/mL to about 100 mg/mL, about 200 μg/mL to about 100 mg/mL, about 500 μg/mL to about 100 mg/mL, about 1 mg/mL to about 100 mg/mL, about 2 mg/mL to about 100 mg/mL, about 5 mg/mL to about 100 mg/mL, about 10 mg/mL to about 100 mg/mL, or about 20 mg/mL to about 100 mg/mL. One suitable method for measuring the concentration of the at least one biologically-produced product in the product stream is running an enzyme-linked immunosorbent assay (ELISA).
In some embodiments, the product stream (e.g., the purified filtrate stream, the formulated product stream) exiting the biomanufacturing system has a relatively high product purity. Product purity generally refers to the degree to which the product is unmixed with non-product materials. For example, in certain embodiments, the product stream has a purity of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, the product stream has a purity in the range of about 50% to about 99%, about 60% to about 99%, about 70% to about 99%, about 75% to about 99%, about 80% to about 99%, about 85% to about 99%, about 90% to about 99%, or about 95% to about 99%. One suitable method of measuring purity is reversed phase liquid chromatography.
In certain embodiments, the product stream exiting the biomanufacturing system has a relatively low concentration of host cell proteins. A host cell generally refers to a biological cell that is engineered to produce a product (e.g., Pichia pastoris), and host cell proteins generally refer to the proteins that are produced by the biological cell that are not the product. In some embodiments, the product stream has a host cell protein concentration of about 1000 ng/(mg product) or less, about 500 ng/(mg product) or less, about 200 ng/(mg product), about 100 ng/(mg product) or less, about 50 ng/(mg product) or less, about 20 ng/(mg product) or less, about 10 ng/(mg product) or less, about 5 ng/(mg product) or less, about 2 ng/(mg product) or less, or about 1 ng/(mg product) or less. In some embodiments, the product stream has a host cell protein concentration in the range of about 1 ng/(mg product) to about 10 ng/(mg product), about 1 ng/(mg product) to about 20 ng/(mg product), about 1 ng/(mg product) to about 50 ng/(mg product), about 1 ng/(mg product) to about 100 ng/(mg product), about 1 ng/(mg product) to about 200 ng/(mg product), about 1 ng/(mg product) to about 500 ng/(mg product), about 1 ng/(mg product) to about 1000 ng/(mg product), about 10 ng/(mg product) to about 50 ng/(mg product), about 10 ng/(mg product) to about 100 ng/(mg product), about 10 ng/(mg product) to about 200 ng/(mg product), about 10 ng/(mg product) to about 500 ng/(mg product), about 10 ng/(mg product) to about 1000 ng/(mg product), about 20 ng/(mg product) to about 100 ng/(mg product), about 20 ng/(mg product) to about 200 ng/(mg product), about 20 ng/(mg product) to about 500 ng/(mg product), about 20 ng/(mg product) to about 1000 ng/(mg product), about 50 ng/(mg product) to about 100 ng/(mg product), about 50 ng/(mg product) to about 200 ng/(mg product), about 50 ng/(mg product) to about 500 ng/(mg product), about 50 ng/(mg product) to about 1000 ng/(mg product), about 100 ng/(mg product) to about 500 ng/(mg product), about 100 ng/(mg product) to about 1000 ng/(mg product), about 200 ng/(mg product) to about 500 ng/(mg product), about 200 ng/(mg product) to about 1000 ng/(mg product), or about 500 ng/(mg product) to about 1000 ng/(mg product). An exemplary method for measuring the host cell protein concentration is ELISA (Cygnus Technologies).
In certain embodiments, the product stream exiting the biomanufacturing system has a concentration of DNA of about 100 ng/(mg product) or less, about 50 ng/(mg product) or less, about 20 ng/(mg product) or less, about 10 ng/(mg product) or less, about 5 ng/(mg product) or less, about 2 ng/(mg product) or less, about 1 ng/(mg product) or less, about 0.5 ng/(mg product) or less, about 0.2 ng/(mg product) or less, about 0.1 ng/(mg product) or less, about 0.05 ng/(mg product) or less, about 0.02 ng/(mg product) or less, about 0.01 ng/(mg product) or less, about 0.005 ng/(mg product) or less, about 0.002 ng/(mg product) or less, or about 0.001 ng/(mg product) or less. In some embodiments, the product stream has a concentration of DNA in the range of about 0.001 ng/(mg product) to about 0.01 ng/(mg product), about 0.001 ng/(mg product) to about 0.05 ng/(mg product), about 0.001 ng/(mg product) to about 0.1 ng/(mg product), about 0.001 ng/(mg product) to about 0.2 ng/(mg product), about 0.001 ng/(mg product) to about 0.5 ng/(mg product), about 0.001 ng/(mg product) to about 1 ng/(mg product), about 0.001 ng/(mg product) to about 10 ng/(mg product), about 0.001 ng/(mg product) to about 20 ng/(mg product), about 0.001 ng/(mg product) to about 50 ng/(mg product), about 0.001 ng/(mg product) to about 100 ng/(mg product), about 0.01 ng/(mg product) to about 0.1 ng/(mg product), about 0.01 ng/(mg product) to about 0.5 ng/(mg product), about 0.01 ng/(mg product) to about 1 ng/(mg product), about 0.01 ng/(mg product) to about 10 ng/(mg product), about 0.01 ng/(mg product) to about 20 ng/(mg product), about 0.01 ng/(mg product) to about 50 ng/(mg product), about 0.01 ng/(mg product) to about 100 ng/(mg product), about 0.1 ng/(mg product) to about 0.5 ng/(mg product), about 0.1 ng/(mg product) to about 1 ng/(mg product), about 0.1 ng/(mg product) to about 10 ng/(mg product), about 0.1 ng/(mg product) to about 50 ng/(mg product), about 0.1 ng/(mg product) to about 100 ng/(mg product), about 1 ng/(mg product) to about 10 ng/(mg product), about 1 ng/(mg product) to about 20 ng/(mg product), about 1 ng/(mg product) to about 50 ng/(mg product), about 1 ng/(mg product) to about 100 ng/(mg product), about 10 ng/(mg product) to about 50 ng/(mg product), about 10 ng/(mg product) to about 100 ng/(mg product), about 20 ng/(mg product) to about 100 ng/(mg product), or about 50 ng/(mg product) to about 100 ng/(mg product). An exemplary method for measuring DNA concentration is the Quant-iT™ PicoGreen® dsDNA Assay Kit (ThermoFisher).
In some embodiments, the product stream has a relatively low concentration of aggregates. In some embodiments, the percentage of aggregates present in the product stream is about 5.00% or less, about 2.00% or less, about 1.50% or less, about 1.00% or less, about 0.50% or less, about 0.20% or less, or about 0.10% or less. In some embodiments, the percentage of aggregates present in the product stream is in the range of about 0.10% to about 0.50%, about 0.10% to about 1.00%, about 0.10% to about 1.50%, about 0.10% to about 2.00%, about 0.10% to about 5.00%, about 0.50% to about 1.00%, about 0.50% to about 1.50%, about 0.50% to about 2.00%, about 0.50% to about 5.00%, about 1.00% to about 2.00%, about 1.00% to about 5.00%, or about 2.00% to about 5.00%. An exemplary method for measuring the percentage of aggregates is size exclusion chromatography.
In some embodiments, the product stream has a relatively high potency. In some embodiments, the product stream has a potency that is at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, or at least about 200% of standard potency as evaluated against a reference product such as WHO standard material. In some embodiments, the product stream has a potency that is about 50% to about 100%, about 50% to about 125%, about 50% to about 150%, about 50% to about 175%, about 50% to about 200%, about 60% to about 100%, about 60% to about 125%, about 60% to about 150%, about 60% to about 175%, about 60% to about 200%, about 70% to about 100%, about 70% to about 125%, about 70% to about 150%, about 70% to about 175%, about 70% to about 200%, about 75% to about 100%, about 75% to about 125%, about 75% to about 150%, about 75% to about 175%, about 75% to about 200%, about 80% to about 99%, about 80% to about 125%, about 80% to about 150%, about 80% to about 175%, about 80% to about 200%, about 85% to about 100%, about 85% to about 125%, about 85% to about 150%, about 85% to about 175%, about 85% to about 200%, about 90% to about 100%, about 90% to about 125%, about 90% to about 150%, about 90% to about 175%, about 90% to about 200%, about 100% to about 125%, about 100% to about 150%, about 100% to about 175%, about 100% to about 200%, or about 150% to about 200% of standard potency. An exemplary method for measuring potency is running a cell-based proliferation assay.
Certain biomanufacturing systems described herein have a relatively small footprint. As used herein, the footprint of a system generally refers to the sum of the surface areas of the bottom surfaces of each surface component (e.g., the surfaces in contact with the floor). In some cases, a relatively small footprint may advantageously facilitate transport of the system (e.g., via a motor vehicle). In certain cases, the biomanufacturing system has a footprint of about 20 m2 or less, about 10 m2 or less, about 5 m2 or less, about 1 m2 or less, or about 0.5 m2 or less. In some embodiments, the biomanufacturing system has a footprint of about 0.5 m2 to about 5 m2, about 0.5 m2 to about 10 m2, about 0.5 m2 to about 20 m2, about 1 m2 to about 5 m2, about 1 m2 to about 10 m2, about 1 m2 to about 20 m2, about 5 m2 to about 10 m2, about 5 m2 to about 20 m2, or about 10 m2 to about 20 m2.
In some embodiments, the biomanufacturing system has a relatively low maximum height. The maximum height of the system may refer to the maximum vertical distance between a bottom surface of the system and a top surface of the system. In some cases, a relatively low maximum height may advantageously facilitate transport of the system (e.g., via a motor vehicle). In some embodiments, the biomanufacturing system has a maximum height of about 3 m or less, about 2 m or less, about 1 m or less, about 0.5 m or less, or about 0.1 m or less. In some embodiments, the biomanufacturing system has a maximum height in the range of about 0.1 m to about 0.5 m, about 0.1 m to about 1 m, about 0.1 m to about 2 m, or about 0.1 m to about 3 m.
In some embodiments, one or more modules of the biomanufacturing system (e.g., bioreactor 902, filter 904, adjustment module 916, purification module 906, formulation module 920) are disposable. In some embodiments, one or more modules is configured for single use. In some embodiments, each module of the biomanufacturing system is disposable. In some embodiments, each module of the biomanufacturing system is configured for single use. In some cases, a single use (e.g., disposable) bioreactor may be associated with certain advantages, such as a lower cross-contamination risk, simplified handling, high flexibility, and time and cost savings (e.g., due to pre-sterilized components). In some embodiments, the volumetric productivity of the biomanufacturign systems described herein may be relatively high per unit volume of the system.
In some embodiments, one or more modules of the biomanufacturing system (e.g., bioreactor 902, filter 904, adjustment module 916, purification module 906, formulation module 920) are configured to be cleaned in place. In some embodiments, a cleaning solution may be directed to flow through one or more fluidic paths within one or more modules of the biomanufacturing system. In some embodiments, the cleaning solution comprises hot water, steam, sodium hydroxide, ozone, hydrogen peroxide, bleach, alcohols (e.g., methanol, ethanol), and/or surfactant solutions. In some embodiments, the cleaning solution may sanitize and/or sterilize the one or more fluidic paths. In some embodiments, the cleaning may facilitate reuse of one or more modules of the biomanufacturing system. In some embodiments, the cleaning may facilitate reuse of all modules of the biomanufacturing system.
In general, the biomanufacturing system described herein does not suffer from one or more limitations of conventional systems. For instance, in conventional approaches, additional process steps to adjust the pH, conductivity, composition, and concentration of eluted fluids, or temporary storage steps, are often inserted between at least some (e.g., each) step of the complete sequence of operations in a unit and/or module. These additional steps add costs, time, or other inefficiencies when seeking to maximize the productivity of the process. The biomanufacturing system described herein have been designed to minimize and/or substantial reduce the total number of steps, units, and/or modules necessary to manufacture a biologically-produced product.
In general, the biomanufacturing system may be configured to manufacture biologically-produced products (e.g., pharmaceutically acceptable formulations comprising biologically-produced products, purified biologically-produced products) using a relatively small number of modules (e.g., between about 2 and about 10, between about 2 and about 8, between about 2 and about 6, between about 2 and about 5).
In general, the biomanufacturing system may be configured to perform one or more process steps (e.g., adjustment, purification, formulation, all process steps in a module) in an automated fashion. One feature of the system, according to certain embodiments, may be ease of configurability so that different biologically-produced products can be obtained on the same system.
In some embodiments, one or more modules of the biomanufacturing system (e.g., bioreactor 902, filter 904, adjustment module 916, purification module 906, formulation module 920) are configured to be modular and/or portable. In some such embodiments, the modularity and/or portability of a module(s) may allow for the replacement and/or removal of a module(s). In certain embodiments, a biomanufacturing system comprising a modular design may have a standardization of sizes so that modules may be easily exchanged and/or replaced.
As described above, the biomanufacturing system may include a process and monitoring control system associated with the overall system and/or a component thereof (e.g., a module). In some embodiments, one or more portions of the process and monitoring control system may be in physical contact with the biomanufacturing system and/or a component thereof (e.g., a module) or otherwise integrated with the biomanufacturing system and/or a component thereof. In some embodiments, at least a portion (e.g., all) of the process and monitoring control system is not in physical contact with the biomanufacturing system and/or a component thereof (e.g., a module) or otherwise spatially distinct and/or physically separate from the biomanufacturing system. In general, the process and monitoring control system can be used to operate (e.g., autonomously) various components of the biomanufacturing system. In general, any calculation methods, steps, simulations, algorithms, systems, and system elements described herein may be implemented and/or controlled using the process and monitoring control system. In some embodiments, an automated biomanufacturing system may comprise software capable of performing different optimization algorithms such as simplex, conjugate gradient, and/or interior point methods. In certain embodiments, an automated biomanufacturing system may allow the user to treat the system as a black box. Automation of a biomanufacturing system may be accomplished by a variety of suitable automation systems.
Certain embodiments are directed to kits comprising one or more modules or other components of the biomanufacturing system. In some instances, the kit comprises bioreactor 902, filter 904, adjustment module 916, purification module 906, and/or formulation module 920. Any of the kits described here may further comprise one or more modules, components, and/or systems for manufacturing the biologically-produced product described herein. For example, a kit may comprise a level sensing system. Further, the kit may also comprise an instruction manual providing guidance for using the kit to manufacture one or more biologically-produced product.
Some embodiments described herein relate to systems for producing granulocyte colony-stimulating factor (G-CSF). According to some embodiments, the system comprises a bioreactor (e.g., a perfusion bioreactor), at least one filter, and a purification module. The bioreactor may comprise a reaction chamber, and, as described above, may have any suitable shape and be formed of any suitable material. In some embodiments, the reaction chamber contains a suspension comprising at least one cell culture medium and at least a first type of biological cells configured to express G-CSF. In some embodiments, the biological cells are yeast cells. In certain cases, the yeast cells are Pichia pastoris cells. In some embodiments, for example during a cell growth phase, the at least one cell culture medium comprises chemically defined media comprising a carbon source or buffered glycerol-complex medium (BMGY). In some embodiments, for example during a G-CSF production phase, the at least one cell culture medium comprises chemically defined media comprising a carbon source and/or other additive for induction of protein expression or buffered methanol-complex media (BMMY).
In some embodiments, the at least one filter of the system comprises a filter probe, a filtration membrane, and/or a ceramic filter. The at least one filter may, in some embodiments, be fluidically connected (e.g., directly fluidically connected) to the bioreactor. For example, in certain embodiments, the at least one filter comprises at least one filter probe at least partially submerged in the suspension in the bioreactor. In some embodiments, the at least one filter is configured to receive an output of the bioreactor and produce at least one filtrate lean in the first type of biological cells relative to the suspension. In some embodiments, the at least one filtrate comprises G-CSF.
In some embodiments, the system further comprises an adjustment module configured to adjust one or more properties (e.g., pH, conductivity, product stability) of an input stream received by the adjustment module (e.g., the at least one filtrate) to produce an adjusted output stream (e.g., an adjusted filtrate). According to some embodiments, the adjustment module is fluidically connected (e.g., directly fluidically connected) to the bioreactor, the at least one filter, and/or the purification module. In some embodiments, the adjustment module comprises a surge tank. In certain cases, the surge tank has a volume of about 50 mL to about 2 L, about 2 L to about 10 L, or about 10 L to about 100 L. In some cases, the surge tank may advantageously facilitate connection of an upstream process associated with a first flow rate and/or pressure and a downstream process associated with a second, different flow rate and/or pressure. For instance, in some embodiments, a surge tank may help to release pressure from the filter.
In certain illustrative embodiments, the adjustment module is configured to increase or decrease the pH of the at least one filtrate to produce a pH-adjusted filtrate. In some embodiments, the pH of the adjusted filtrate is compatible with a first column of the purification module. In certain cases, adjusting the pH of the at least one filtrate may facilitate effective capture of G-CSF on the first column of the purification module. In addition, adjusting the pH of the at least one filtrate may advantageously reduce the amount of product aggregates, increase product yield, increase G-CSF stability, and/or maximize product quality. In some embodiments, the pH-adjusted filtrate has a pH of about 4.0, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, or about 9.0.
In some embodiments, the adjustment module is configured to minimize hold time after adjustment. Minimizing hold time after adjustment may, in some cases, advantageously maximize product quality. In certain embodiments, the hold time of the adjusted filtrate is about 24 hours or less, about 18 hours or less, about 12 hours or less, about 6 hours or less, about 1 hour or less, about 30 minutes or less, or about 10 minutes or less. In some embodiments, the hold time of the adjusted filtrate is in the range of about 10 minutes to about 30 minutes, about 10 minutes to about 1 hour, about 10 minutes to about 6 hours, about 10 minutes to about 12 hours, about 10 minutes to about 18 hours, about 10 minutes to about 24 hours, about 30 minutes to about 1 hour, about 30 minutes to about 6 hours, about 30 minutes to about 12 hours, about 30 minutes to about 18 hours, about 30 minutes to about 24 hours, about 1 hour to about 6 hours, about 1 hour to about 12 hours, about 1 hour to about 18 hours, about 1 hour to about 24 hours, about 6 hours to about 12 hours, about 6 hours to about 18 hours, or about 6 hours to about 24 hours.
In some embodiments, the purification module is configured to remove at least a first type of impurity, a second type of impurity, and a third type of impurity from an input stream received by the purification module (e.g., the at least one filtrate, the adjusted filtrate) to produce a purified filtrate. In some embodiments, the purification module is fluidically connected (e.g., directly fluidically connected) to the at least one filter and/or the adjustment module.
In some embodiments, the purification module comprises a first partitioning unit configured to remove at least the first type of impurity, a second partitioning unit configured to remove at least the second type of impurity, and a third partitioning unit configured to remove at least the third type of impurity. In certain embodiments, the design framework described above may be used to generate and evaluate candidate sequences of partitioning steps. Using the design framework, for example, a sequence of chromatography columns and associated conditions suitable for capturing and purifying G-CSF may be identified.
In some embodiments, the purification module comprises a first column comprising a multimodal cation exchange resin. In some embodiments, the first column is configured to remove at least the first type of impurity to produce a first partitioned filtrate that comprises G-CSF and is lean in the first type of impurity relative to the first filtrate. According to certain embodiments, the multimodal cation exchange resin comprises Capto MMC ImpRes, Capto MMC, Nuvia cPrime, Toyopearl MX-Trp-650M, CMM HyperCel, and/or Eshmuno HCX.
In some embodiments, the purification module further comprises a second column comprising an anion exchange resin (e.g., a salt-tolerant anion exchange resin). The second column may be fluidically connected (e.g., directly fluidically connected) to the first column, according to certain embodiments. In some embodiments, the second column is configured to remove at least the second type of impurity to produce a second partitioned filtrate that comprises G-CSF and is lean in the second type of impurity relative to the first partitioned filtrate. In certain cases, the anion exchange resin comprises HyperCel STAR AX and/or Toyopearl NH2-750F.
In some embodiments, the purification module further comprises a third column comprising an HCIC resin. The third column is fluidically connected (e.g., directly fluidically connected) to the second column, according to certain embodiments. In some embodiments, the third column is configured to remove at least the third type of impurity to produce a third partitioned filtrate that comprises G-CSF and is lean in the third type of impurity relative to the second partitioned filtrate. In certain cases, the HCIC resin comprises MEP HyperCel, PPA HyperCel, and/or HEA HyperCel.
In some embodiments, the system further comprises a formulation module configured to produce a formulated product stream. In some embodiments, the formulation module is fluidically connected (e.g., directly fluidically connected) to the purification module. In some embodiments, the formulation module comprises a filtration unit, a viral filtration unit, a dilution adjustment unit, and/or a product packaging unit. According to some embodiments, the filtration unit comprises a tangential flow filtration device.
In some embodiments, the product stream (e.g., the purified filtrate stream from the purification module, the formulated product stream from the formulation module) has a relatively high concentration of G-CSF. In certain embodiments, the product stream has a G-CSF concentration of at least about 0.05 mg/mL, at least about 0.1 mg/mL, at least about 0.2 mg/mL, at least about 0.3 mg/mL, at least about 0.4 mg/mL, at least about 0.5 mg/mL, at least about 1 mg/mL, at least about 5 mg/mL, at least about 10 mg/mL, at least about 25 mg/mL, at least about 50 mg/mL, at least about 75 mg/mL, or at least about at least about 90 mg/mL. In some embodiments, the product stream has a G-CSF concentration in the range of about 0.05 mg/mL to about 100 mg/mL, about 0.5 mg/mL to about 100 mg/mL, or about 1 mg/mL to about 100 mg/mL. In certain embodiments, the product stream has a G-CSF concentration in the range of about 0.05 mg/mL to about 0.2 mg/mL, about 0.05 mg/mL to about 0.3 mg/mL, about 0.05 mg/mL to about 0.4 mg/mL, about 0.05 mg/mL to about 0.5 mg/mL, about 0.1 mg/mL to about 0.3 mg/mL, about 0.1 mg/mL to about 0.4 mg/mL, about 0.1 mg/mL to about 0.5 mg/mL, about 0.2 mg/mL to about 0.3 mg/mL, about 0.2 mg/mL to about 0.4 mg/mL, about 0.2 mg/mL to about 0.5 mg/mL, or about 0.3 mg/mL to about 0.5 mg/mL.
In some embodiments, the product stream has a relatively high product yield. In certain embodiments, the product stream has a product yield of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. In certain embodiments, the product stream has a product yield in the range of about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 99%, about 60% to about 80%, about 60% to about 90%, about 60% to about 95%, about 60% to about 99%, about 70% to about 90%, about 70% to about 95%, about 70% to about 99%, about 80% to about 95%, about 80% to about 99%, or about 90% to about 99%.
In some embodiments, the product stream has a relatively low concentration of impurities. In certain embodiments, for example, the product stream has a relatively low concentration of host cell proteins. In some embodiments, the product stream has a host cell protein concentration of about 100 ng/(mg G-CSF) or less, about 50 ng/(mg G-CSF) or less, about 20 ng/(mg G-CSF) or less, about 10 ng/(mg G-CSF) or less, about 8 ng/(mg G-CSF) or less, or about 5 ng/(mg G-CSF) or less. In some embodiments, the product stream has a host cell protein concentration in the range of about 0 ng/(mg G-CSF) to about 5 ng/(mg G-CSF), about 0 ng/(mg G-CSF) to about 8 ng/(mg G-CSF), about 0 ng/(mg G-CSF) to about 10 ng/(mg G-CSF), about 0 ng/(mg G-CSF) to about 20 ng/(mg G-CSF), about 0 ng/(mg G-CSF) to about 50 ng/(mg G-CSF), about 0 ng/(mg G-CSF) to about 100 ng/(mg G-CSF), about 5 ng/(mg G-CSF) to about 8 ng/(mg G-CSF), about 5 ng/(mg G-CSF) to about 10 ng/(mg G-CSF), about 5 ng/(mg G-CSF) to about 20 ng/(mg G-CSF), about 5 ng/(mg G-CSF) to about 50 ng/(mg G-CSF), about 5 ng/(mg G-CSF) to about 100 ng/(mg G-CSF), about 10 ng/(mg G-CSF) to about 20 ng/(mg G-CSF), about 10 ng/(mg G-CSF) to about 50 ng/(mg G-CSF), or about 10 ng/(mg G-CSF) to about 100 ng/(mg G-CSF).
In certain embodiments, the product stream has a relatively low concentration of DNA. For example, in some embodiments, the product stream has a DNA concentration of about 100 ng/(mg G-CSF) or less, about 50 ng/(mg G-CSF) or less, about 20 ng/(mg G-CSF) or less, about 10 ng/(mg G-CSF) or less, about 5 ng/(mg G-CSF) or less, about 1 ng/(mg G-CSF) or less, about 0.5 ng/(mg G-CSF) or less, about 0.1 ng/(mg G-CSF) or less, about 0.05 ng/(mg G-CSF) or less, about 0.01 ng/(mg G-CSF) or less, or about 0 ng/(mg G-CSF) (e.g., not detectable). In some embodiments, the product stream has a DNA concentration in the range of about 0 ng/(mg G-CSF) to about 0.01 ng/(mg G-CSF), about 0 ng/(mg G-CSF) to about 0.05 ng/(mg G-CSF), about 0 ng/(mg G-CSF) to about 0.1 ng/(mg G-CSF), about 0 ng/(mg G-CSF) to about 0.5 ng/(mg G-CSF), about 0 ng/(mg G-CSF) to about 1 ng/(mg G-CSF), about 0 ng/(mg G-CSF) to about 5 ng/(mg G-CSF), about 0 ng/(mg G-CSF) to about 10 ng/(mg G-CSF), about 0 ng/(mg G-CSF) to about 20 ng/(mg G-CSF), about 0 ng/(mg G-CSF) to about 50 ng/(mg G-CSF), about 0 ng/(mg G-CSF) to about 100 ng/(mg G-CSF).
In some embodiments, the product stream contains a relatively low amount of aggregates (e.g., aggregates of the product). In some embodiments, the product stream has an aggregate content of about 2% or less, about 1% or less, about 0.90% or less, about 0.80% or less, about 0.70% or less, about 0.60% or less, or about 0.50% or less, about 0.40% or less, about 0.30% or less, about 0.20% or less, or about 0.10% or less. In some embodiments, the product stream has an aggregate content in the range of about 0.10% to about 0.20%, about 0.10% to about 0.30%, about 0.10% to about 0.40%, about 0.10% to about 0.50%, about 0.10% to about 0.60%, about 0.10% to about 0.70%, about 0.10% to about 0.80%, about 0.10% to about 0.90%, about 0.10% to about 1%, or about 0.10% to about 2%.
In some embodiments, the system is configured to be continuously operated. In certain embodiments, for example, the bioreactor is configured to receive at least one feed stream comprising the at least one cell culture medium. In some embodiments, the at least one filtrate is an at least one filtrate stream. In some embodiments, the purified filtrate is a purified filtrate stream.
In some embodiments, the reactor chamber of the bioreactor has a volume of about 1 L or less. In certain of these embodiments, an input stream to the system (e.g., the at least one feed stream) and an output stream of the system (e.g., the purified filtrate stream, the formulated product stream) each have a flow rate of at least about 0.1 mL/min, at least about 0.5 mL/min, at least about 1.0 mL/min, at least about 1.5 mL/min, or at least about 2 mL/min over a period of at least about 1 day. In some embodiments, the input stream and the output stream each have a flow rate in the range of 0.1 mL/min to about 0.5 mL/min, about 0.1 mL/min to about 1.0 mL/min, about 0.1 mL/min to about 1.5 mL/min, about 0.1 mL/min to about 2 mL/min, about 0.5 mL/min to about 1.0 mL/min, about 0.5 mL/min to about 1.5 mL/min, about 0.5 mL/min to about 2 mL/min, or about 1 mL/min to about 2 mL/min over a period of at least about 1 day. In some embodiments, the system is configured to produce at least about 1 mg, at least about 5 mg, at least about 10 mg, at least about 20 mg, at least about 50 mg, at least about 100 mg, at least about 200 mg, at least about 500 mg, at least about 1 g, at least about 2 g, or at least about 5 g of G-CSF per day. In some embodiments, the system is configured to produce an amount of G-CSF in the range of about 1 mg to about 5 mg, about 1 mg to about 10 mg, about 1 mg to about 20 mg, about 1 mg to about 50 mg, about 1 mg to about 100 mg, about 1 mg to about 500 mg, about 1 mg to about 1 g, about 1 mg to about 2 g, about 1 mg to about 5 g, about 10 mg to about 20 mg, about 10 mg to about 50 mg, about 10 mg to about 100 mg, about 10 mg to about 500 mg, about 10 mg to about 1 g, about 10 mg to about 2 g, about 10 mg to about 5 g, about 50 mg to about 100 mg, about 50 mg to about 500 mg, about 50 mg to about 1 g, about 50 mg to about 2 g, about 50 mg to about 5 g, about 100 mg to about 500 mg, about 100 mg to about 1 g, about 100 mg to about 2 g, about 100 mg to about 5 g, about 500 mg to about 1 g, about 500 mg to about 2 g, about 500 mg to about 5 g, or about 1 g to about 5 g per day.
In some embodiments, the reactor chamber of the bioreactor has a volume of about 1 L to about 10 L. In certain of these embodiments, an input stream to the system (e.g., the at least one feed stream) and an output stream of the system (e.g., the purified filtrate stream, the formulated product stream) each have a flow rate of at least about 0.5 mL/min, at least about 1 mL/min, at least about 5 mL/min, at least about 10 mL/min, or at least about 20 mL/min over a period of at least about 1 day. In certain embodiments, the input stream and the output stream each have a flow rate in the range of about 0.5 mL/min to about 1 mL/min, about 0.5 mL/min to about 5 mL/min, about 0.5 mL/min to about 10 mL/min, about 0.5 mL/min to about 20 mL/min, about 1 mL/min to about 5 mL/min, about 1 mL/min to about 10 mL/min, about 1 mL/min to about 20 mL/min, about 5 mL/min to 10 mL/min, about 5 mL/min to about 20 mL/min, or about 10 mL/min to about 20 mL/min over a period of at least about 1 day. In some of these embodiments, the system is configured to produce at least about 50 mg, at least about 100 mg, at least about 200 mg, at least about 500 mg, at least about 1 g, at least about 5 g, at least about 10 g, or at least about 50 g of G-CSF per day. In some embodiments, the system is configured to produce an amount of G-CSF in the range of about 50 mg to about 100 mg, about 50 mg to about 200 mg, about 50 mg to about 500 mg, about 50 mg to about 1 g, about 50 mg to about 5 g, about 50 mg to about 10 g, about 50 mg to about 50 g, about 100 mg to about 500 mg, about 100 mg to about 1 g, about 100 mg to about 5 g, about 100 mg to about 10 g, about 100 mg to about 50 g, about 500 mg to about 1 g, about 500 mg to about 5 g, about 500 mg to about 10 g, about 500 mg to about 50 g, about 1 g to about 10 g, about 1 g to about 50 g, or about 10 g to about 50 g per day.
In some embodiments, the reactor chamber of the bioreactor has a volume of about 10 L to about 50 L. In certain of these embodiments, an input stream to the system (e.g., the at least one feed stream) and an output stream of the system (e.g., the purified filtrate stream, the formulated product stream) each have a flow rate of at least about 5 mL/min, at least about 10 mL/min, at least about 20 mL/min, at least about 50 mL/min, at least about 100 mL/min, at least about 150 mL/min, or at least about 200 mL/min over a period of at least about 1 day. In some embodiments, the input stream and the output stream each have a flow rate in the range of about 5 mL/min to about 10 mL/min, about 5 mL/min to about 20 mL/min, about 5 mL/min to about 50 mL/min, about 5 mL/min to about 100 mL/min, about 5 mL/min to about 150 mL/min, about 5 mL/min to about 200 mL/min, about 10 mL/min to about 20 mL/min, about 10 mL/min to about 50 mL/min, about 10 mL/min to about 100 mL/min, about 10 mL/min to about 150 mL/min, about 10 mL/min to about 200 mL/min, about 50 mL/min to about 100 mL/min, about 50 mL/min to about 150 mL/min, about 50 mL/min to about 200 mL/min, or about 100 mL/min to about 200 mL/min over a period of at least about 1 day. In some of these embodiments, the system is configured to produce at least about 500 mg, at least about 1 g, at least about 2 g, at least about 5 g, at least about 10 g, at least about 50 g, at least about 100 g, at least about 200 g, or at least about 500 g of G-CSF per day. In some embodiments, the system is configured to produce an amount of G-CSF in the range of about 500 mg to about 1 g, about 500 mg to about 2 g, about 500 mg to about 5 g, about 500 mg to about 10 g, about 500 mg to about 50 g, about 500 mg to about 100 g, about 500 mg to about 200 g, about 500 mg to about 500 g, about 1 g to about 5 g, about 1 g to about 10 g, about 1 g to about 50 g, about 1 g to about 100 g, about 1 g to about 500 g, about 10 g to about 50 g, about 10 g to about 100 g, about 10 g to about 500 g, or about 100 g to about 500 g per day. Some embodiments described herein relate to methods for producing G-CSF. In certain embodiments, the method comprises supplying a growth cell medium to a bioreactor (e.g., a perfusion bioreactor). In some embodiments, the method further comprises incubating a first type of biological cells in the growth cell culture medium for a period of at least one day. In some embodiments, the method further comprises at least partially removing the growth cell culture medium from the bioreactor. In some embodiments, the method further comprises supplying at least one cell culture medium (e.g., a production cell culture medium) to the bioreactor. In some embodiments, the method comprises producing, within the bioreactor, a suspension comprising the at least one cell culture medium and at least a first type of biological cells expressing G-CSF.
In some embodiments, the method further comprises causing at least a portion of the suspension to flow through at least one filter to produce at least one filtrate lean in the first type of biological cells. In some embodiments, the at least one filtrate comprises G-CSF.
In some embodiments, the method comprises flowing the at least one filtrate to an adjustment module (e.g., from the at least one filter to the adjustment module). In some embodiments, the method further comprises adjusting, within the adjustment module, one or more properties (e.g., pH, conductivity, product stability) of the at least one filtrate. In certain embodiments, the pH of the at least one filtrate is increased or decreased (e.g., to be compatible with the first column of the purification module). In some embodiments, increasing the pH of the at least one filtrate comprises adding a base to the at least one filtrate. In some embodiments, decreasing the pH of the at least one filtrate comprises adding an acid to the at least one filtrate. In some embodiments, the method further comprises flowing the at least one filtrate and/or the adjusted filtrate through a first column comprising a multimodal cation exchange resin. In certain embodiments, the multimodal cation exchange resin comprises Capto MMC ImpRes, Capto MMC, Nuvia cPrime, Toyopearl MX-Trp-650M, CMM HyperCel, and/or Eshmuno HCX. In certain non-limiting embodiments, the multimodal cation exchange resin comprises a Capto MMC ImpRes resin.
In certain embodiments, the first column is operated in bind-elute mode. In some embodiments, the method further comprises flowing a first mobile phase material through the first column. In some embodiments, the first mobile phase material is configured to promote binding of G-CSF to the multimodal cation exchange resin. In some embodiments, the first mobile phase material comprises sodium citrate, sodium phosphate, sodium chloride, sodium acetate, Tris-HCl, glycine, and/or histidine. According to certain embodiments, the first mobile phase material comprises 20 mM sodium phosphate and/or 20 mM sodium citrate. In some embodiments, the first mobile phase material has a pH of about 4.0, about 4.5, about 5.0, about 5.5, or about 6.0. In some embodiments, the method further comprises flowing the at least one filtrate and/or the adjusted filtrate through the first column and, subsequently, flowing a second mobile phase material through the first column. In some embodiments, the second mobile phase material is configured to wash one or more impurities from the multimodal cation exchange resin. In some embodiments, the second mobile phase material comprises sodium citrate, sodium phosphate, sodium chloride, sodium acetate, Tris-HCl, glycine, and/or histidine. According to certain embodiments, the second mobile phase material comprises 20 mM sodium phosphate and/or 20 mM sodium citrate. In some embodiments, the second mobile phase material has a pH of about 4.5, about 5.0, about 5.5, about 5.8, about 6.0, about 6.5, or about 7.0. In some embodiments, the second mobile phase material has a salt (e.g., sodium chloride) concentration of about 100 mM, about 125 mM, about 150 mM, about 175 mM, or about 200 mM. In some embodiments, the method further comprises subsequently flowing a third mobile phase material through the first column. In some embodiments, the third mobile phase material is configured to elute G-CSF from the multimodal cation exchange resin. In some embodiments, the third mobile phase material comprises sodium citrate, sodium phosphate, sodium chloride, sodium acetate, Tris-HCl, glycine, and/or histidine. According to certain embodiments, the third mobile phase material comprises 20 mM sodium phosphate and/or 20 mM sodium citrate. In some embodiments, the third mobile phase material has a pH of about 6.0, 6.5, 7.0, 7.5, or 8.0. In some embodiments, the third mobile phase material has a salt (e.g., sodium chloride) concentration of about 100 mM, about 125 mM, about 150 mM, about 175 mM, or about 200 mM. In some embodiments, the method further comprises collecting one or more first fractions comprising G-CSF from an outflow of the first column. In some embodiments, the one or more first fractions are lean in the first type of impurity relative to the at least one filtrate or the adjusted filtrate. In some embodiments, the one or more first fractions have a concentration of the first type of impurity that is at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 99% less than the concentration of the first type of impurity in the at least one filtrate or the adjusted filtrate.
In some embodiments, the method further comprises flowing the one or more first fractions through a second column comprising an anion exchange resin. In some embodiments, the anion exchange resin comprises a HyperCel STAR AX resin and/or a Toyopearl NH2-750F resin. In certain embodiments, the second column is operated in flow-through mode. In some embodiments, the method comprises flowing through a first mobile phase material through the second column. In some embodiments, the first mobile phase material comprises sodium citrate, sodium phosphate, sodium chloride, sodium acetate, Tris-HCl, glycine, and/or histidine. According to certain embodiments, the first mobile phase material comprises 20 mM sodium phosphate and/or 20 mM sodium citrate. In some embodiments, the first mobile phase material has a pH of about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0. In some embodiments, the first mobile phase material has a salt (e.g., sodium chloride) concentration of about 100 mM, about 125 mM, about 150 mM, about 175 mM, or about 200 mM. In some embodiments, the method further comprises collecting one or more second fractions comprising G-CSF from an outflow of the second column. In some embodiments, the one or more second fractions are lean in the second type of impurity relative to the first fractions. In some embodiments, the one or more second fractions have a concentration of the second type of impurity that is at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 99% less than the concentration of the second type of impurity in the first fractions.
In some embodiments, the method further comprises flowing the one or more second fractions through a third column comprising an HCIC resin. In some embodiments, the HCIC resin comprises an MEP HyperCel resin, a PPA HyperCel resin, and/or an HEA HyperCel resin. In some embodiments, the third column is operated in bind-elute mode. In some embodiments, the method further comprises flowing a first mobile phase material through the third column prior to flowing the second fractions through the third column. In some embodiments, the first mobile phase material is configured to promote binding of G-CSF to the HCIC resin. In some embodiments, the first mobile phase material has a pH of about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0. In some embodiments, the first mobile phase material has a salt concentration of about 100 mM, about 125 mM, about 150 mM, about 175 mM, or about 200 mM. In some embodiments, the method further comprises flowing a second mobile phase material through the third column after flowing the second fractions through the third column. In some embodiments, the second mobile phase material has a pH of about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5. In some embodiments, the second mobile phase has a salt concentration less than about 200 mM, about 175 mM, about 150 mM, about 125 mM, or about 100 mM. In some embodiments, the method further comprises flowing a third mobile phase material through the third column. In some embodiments, the third mobile phase material is configured to elute G-CSF from the third column. In some embodiments, the third mobile phase material has a pH of about 2.0, about 2.5, about 3.0, about 3.5, or about 4.0. In some embodiments, the third mobile phase material has a salt concentration less than about 200 mM, about 175 mM, about 150 mM, about 125 mM, or about 100 mM. In some embodiments, the method further comprises collecting one or more third fractions comprising G-CSF from an outflow of the third column. In some embodiments, the one or more third fractions are lean in a third type of impurity relative to the second fractions. In some embodiments, the one or more third fractions have a concentration of the third type of impurity that is at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 99% less than the concentration of the third type of impurity in the second fractions. In some embodiments, the third fractions are collected as a biologically-produced product stream.
In some embodiments, the method further comprises flowing the third fractions to a formulation module to produce a formulated product stream. In some embodiments, flowing the purified filtrate through the formulation module comprises flowing the purified filtrate through a tangential flow filtration device. In some embodiments, flowing the purified filtrate through the formulation module comprises flowing the purified filtrate through a viral filtration unit. In some embodiments, the formulated product stream is lean in one or more viruses relative to the purified filtrate stream. In some embodiments, flowing the purified filtrate through the formulation module comprises flowing the purified filtrate through a dilution adjustment unit. In some embodiments, flowing the purified filtrate through the dilution adjustment unit comprises adding a diluent to the purified filtrate. In some embodiments, flowing the purified filtrate through the formulation module comprises depositing one or more portions of the purified filtrate stream into one or more containers (e.g., bags, vials, syringes, bottles). In some embodiments, the one or more containers are aseptic and/or sterile containers.
hGH-Specific Systems and Methods
Some embodiments described herein relate to systems for producing human growth hormone (hGH). According to some embodiments, the system comprises a bioreactor (e.g., a perfusion bioreactor). The bioreactor may comprise a reaction chamber, and, as described above, may have any suitable shape and be formed of any suitable material. In some embodiments, the reaction chamber contains a suspension comprising at least one cell culture medium and at least a first type of biological cells configured to express hGH. In some embodiments, the biological cells are yeast cells. In some embodiments, the yeast cells are Pichia pastoris cells. In some embodiments, for example during a cell growth phase, the at least one cell culture medium comprises chemically defined media comprising a carbon source or buffered glycerol-complex medium (BMGY). In some embodiments, for example during an hGH production phase, the at least one cell culture medium comprises chemically defined media comprising a carbon source and/or other additive for induction of protein expression or buffered methanol-complex media (BMMY).
In some embodiments, the system further comprises at least one filter. In certain embodiments, the at least one filter comprises a filter probe, a filtration membrane, and/or a ceramic filter. The at least one filter may, in some embodiments, be fluidically connected (e.g., directly fluidically connected) to the bioreactor. For example, the at least one filter may comprise at least one filter probe at least partially submerged in the suspension in the bioreactor. In some embodiments, the at least one filter is configured to receive an output of the bioreactor and produce at least one filtrate lean in the first type of biological cells relative to the suspension. In some embodiments, the at least one filtrate comprises hGH.
In some embodiments, the system further comprises an adjustment module configured to adjust one or more properties (e.g., pH, conductivity, product stability) of an input stream received by the adjustment module (e.g., the at least one filtrate) to produce an adjusted output stream (e.g., an adjusted filtrate). In some embodiments, the adjustment module is fluidically connected (e.g., directly fluidically connected) to the bioreactor, the at least one filter, and/or the purification module. In some embodiments, the adjustment module comprises a surge tank. In certain cases, the surge tank has a volume of about 50 mL to about 2 L, about 2 L to about 10 L, or about 10 L to about 100 L. In some cases, the surge tank may advantageously facilitate connection of an upstream process associated with a first flow rate and/or pressure and a downstream process associated with a second, different flow rate and/or pressure. For instance, in some embodiments, a surge tank may help to release pressure from the filter.
In certain illustrative embodiments, the adjustment module is configured to increase or decrease the pH of the at least one filtrate to produce a pH-adjusted filtrate. In some embodiments, the pH of the adjusted filtrate is compatible with a first column of the purification module. In certain cases, adjusting the pH of the at least one filtrate may facilitate effective capture of hGH on the first column of the purification module. In addition, adjusting the pH of the at least one filtrate may advantageously reduce the amount of product aggregates, increase product yield, increase hGH stability, and/or increase product quality. In some embodiments, the pH-adjusted filtrate has a pH of about 4.0, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, or about 9.0.
In some embodiments, the adjustment module is configured to minimize hold time after adjustment. Minimizing hold time after adjustment may, in some cases, advantageously maximize product quality. In certain embodiments, the hold time of the adjusted filtrate is about 12 hours or less, about 6 hours or less, about 1 hour or less, about 30 minutes or less, or about 10 minutes or less. In some embodiments, the hold time of the adjusted filtrate is in the range of about 10 minutes to about 30 minutes, about 10 minutes to about 1 hour, about 10 minutes to about 6 hours, about 10 minutes to about 12 hours, about 10 minutes to about 18 hours, about 10 minutes to about 24 hours, about 30 minutes to about 1 hour, about 30 minutes to about 6 hours, about 30 minutes to about 12 hours, about 30 minutes to about 18 hour, about 30 minutes to about 24 hours, about 1 hour to about 6 hours, about 1 hour to about 12 hours, about 1 hour to about 18 hours, about 1 hour to about 24 hours, about 6 hour to about 12 hours, about 6 hour to about 18 hours, or about 6 hours to about 24 hours.
In some embodiments, the system further comprises a purification module configured to remove at least a first type of impurity, a second type of impurity, and a third type of impurity from an input stream received by the purification module (e.g., the at least one filtrate, the adjusted filtrate) to produce a purified filtrate. In some embodiments, the purification module is fluidically connected (e.g., directly fluidically connected) to the at least one filter and/or the adjustment module.
In some embodiments, the purification module comprises a first partitioning unit configured to remove at least the first type of impurity, a second partitioning unit configured to remove at least the second type of impurity, and a third partitioning unit configured to remove at least the third type of impurity. In certain embodiments, the design framework described above may be used to generate and evaluate candidate sequences of partitioning steps. Using the design framework, for example, a sequence of chromatography columns and associated conditions suitable for capturing and purifying hGH may be identified.
In some embodiments, the purification module comprises a first column comprising a multimodal cation exchange resin. In some embodiments, the first column is configured to remove at least the first type of impurity to produce a first partitioned filtrate lean in the first type of impurity relative to the first filtrate. In some embodiments, the first partitioned filtrate comprises hGH. In some embodiments, the multimodal cation exchange resin comprises Capto MMC, Capto MMC ImpRes, Nuvia cPrime, Toyopearl MX-Trp-650M, CMM HyperCel, and/or Eshmuno HCX.
In some embodiments, the purification module further comprises a second column comprising an anion exchange resin. In some embodiments, the second column is fluidically connected (e.g., directly fluidically connected) to the first column. In some embodiments, the second column is configured to remove at least the second type of impurity to produce a second partitioned filtrate lean in the second type of impurity relative to the first partitioned filtrate. In some embodiments, the second partitioned filtrate comprises hGH. In some embodiments, the anion exchange resin comprises HyperCel STAR AX and/or Toyopearl NH2-750F.
In some embodiments, the purification module optionally comprises a third column comprising an HCIC resin. In some embodiments, the third column is fluidically connected (e.g., directly fluidically connected) to the second column. In some embodiments, the third column is configured to remove at least the third type of impurity to produce a third partitioned filtrate lean in the third type of impurity relative to the second partitioned filtrate. In some embodiments, the third partitioned filtrate comprises hGH. In some embodiments, the HCIC resin comprises MEP HyperCel, PPA HyperCel, and/or HEA HyperCel.
In some embodiments, the system further comprises a formulation module configured to produce a formulated product stream. In some embodiments, the formulation module is fluidically connected (e.g., directly fluidically connected) to the purification module. In some embodiments, the formulation module comprises a filtration unit, a viral filtration unit, and/or a product packaging unit. In some embodiments, the filtration unit comprises a tangential flow filtration device.
In some embodiments, the product stream (e.g., the purified filtrate stream from the purification module, the formulated product stream from the formulation module) has a relatively high concentration of hGH. In certain embodiments, the product stream has an hGH concentration of at least about 0.05 mg/mL, at least about 0.1 mg/mL, at least about 0.2 mg/mL, at least about 0.3 mg/mL, at least about 0.4 mg/mL, at least about 0.5 mg/mL, at least about 0.6 mg/mL, at least about 0.7 mg/mL, at least about 0.8 mg/mL, at least about 0.9 mg/mL, at least about 1 mg/mL, at least about 5 mg/mL, at least about 10 mg/mL, at least about 25 mg/mL, at least about 50 mg/mL, at least about 75 mg/mL, or at least about at least about 90 mg/mL. In some embodiments, the product stream has a hGH concentration in the range of about 0.05 mg/mL to about 100 mg/mL, about 0.5 mg/mL to about 100 mg/mL, or about 1 mg/mL to about 100 mg/mL. In certain embodiments, the product stream has an hGH concentration in the range of about 0.05 mg/mL to about 0.5 mg/mL, about 0.05 mg/mL to about 0.6 mg/mL, about 0.05 mg/mL to about 0.7 mg/mL, about 0.05 mg/mL to about 0.8 mg/mL, about 0.05 mg/mL to about 0.9 mg/mL, about 0.05 mg/mL to about 1.0 mg/mL, about 0.1 mg/mL to about 0.5 mg/mL, about 0.1 mg/mL to about 0.6 mg/mL, about 0.1 mg/mL to about 0.7 mg/mL, about 0.1 mg/mL to about 0.8 mg/mL, about 0.1 mg/mL to about 0.9 mg/mL, about 0.1 mg/mL to about 1 mg/mL, about 0.5 mg/mL to about 0.7 mg/mL, about 0.5 mg/mL to about 0.8 mg/mL, about 0.5 mg/mL to about 0.9 mg/mL, about 0.5 mg/mL to about 1 mg/mL, about 0.6 mg/mL to about 0.8 mg/mL, about 0.6 mg/mL to about 0.9 mg/mL, about 0.6 mg/mL to about 1 mg/mL, or about 0.8 mg/mL to about 1 mg/mL.
In some embodiments, the product stream has a relatively high product yield. In certain embodiments, the product stream has a product yield of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. In certain embodiments, the product stream has a product yield in the range of about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 99%, about 60% to about 80%, about 60% to about 90%, about 60% to about 95%, about 60% to about 99%, about 70% to about 90%, about 70% to about 95%, about 70% to about 99%, about 80% to about 95%, about 80% to about 99%, or about 90% to about 99%.
In some embodiments, the product stream (e.g., the purified filtrate stream from the purification module, the formulated pharmaceutical product stream from the formulation module) has a relatively low concentration of impurities. In certain embodiments, for example, the product stream has a relatively low concentration of host cell proteins. In some embodiments, the product stream has a host cell protein concentration of about 50 ng/(mg hGH) or less, about 20 ng/(mg hGH) or less, about 15 ng/(mg hGH) or less, about 13.72 ng/(mg hGH) or less, about 10 ng/(mg hGH) or less, or about 5 ng/(mg hGH) or less. In some embodiments, the product stream has a host cell protein concentration in the range of about 0 ng/(mg hGH) to about 5 ng/(mg hGH), about 0 ng/(mg hGH) to about 10 ng/(mg hGH), about 0 ng/(mg hGH) to about 13.72 ng/(mg hGH), about 0 ng/(mg hGH) to about 15 ng/(mg hGH), about 0 ng/(mg hGH) to about 20 ng/(mg hGH), about 0 ng/(mg hGH) to about 50 ng/(mg hGH), about 5 ng/(mg hGH) to about 10 ng/(mg hGH), about 5 ng/(mg hGH) to about 13.72 ng/(mg hGH), about 5 ng/(mg hGH) to about 15 ng/(mg hGH), about 5 ng/(mg hGH) to about 20 ng/(mg hGH), or about 5 ng/(mg hGH) to about 50 ng/(mg hGH).
In certain embodiments, the product stream has a relatively low concentration of DNA. For example, in some embodiments, the product stream has a DNA concentration of about 500 ng/(mg hGH) or less, about 200 ng/(mg hGH) or less, about 100 ng/(mg hGH) or less, about 77 ng/(mg hGH) or less, about 50 ng/(mg hGH) or less, about 20 ng/(mg hGH) or less, or about 10 ng/(mg hGH) or less. In some embodiments, the product stream has a DNA concentration in the range of about 10 ng/(mg hGH) to about 50 ng/(mg hGH), about 10 ng/(mg hGH) to about 100 ng/(mg hGH), about 10 ng/(mg hGH) to about 200 ng/(mg hGH), about 10 ng/(mg hGH) to about 500 ng/(mg hGH), about 50 ng/(mg hGH) to about 100 ng/(mg hGH), about 50 ng/(mg hGH) to about 200 ng/(mg hGH), or about 50 ng/(mg hGH) to about 500 ng/(mg hGH).
In some embodiments, the product stream contains a relatively low amount of aggregates (e.g., aggregates of the product). In some embodiments, the product stream has an aggregate content of about 2% or less, about 1% or less, about 0.5% or less, about 0.48% or less, about 0.4% or less, about 0.3% or less, about 0.2% or less, or about 0.1% or less. In some embodiments, the product stream has an aggregate content in the range of about 0.10% to about 0.20%, about 0.10% to about 0.30%, about 0.10% to about 0.40%, about 0.10% to about 0.50%, about 0.10% to about 0.60%, about 0.10% to about 0.70%, about 0.10% to about 0.80%, about 0.10% to about 0.90%, about 0.10% to about 1%, or about 0.10% to about 2%.
In some embodiments, the system is configured to be continuously operated. In certain embodiments, for example, the bioreactor is configured to receive at least one feed stream comprising the at least one cell culture medium. In some embodiments, the at least one filtrate is an at least one filtrate stream. In some embodiments, the purified filtrate is a purified filtrate stream.
In some embodiments, the reactor chamber of the bioreactor has a volume of about 1 L or less. In certain of these embodiments, an input stream to the system (e.g., the at least one feed stream) and an output stream of the system (e.g., the purified filtrate stream, the formulated product stream) each have a flow rate of at least about 0.1 mL/min, at least about 0.5 mL/min, at least about 1.0 mL/min, at least about 1.5 mL/min, or at least about 2 mL/min over a period of at least about 1 day. In some embodiments, the input stream and the output stream each have a flow rate in the range of 0.1 mL/min to about 0.5 mL/min, about 0.1 mL/min to about 1.0 mL/min, about 0.1 mL/min to about 1.5 mL/min, about 0.1 mL/min to about 2 mL/min, about 0.5 mL/min to about 1.0 mL/min, about 0.5 mL/min to about 1.5 mL/min, about 0.5 mL/min to about 2 mL/min, or about 1 mL/min to about 2 mL/min over a period of at least about 1 day. In some embodiments, the system is configured to produce at least about 1 mg, at least about 5 mg, at least about 10 mg, at least about 20 mg, at least about 50 mg, at least about 100 mg, at least about 500 mg, at least about 1 g, at least about 2 g, at least about 3 g, at least about 4 g, or at least about 50 mg of hGH per day. In some embodiments, the system is configured to produce an amount of hGH in the range of about 1 mg to about 5 mg, about 1 mg to about 10 mg, about 1 mg to about 20 mg, about 1 mg to about 50 mg, about 1 mg to about 100 mg, about 1 mg to about 500 mg, about 1 mg to about 1 g, about 1 mg to about 2 g, about 1 mg to about 3 g, about 1 mg to about 4 g, about 1 mg to about 5 g, about 5 mg to about 10 mg, about 5 mg to about 20 mg, about 5 mg to about 50 mg, about 5 mg to about 100 mg, about 5 mg to about 500 mg, about 5 mg to about 1 g, about 10 mg to about 20 mg, about 10 mg to about 50 mg, about 10 mg to about 100 mg, about 10 mg to about 500 mg, about 10 mg to about 1 g, about 10 mg to about 2 g, about 10 mg to about 3 g, about 10 mg to about 4 g, about 10 mg to about 5 g, about 20 mg to about 50 mg, about 20 mg to about 100 mg, about 20 mg to about 500 mg, about 20 mg to about 1 g, about 20 mg to about 2 g, about 20 mg to about 3 g, about 20 mg to about 4 g, or about 20 mg to about 50 mg per day, about 100 mg to about 500 mg, about 100 mg to about 1 g, about 100 mg to about 2 g, about 100 mg to about 3 g, about 100 mg to about 4 g, about 100 mg to about 5 g, about 500 mg to about 1 g, about 500 mg to about 2 g, about 500 mg to about 3 g, about 500 mg to about 4 g, about 500 mg to about 5 g, about 1 g to about 2 g, about 1 g to about 3 g, about 1 g to about 4 g, or about 1 g to about 5 g.
In some embodiments, the reactor chamber of the bioreactor has a volume of about 1 L to about 10 L. In certain of these embodiments, an input stream to the system (e.g., the at least one feed stream) and an output stream of the system (e.g., the purified filtrate stream, the formulated product stream) each have a flow rate of at least about 0.5 mL/min, at least about 1 mL/min, at least about 5 mL/min, at least about 10 mL/min, or at least about 20 mL/min over a period of at least about 1 day. In certain embodiments, the input stream and the output stream each have a flow rate in the range of about 0.5 mL/min to about 1 mL/min, about 0.5 mL/min to about 5 mL/min, about 0.5 mL/min to about 10 mL/min, about 0.5 mL/min to about 20 mL/min, about 1 mL/min to about 5 mL/min, about 1 mL/min to about 10 mL/min, about 1 mL/min to about 20 mL/min, about 5 mL/min to 10 mL/min, about 5 mL/min to about 20 mL/min, or about 10 mL/min to about 20 mL/min over a period of at least about 1 day. In some of these embodiments, the system is configured to produce at least about 50 mg, at least about 100 mg, at least about 200 mg, at least about 500 mg, at least about 1 g, at least about 5 g, at least about 10 g, or at least about 500 mg of hGH per day. In some embodiments, the system is configured to produce an amount of hGH in the range of about 50 mg to about 100 mg, about 50 mg to about 200 mg, about 50 mg to about 500 mg, 50 mg to about 1 g, about 50 mg to about 5 g, about 50 mg to about 10 g, about 50 mg to about 50 g, about 100 mg to about 200 mg, about 100 mg to about 500 mg, about 100 mg to about 1 g, about 100 mg to about 5 g, about 100 mg to about 10 g, about 100 mg to about 50 g, or about 200 mg to about 500 mg, about 200 mg to about 1 g, about 200 mg to about 5 g, about 200 mg to about 10 g, or about 200 mg to about 500 mg, about 500 mg to about 1 g, about 500 mg to about 5 g, about 500 mg to about 10 g, about 500 mg to about 50 g, about 1 g to about 5 g, about 1 g to about 10 g, about 1 g to about 50 g, about 5 g to about 10 g, about 5 g to about 50 g, or about 10 g to about 50 g per day.
In some embodiments, the reactor chamber of the bioreactor has a volume of about 10 L to about 50 L. In certain of these embodiments, an input stream to the system (e.g., the at least one feed stream) and an output stream of the system (e.g., the purified filtrate stream, the formulated product stream) each have a flow rate of at least about 5 mL/min, at least about 10 mL/min, at least about 20 mL/min, at least about 50 mL/min, at least about 100 mL/min, at least about 150 mL/min, or at least about 200 mL/min over a period of at least about 1 day. In some embodiments, the input stream and the output stream each have a flow rate in the range of about 5 mL/min to about 10 mL/min, about 5 mL/min to about 20 mL/min, about 5 mL/min to about 50 mL/min, about 5 mL/min to about 100 mL/min, about 5 mL/min to about 150 mL/min, about 5 mL/min to about 200 mL/min, about 10 mL/min to about 20 mL/min, about 10 mL/min to about 50 mL/min, about 10 mL/min to about 100 mL/min, about 10 mL/min to about 150 mL/min, about 10 mL/min to about 200 mL/min, about 50 mL/min to about 100 mL/min, about 50 mL/min to about 150 mL/min, about 50 mL/min to about 200 mL/min, or about 100 mL/min to about 200 mL/min over a period of at least about 1 day. In some of these embodiments, the system is configured to produce at least about 500 mg, at least about 1 g, at least about 2 g, at least about 5 g, at least about 10 g, at least about 50 g, at least about 100 g, or at least about 250 g of hGH per day. In some embodiments, the system is configured to produce an amount of hGH in the range of about 500 mg to about 1 g, about 500 mg to about 2 g, about 500 mg to about 5 g, about 500 mg to about 10 g, about 500 mg to about 50 g, about 500 mg to about 100 g, about 500 mg to about 250 g, about 1 g to about 5 g, about 1 g to about 10 g, about 1 g to about 50 g, about 1 g to about 100 g, about 1 g to about 250 g, about 10 g to about 50 g, about 10 g to about 100 g, or about 2 10 g to about 250 g, about 50 g to about 100 g, about 50 g to about 250 g, or about 100 g to about 250 g per day.
Some embodiments described herein relate to methods for producing hGH. In certain embodiments, the method comprises supplying a growth cell medium to a bioreactor (e.g., a perfusion bioreactor). In some embodiments, the method further comprises incubating a first type of biological cells in the growth cell culture medium for a period of at least one day. In some embodiments, the method further comprises at least partially removing the growth cell culture medium from the bioreactor. In some embodiments, the method comprises supplying at least one cell culture medium (e.g., a production cell culture medium) to the bioreactor. In some embodiments, the method comprises producing, within the bioreactor, a suspension comprising the at least one cell culture medium and at least a first type of biological cells comprising hGH.
In some embodiments, the method further comprises causing at least a portion of the suspension to flow through at least one filter to produce at least one filtrate lean in the first type of biological cells. In some embodiments, the at least one filtrate comprises hGH.
In some embodiments, the method comprises flowing the at least one filtrate to an adjustment module (e.g., from the at least one filter to the adjustment module). In some embodiments, the method further comprises adjusting, within the adjustment module, one or more properties (e.g., pH, conductivity, product stability) of the at least one filtrate. In certain embodiments, the pH of the at least one filtrate is increased or decreased (e.g., to be compatible with the first column of the purification module). In some embodiments, increasing the pH of the at least one filtrate comprises adding a base to the at least one filtrate. In some embodiments, decreasing the pH of the at least one filtrate comprises adding an acid to the at least one filtrate.
In some embodiments, the method further comprises flowing the at least one filtrate and/or the adjusted filtrate through a first column comprising a multimodal cation exchange resin. In certain embodiments, the multimodal cation exchange resin comprises Capto MMC ImpRes, Capto MMC, Nuvia cPrime, Toyopearl MX-Trp-650M, CMM HyperCel, and/or Eshmuno HCX. In certain embodiments, the multimodal cation exchange resin is a Capto MMC resin.
In certain embodiments, the first column is operated in bind-elute mode. In some embodiments, the method further comprises flowing a first mobile phase material through the first column. In some embodiments, the first mobile phase material is configured to promote binding of hGH to the multimodal cation exchange resin. In some embodiments, the first mobile phase material comprises sodium citrate, sodium phosphate, sodium chloride, sodium acetate, Tris-HCl, glycine, and/or histidine. According to certain embodiments, the first mobile phase material comprises 20 mM sodium phosphate and/or 20 mM sodium citrate. In some embodiments, the first mobile phase material has a pH of about 4.0, about 4.5, about 5.0. about 5.5, or about 6.0. In some embodiments, the method further comprises flowing the at least one filtrate and/or the adjusted filtrate through the first column and, subsequently, flowing a second mobile phase material through the first column. In some embodiments, the second mobile phase material is configured to wash one or more impurities from the multimodal cation exchange resin. In some embodiments, the second mobile phase material comprises sodium citrate, sodium phosphate, sodium chloride, sodium acetate, Tris-HCl, glycine, and/or histidine. According to certain embodiments, the second mobile phase material comprises 20 mM sodium phosphate and/or 20 mM sodium citrate. In some embodiments, the second mobile phase material has a pH of about 4.0, about 4.5, about 5.0, about 5.5, or about 6.0. In some embodiments, the second mobile phase material has a sodium chloride concentration of about 400 mM, about 450 mM, about 475 mM, about 500 mM, about 525 mM, about 550 mM, or about 600 mM. In some embodiments, the method further comprise subsequently flowing a third mobile phase material through the first column. In some embodiments, the third mobile phase material is configured to elute hGH from the multimodal cation exchange resin. In some embodiments, the third mobile phase material comprises sodium citrate, sodium phosphate, sodium chloride, sodium acetate, Tris-HCl, glycine, and/or histidine. According to certain embodiments, the third mobile phase material comprises 20 mM sodium phosphate and/or 20 mM sodium citrate. In some embodiments, the third mobile phase material has a pH of about 5.0, about 5.5, about 6.0, about 6.5, or about 7.0. In some embodiments, the third mobile phase material has a sodium chloride concentration of about 50 mM, about 75 mM, about 100 mM, about 125 mM, or about 150 mM. In some embodiments, the method further comprises collecting one or more first fractions comprising hGH from an outflow of the first column. In some embodiments, the one or more first fractions are lean in the first type of impurity relative to the at least one filtrate or the adjusted filtrate. In some embodiments, the one or more first fractions have a concentration of the first type of impurity that is at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 99% less than the concentration of the first type of impurity in the at least one filtrate or the adjusted filtrate.
In some embodiments, the method further comprises flowing the one or more first fractions through a second column comprising an anion exchange resin. In some embodiments, the anion exchange resin comprises a HyperCel STAR AX resin and/or a Toyopearl NH2-750F resin. In certain embodiments, the second column is operated in flow-through mode. In some embodiments, the method comprises flowing through a first mobile phase material through the second column. In some embodiments, the first mobile phase material comprises sodium citrate, sodium phosphate, sodium chloride, sodium acetate, Tris-HCl, glycine, and/or histidine. According to certain embodiments, the first mobile phase material comprises 20 mM sodium phosphate and/or 20 mM sodium citrate. In some embodiments, the first mobile phase material has a pH of about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0. In some embodiments, the first mobile phase material has a salt (e.g., sodium chloride) concentration of about 100 mM, about 125 mM, about 150 mM, about 175 mM, or about 200 mM. In some embodiments, the method further comprises collecting one or more second fractions comprising hGH from an outflow of the second column. In some embodiments, the one or more second fractions are lean in the second type of impurity relative to the first fractions. In some embodiments, the one or more second fractions have a concentration of the second type of impurity that is at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 99% less than the concentration of the second type of impurity in the first fractions.
In some embodiments, the method further comprises flowing the one or more second fractions through a third column comprising an HCIC resin. In some embodiments, the HCIC resin comprises an MEP HyperCel resin, a PPA HyperCel resin, and/or an HEA HyperCel resin. In some embodiments, the third column is operated in bind-elute mode. In some embodiments, the method further comprises flowing a first mobile phase material through the third column prior to flowing the second fractions through the third column. In some embodiments, the first mobile phase material is configured to promote binding of hGH to the HCIC resin. In some embodiments, the first mobile phase material has a pH of about 5.0, about 5.5, about 6.0, about 6.5, or about 7.0. In some embodiments, the first mobile phase material has a sodium chloride concentration of about 50 mM, about 75 mM, about 100 mM, about 125 mM, or about 150 mM. In some embodiments, the method further comprises flowing a second mobile phase material through the third column after flowing the second fractions through the third column. In some embodiments, the second mobile phase material has a pH of about 4.0, about 4.5, about 5.1, about 5.5, or about 6.0. In some embodiments, the second mobile phase material has a sodium chloride concentration less than about 200 nm, about 150 nm, about 100 nm, or about 50 nm. In some embodiments, the method further comprises flowing a third mobile phase material through the third column. In some embodiments, the third mobile phase material is configured to elute hGH from the third column. In some embodiments, the third mobile phase material has a pH of about 2.0, about 2.5, about 3.0, about 3.5, or about 4.0. In some embodiments, the third mobile phase material has a sodium chloride concentration less than about 200 mM, about 150 mM, about 100 mM, or about 50 mM. In some embodiments, the method further comprises collecting one or more third fractions comprising hGH from an outflow of the third column. In some embodiments, the one or more third fractions are lean in a third type of impurity relative to the second fractions. In some embodiments, the one or more third fractions have a concentration of the third type of impurity that is at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 99% less than the concentration of the third type of impurity in the second fractions. In some embodiments, the third fractions are collected as a biologically-produced product stream.
In some embodiments, the method further comprises flowing the third fractions to a formulation module to produce a formulated product stream. In some embodiments, flowing the purified filtrate through the formulation module comprises flowing the purified filtrate through a tangential flow filtration device. In some embodiments, flowing the purified filtrate through the formulation module comprises flowing the purified filtrate through a viral filtration unit. In some embodiments, the formulated product stream is lean in one or more viruses relative to the purified filtrate stream. In some embodiments, flowing the purified filtrate through the formulation module comprises depositing one or more portions of the purified filtrate stream into one or more containers (e.g., bags, vials, syringes, bottles). In some embodiments, the one or more containers are aseptic and/or sterile containers.
Some embodiments described herein relate to systems for producing interferon α-2b (IFN-α2b). In some embodiments, the system comprises a bioreactor (e.g., a perfusion bioreactor). In some embodiments, the bioreactor comprises a reaction chamber containing suspension comprising at least one cell culture medium and at least a first type of biological cells configured to express IFN-α2b. In some embodiments, the biological cells are yeast cells. In some embodiments, the yeast cells are Pichia pastoris cells. In some embodiments, for example during a cell growth phase, the at least one cell culture medium comprises chemically defined media comprising a carbon source or buffered glycerol-complex medium (BMGY). In some embodiments, for example during an IFN-α2b production phase, the at least one cell culture medium comprises chemically defined media comprising a carbon source and/or other additive for induction of protein expression or buffered methanol-complex media (BMMY).
In some embodiments, the system further comprises a filter. In some embodiments, the at least one filter of the system comprises a filter probe, a filtration membrane, and/or a ceramic filter. The at least one filter may, in some embodiments, be fluidically connected (e.g., directly fluidically connected) to the bioreactor. For example, the at least one filter may comprise a filter probe at least partially submerged in the suspension in the bioreactor. In some embodiments, the at least one filter is configured to receive an output of the bioreactor and produce at least one filtrate lean in the first type of biological cells relative to the suspension. In some embodiments, the at least one filtrate comprises IFN-α2b.
In some embodiments, the system further comprises an adjustment module configured to adjust one or more properties (e.g., pH, conductivity, product stability) of an input stream received by the adjustment module (e.g., the at least one filtrate) to produce an adjusted output stream (e.g., an adjusted filtrate). In some embodiments, the adjustment module is fluidically connected (e.g., directly fluidically connected) to the bioreactor, the at least one filter, and/or the purification module. In some embodiments, the adjustment module comprises a surge tank. In certain cases, the surge tank has a volume of about 50 mL to about 2 L, about 2 L to about 10 L, or about 10 L to about 100 L. In some cases, the surge tank may advantageously facilitate connection of an upstream process associated with a first flow rate and/or pressure and a downstream process associated with a second, different flow rate and/or pressure.
In some embodiments, the adjustment module is configured to increase or decrease the pH of the at least one filtrate to produce a pH-adjusted filtrate. In some embodiments, the pH of the adjusted filtrate is compatible with a first column of the purification module. In certain cases, adjusting the pH of the at least one filtrate may facilitate effective capture of IFN-α2b on the first column of the purification module. In addition, adjusting the pH of the at least one filtrate may advantageously reduce the amount of product aggregates, increase product yield, increase IFN-α2b stability, and/or increase product quality. In some embodiments, the pH-adjusted filtrate has a pH in the range of about 4.0 to about 9.0 (e.g., about 4.0 to about 8.0, about 4.0 to about 7.0, about 6.0 to about 8.0). In some embodiments, the pH-adjusted filtrate has a pH of about 4.0, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, or about 9.0.
In some embodiments, the adjustment module is configured to minimize hold time after adjustment. Minimizing hold time after adjustment may, in some cases, advantageously maximize product quality. In certain embodiments, the hold time of the adjusted filtrate is about 12 hours or less, about 6 hours or less, about 1 hour or less, about 30 minutes or less, or about 10 minutes or less. In some embodiments, the hold time of the adjusted filtrate is in the range of about 10 minutes to about 30 minutes, about 10 minutes to about 1 hour, about 10 minutes to about 6 hours, about 10 minutes to about 12 hours, about 10 minutes to about 18 hours, about 10 minutes to about 24 hours, about 30 minutes to about 1 hour, about 30 minutes to about 6 hours, about 30 minutes to about 12 hours, about 30 minutes to about 18 hour, about 30 minutes to about 24 hours, about 1 hour to about 6 hours, about 1 hour to about 12 hours, about 1 hour to about 18 hours, about 1 hour to about 24 hours, about 6 hour to about 12 hours, about 6 hour to about 18 hours, or about 6 hours to about 24 hours.
In some embodiments, the system further comprises a purification module configured to remove at least a first type of impurity, a second type of impurity, and a third type of impurity from an input stream received by the purification module (e.g., the at least one filtrate, the adjusted filtrate) to produce a purified filtrate. In some embodiments, the purification module is fluidically connected (e.g., directly fluidically connected) to the at least one filter and/or the adjustment module.
In some embodiments, the purification module comprises a first partitioning unit configured to remove at least the first type of impurity, a second partitioning unit configured to remove at least the second type of impurity, and a third partitioning unit configured to remove at least the third type of impurity. In certain embodiments, the design framework described above may be used to generate and evaluate candidate sequences of partitioning steps. Using the design framework, for example, a sequence of chromatography columns and associated conditions suitable for capturing and purifying IFN-α2b may be identified.
In some embodiments, the purification module comprises a first column comprising a multimodal cation exchange resin. In some embodiments, the first column is configured to remove at least the first type of impurity to produce a first partitioned filtrate lean in the first type of impurity relative to the at least one filtrate or the adjusted filtrate. In some embodiments, the first partitioned filtrate comprises IFN-α2b. According to certain embodiments, the multimodal cation exchange resin comprises Capto MMC ImpRes, Capto MMC, Nuvia cPrime, Toyopearl MX-Trp-650M, CMM HyperCel, and/or Eshmuno HCX. In some embodiments, the multimodal cation exchange resin comprises a Capto MMC ImpRes resin.
In some embodiments, the purification module further comprises a second column comprising an HCIC resin. In some embodiments, the second column is fluidically connected (e.g., directly fluidically connected) to the first column. In some embodiments, the second column is configured to remove at least the second type of impurity to produce a second partitioned filtrate lean in the second type of impurity relative to the first partitioned filtrate. In some embodiments, the second partitioned filtrate comprises IFN-α2b. In certain cases, the HCIC resin comprises MEP HyperCel, PPA HyperCel, and/or HEA HyperCel. In some embodiments, the HCIC resin comprises an MEP HyperCel resin or an HEA HyperCel resin.
In some embodiments, the purification module further comprises a third column comprising a cation exchange resin. In some embodiments, the third column is fluidically connected (e.g., directly fluidically connected) to the second column. In some embodiments, the third column is configured to remove at least the third type of impurity to produce a third partitioned filtrate lean in the third type of impurity relative to the second partitioned filtrate. In some embodiments, the third partitioned filtrate comprises IFN-α2b. In some embodiments, the cation exchange resin comprises an SP Sepharose HP resin or a Toyopearl MX-Trp-650M resin.
As another example, in some embodiments, the purification module comprises a first column comprising a multimodal cation exchange resin. In some embodiments, the first column is configured to remove at least the first type of impurity to produce a first partitioned filtrate lean in the first type of impurity relative to the at least one filtrate or the adjusted filtrate. In some embodiments, the first partitioned filtrate comprises IFN-α2b. According to certain embodiments, the multimodal cation exchange resin comprises Capto MMC ImpRes, Capto MMC, CMM HyperCel, and/or Eshmuno HCX. In some embodiments, the multimodal cation exchange resin comprises a Capto MMC ImpRes resin.
The purification module may further comprise a second column comprising a flow-through resin. In some embodiments, the second column is fluidically connected (e.g., directly fluidically connected) to the first column. In some embodiments, the second column is configured to remove at least the second type of impurity to produce a second partitioned filtrate lean in the second type of impurity relative to the first partitioned filtrate. In some embodiments, the second partitioned filtrate comprises IFN-α2b. In certain cases, the flow-through resin comprises Q Sepharose HP resin, HyperCel STAR AX resin, and/or Toyopearl NH2-750F resin. In some embodiments, the flow-through resin comprises a Q Sepharose HP resin.
The purification module may further comprise a third column comprising an anion exchange resin. In some embodiments, the third column is fluidically connected (e.g., directly fluidically connected) to the second column. In some embodiments, the third column is configured to remove at least the third type of impurity to produce a third partitioned filtrate lean in the third type of impurity relative to the second partitioned filtrate. In some embodiments, the third partitioned filtrate comprises IFN-α2b. In some embodiments, the anion exchange resin comprises a Capto Adhere resin.
In other embodiments, the purification module comprises a first partitioning unit configured to remove at least the first type of impurity and a second partitioning unit configured to remove at least the second type of impurity. In some such cases, the first column may comprise a multimodal cation exchange resin, as described herein, and the second column may comprise an anion exchange resin, as described herein.
Regardless of the purification module used, in some embodiments, the system further comprises a formulation module configured to produce a formulated product stream. In some embodiments, the formulation module is fluidically connected (e.g., directly fluidically connected) to the purification module. In some embodiments, the formulation module comprises a filtration unit, a viral filtration unit, and/or a product packaging unit. In some embodiments, the filtration unit comprises a tangential flow filtration device.
In some embodiments, the product stream (e.g., the purified filtrate stream from the purification module, the formulated product stream from the formulation module) has a relatively high concentration of IFN. In certain embodiments, the product stream has a product concentration of at least about 0.01 mg/mL, at least about 0.02 mg/mL, at least about 0.03 mg/mL, at least about 0.04 mg/mL, at least about 0.05 mg/mL, at least about 0.1 mg/mL, at least about 0.5 mg/mL, at least about 1 mg/mL, at least about 5 mg/mL, at least about 10 mg/mL, at least about 25 mg/mL, at least about 50 mg/mL, at least about 75 mg/mL, or at least about at least about 90 mg/mL. In some embodiments, the product stream has a IFN concentration in the range of about 0.05 mg/mL to about 100 mg/mL, about 0.5 mg/mL to about 100 mg/mL, or about 1 mg/mL to about 100 mg/mL. In certain embodiments, the product stream has a product concentration in the range of about 0.01 mg/mL to about 0.1 mg/mL, about 0.02 mg/mL to about 0.1 mg/mL, about 0.03 mg/mL to about 0.1 mg/mL, about 0.04 mg/mL to about 0.1 mg/mL, or about 0.05 mg/mL to about 0.1 mg/mL.
In some embodiments, the product stream has a relatively high product yield. In certain embodiments, the product stream has a product yield of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. In certain embodiments, the product stream has a product yield in the range of about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 99%, about 60% to about 80%, about 60% to about 90%, about 60% to about 95%, about 60% to about 99%, about 70% to about 90%, about 70% to about 95%, about 70% to about 99%, about 80% to about 95%, about 80% to about 99%, or about 90% to about 99%.
In some embodiments, the product stream has a purity of at least about 50%, at least about 60%, at least about 70%, at least about 77%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, the product stream has a purity in the range of about 50% to about 99%, about 60% to about 99%, about 70% to about 99%, about 77% to about 99%, about 80% to about 99%, or about 90% to about 99%. For example, in some embodiments in which the purification module comprises a flow-through resin, the product stream has a product yield of at least about 65% (e.g., in the range of about 65% to about 99%, at least about 70%, in the range of about 70% to about 99%).
In some embodiments, the product stream has a DNA concentration of about 1 ng/(mg IFN-α2b) or less, about 0.8 ng/(mg IFN-α2b) or less, about 0.6 ng/(mg IFN-α2b) or less, about 0.51 ng/(mg IFN-α2b) or less, or about 0.2 ng/(mg IFN-α2b) or less. In some embodiments, the product stream has a DNA concentration in the range of about 0.0 ng/(mg IFN-α2b) to about 0.2 ng/(mg IFN-α2b), 0.0 ng/(mg IFN-α2b) to about 0.51 ng/(mg IFN-α2b), about 0.0 ng/(mg IFN-α2b) to about 0.6 ng/(mg IFN-α2b), about 0.0 ng/(mg IFN-α2b) to about 0.8 ng/(mg IFN-α2b), about 0.0 ng/(mg IFN-α2b) to about 1 ng/(mg IFN-α2b), about 0.2 ng/(mg IFN-α2b) to about 0.6 ng/(mg IFN-α2b), about 0.2 ng/(mg IFN-α2b) to about 0.8 ng/(mg IFN-α2b), or about 0.2 ng/(mg IFN-α2b) to about 1 ng/(mg IFN-α2b)
In some embodiments, the product stream has an aggregate content of about 2% or less, about 1% or less, about 0.5% or less, about 0.1% or less, about 0.07% or less, or about 0.05% or less. In some embodiments, the product stream has an aggregate content in the range of about 0.05% to about 0.1%, about 0.05% to about 0.5%, about 0.05% to about 1%, or about 0.05% to about 2%.
In some embodiments, the system is configured to be continuously operated. In certain embodiments, for example, the bioreactor is configured to receive at least one feed stream comprising the at least one cell culture medium. In some embodiments, the at least one filtrate is an at least one filtrate stream. In some embodiments, the purified filtrate is a purified filtrate stream.
In some embodiments, the reactor chamber of the bioreactor has a volume of about 1 L or less. In certain of these embodiments, an input stream to the system (e.g., the at least one feed stream) and an output stream of the system (e.g., the purified filtrate stream, the formulated product stream) each have a flow rate of at least about 0.1 mL/min, at least about 0.5 mL/min, at least about 1.0 mL/min, at least about 1.5 mL/min, or at least about 2 mL/min over a period of at least about 1 day. In some embodiments, the input stream and the output stream each have a flow rate in the range of 0.1 mL/min to about 0.5 mL/min, about 0.1 mL/min to about 1.0 mL/min, about 0.1 mL/min to about 1.5 mL/min, about 0.1 mL/min to about 2 mL/min, about 0.5 mL/min to about 1.0 mL/min, about 0.5 mL/min to about 1.5 mL/min, about 0.5 mL/min to about 2 mL/min, or about 1 mL/min to about 2 mL/min over a period of at least about 1 day. In some embodiments, the system is configured to produce at least about 1 mg, at least about 5 mg, at least about 10 mg, at least about 20 mg, at least about 100 mg, at least about 500 mg, at least about 1 g, at least about 2 g, at least about 3 g, at least about 4 g, or at least about 50 mg of IFN-α2b per day. In some embodiments, the system is configured to produce an amount of IFN-α2b in the range of about 1 mg to about 5 mg, about 1 mg to about 10 mg, about 1 mg to about 20 mg, about 1 mg to about 50 mg, about 1 mg to about 100 mg, about 1 mg to about 500 mg, about 1 mg to about 1 g, about 1 mg to about 5 g, about 5 mg to about 10 mg, about 5 mg to about 20 mg, about 5 mg to about 50 mg, about 5 mg to about 100 mg, about 5 mg to about 500 mg, about 5 mg to about 1 g, about 5 mg to about 5 g, about 10 mg to about 20 mg, about 10 mg to about 50 mg, about 10 mg to about 100 mg, about 10 mg to about 500 mg, about 10 mg to about 1 g, about 10 mg to about 5 g, about 20 mg to about 100 mg, about 20 mg to about 500 mg, about 20 mg to about 1 g, or about 20 mg to about 50 mg, about 50 mg to about 100 mg, about 50 mg to about 500 mg, about 50 mg to about 1 g, about 50 mg to about 5 g, about 100 mg to about 500 mg, about 100 mg to about 1 g, about 100 mg to about 5 g, about 500 mg to about 1 g, about 500 mg to about 5 g, or about 1 g to about 5 g per day.
In some embodiments, the reactor chamber of the bioreactor has a volume of about 1 L to about 10 L. In certain of these embodiments, an input stream to the system (e.g., the at least one feed stream) and an output stream of the system (e.g., the purified filtrate stream, the formulated product stream) each have a flow rate of at least about 0.5 mL/min, at least about 1 mL/min, at least about 5 mL/min, at least about 10 mL/min, or at least about 20 mL/min over a period of at least about 1 day. In certain embodiments, the input stream and the output stream each have a flow rate in the range of about 0.5 mL/min to about 1 mL/min, about 0.5 mL/min to about 5 mL/min, about 0.5 mL/min to about 10 mL/min, about 0.5 mL/min to about 20 mL/min, about 1 mL/min to about 5 mL/min, about 1 mL/min to about 10 mL/min, about 1 mL/min to about 20 mL/min, about 5 mL/min to 10 mL/min, about 5 mL/min to about 20 mL/min, or about 10 mL/min to about 20 mL/min over a period of at least about 1 day. In some of these embodiments, the system is configured to produce at least about 50 mg, at least about 100 mg, at least about 200 mg, at least about 500 mg, at least about 1 g, at least about 5 g, at least about 10 g, or at least about 50 g of IFN-α2b per day In some of these embodiments, the system is configured to produce at least about 50 mg, at least about 100 mg, at least about 200 mg, at least about 500 mg, at least about 1 g, at least about 5 g, at least about 10 g, or at least about 500 mg of IFN-α2b per day. In some embodiments, the system is configured to produce an amount of IFN-α2b in the range of about 50 mg to about 100 mg, about 50 mg to about 200 mg, about 50 mg to about 500 mg, about 50 mg to about 1 g, about 50 mg to about 5 g, about 50 mg to about 10 g, about 50 mg to about 50 g, about 100 mg to about 200 mg, about 100 mg to about 500 mg, about 100 mg to about 1 g, about 100 mg to about 5 g, about 100 mg to about 10 g, about 100 mg to about 50 g, about 200 mg to about 1 g, about 200 mg to about 5 g, about 200 mg to about 10 g, or about 200 mg to about 500 mg, about 500 mg to about 1 g, about 500 mg to about 5 g, about 500 mg to about 10 g, about 500 mg to about 50 g, about 1 g to about 5 g, about 1 g to about 10 g, about 1 g to about 50 g, about 5 g to about 10 g, about 5 g to about 50 g, or about 10 g to about 50 g per day.
In some embodiments, the reactor chamber of the bioreactor has a volume of about 10 L to about 50 L. In certain of these embodiments, an input stream to the system (e.g., the at least one feed stream) and an output stream of the system (e.g., the purified filtrate stream, the formulated product stream) each have a flow rate of at least about 5 mL/min, at least about 10 mL/min, at least about 20 mL/min, at least about 50 mL/min, at least about 100 mL/min, at least about 150 mL/min, or at least about 200 mL/min over a period of at least about 1 day. In some embodiments, the input stream and the output stream each have a flow rate in the range of about 5 mL/min to about 10 mL/min, about 5 mL/min to about 20 mL/min, about 5 mL/min to about 50 mL/min, about 5 mL/min to about 100 mL/min, about 5 mL/min to about 150 mL/min, about 5 mL/min to about 200 mL/min, about 10 mL/min to about 20 mL/min, about 10 mL/min to about 50 mL/min, about 10 mL/min to about 100 mL/min, about 10 mL/min to about 150 mL/min, about 10 mL/min to about 200 mL/min, about 50 mL/min to about 100 mL/min, about 50 mL/min to about 150 mL/min, about 50 mL/min to about 200 mL/min, or about 100 mL/min to about 200 mL/min over a period of at least about 1 day. In some of these embodiments, the system is configured to produce at least about 500 mg, at least about 1 g, at least about 2 g, at least about 5 g, at least about 10 g, at least about 50 g, at least about 100 g, or at least about 10 250 g of IFN-α2b per day. In some embodiments, the system is configured to produce an amount of IFN-α2b in the range of about 500 mg to about 1 g, about 500 mg to about 2 g, about 500 mg to about 5 g, about 500 mg to about 10 g, about 500 mg to about 50 g, about 500 mg to about 100 g, about 500 mg to about 250 g, about 1 g to about 5 g, about 1 g to about 10 g, about 1 g to about 50 g, about 1 g to about 100 g, about 5 g to about 250 g, about 5 g to about 10 g, about 5 g to about 50 g, about 5 g to about 100 g, or about 52 g to about 250 g, about 10 g to about 100 g, about 10 g to about 250 g, about 50 g to about 100 g, about 50 g to about 250 g, or about 100 g to about 250 g per day.
Some embodiments described herein relate to methods for producing IFN-α2b. In certain embodiments, the method comprises supplying a growth cell medium to a bioreactor (e.g., a perfusion bioreactor). In some embodiments, the method further comprises incubating a first type of biological cells in the growth cell culture medium for a period of at least one day. In some embodiments, the method further comprises at least partially removing the growth cell culture medium from the bioreactor. In some embodiments, the method comprises supplying at least one cell culture medium (e.g., a production cell culture medium) to the bioreactor. In some embodiments, the method comprises producing, within the bioreactor, a suspension comprising the at least one cell culture medium and at least a first type of biological cells comprising IFN-α2b. In some embodiments, the method further comprises causing at least a portion of the suspension to flow through at least one filter to produce at least one filtrate lean in the first type of biological cells. In some embodiments, the at least one filtrate comprises IFN-α2b.
In some embodiments, the method comprises flowing the at least one filtrate to an adjustment module (e.g., from the at least one filter to the adjustment module). In some embodiments, the method further comprises adjusting, within the adjustment module, one or more properties (e.g., pH, conductivity, product stability) of the at least one filtrate. In certain embodiments, the pH of the at least one filtrate is increased or decreased (e.g., to be compatible with the first column of the purification module). In some embodiments, increasing the pH of the at least one filtrate comprises adding a base to the at least one filtrate. In some embodiments, decreasing the pH of the at least one filtrate comprises adding an acid to the at least one filtrate.
In some embodiments, the method further comprises flowing the at least one filtrate and/or the adjusted filtrate through a first column comprising a multimodal cation exchange resin. In certain embodiments, the multimodal cation exchange resin is a Capto MMC ImpRes resin. In some embodiments, the method further comprises collecting one or more first fractions comprising IFN-α2b from an outflow of the first column. In some embodiments, the one or more first fractions are lean in the first type of impurity relative to the at least one filtrate or the adjusted filtrate. In some embodiments, the one or more first fractions have a concentration of the first type of impurity that is at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 99% less than the concentration of the first type of impurity in the at least one filtrate or the adjusted filtrate.
In some embodiments, the method further comprises flowing the one or more first fractions through a second column. The method may further comprise collecting one or more second fractions comprising IFN-α2b from an outflow of the second column. In some such cases, the method may further comprise flowing the one or more second fractions through a third column. In some instances, the method may also comprise collecting one or more third fractions comprising IFN-α2b from an outflow of the third column. For example, in some embodiments, the method further comprises flowing the one or more first fractions through a second column comprising an HCIC resin. In some embodiments, the HCIC comprises an MEP HyperCel resin or an HEA HyperCel resin. In some embodiments, the method further comprises collecting one or more second fractions comprising IFN-α2b from an outflow of the second column. In some embodiments, the one or more second fractions are lean in the second type of impurity relative to the first fractions. In some embodiments, the one or more second fractions have a concentration of the second type of impurity that is at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 99% less than the concentration of the second type of impurity in the first fractions.
In some embodiments, the method further comprises flowing the one or more second fractions through a third column comprising a cation exchange resin. In some embodiments, the cation exchange resin comprises an SP Sepharose HP resin or a Toyopearl MX-Trp-650M resin. In some embodiments, the method further comprises collecting one or more third fractions comprising IFN-α2b from an outflow of the third column. In some embodiments, the one or more third fractions are lean in a third type of impurity relative to the second fractions. In some embodiments, the one or more third fractions have a concentration of the third type of impurity that is at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 99% less than the concentration of the third type of impurity in the second fractions. In some embodiments, the third fractions are collected as a biologically-produced product stream.
As another example, in some embodiments, the method further comprises flowing the one or more first fractions through a second column comprising a flow-through resin. In some embodiments, the flow-through resin comprises a Q Sepharose HP resin. In some embodiments, the method further comprises collecting one or more second fractions comprising IFN-α2b from an outflow of the second column. In some embodiments, the one or more second fractions are lean in the second type of impurity relative to the first fractions. In some embodiments, the one or more second fractions have a concentration of the second type of impurity that is at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 99% less than the concentration of the second type of impurity in the first fractions. In some embodiments, the method further comprises flowing the one or more second fractions through a third column comprising an anion exchange resin. In some embodiments, the anion exchange resin comprises a Capto Adhere resin. In some embodiments, the method further comprises collecting one or more third fractions comprising IFN-α2b from an outflow of the third column. In some embodiments, the one or more third fractions are lean in a third type of impurity relative to the second fractions. In some embodiments, the one or more third fractions have a concentration of the third type of impurity that is at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 99% less than the concentration of the third type of impurity in the second fractions. In some embodiments, the third fractions are collected as a biologically-produced product stream.
In some embodiments, the method further comprises flowing the third fractions to a formulation module to produce a formulated product stream. In some embodiments, flowing the purified filtrate through the formulation module comprises flowing the purified filtrate through a tangential flow filtration device. In some embodiments, flowing the purified filtrate through the formulation module comprises flowing the purified filtrate through a viral filtration unit. In some embodiments, the formulated product stream is lean in one or more viruses relative to the purified filtrate stream. In some embodiments, flowing the purified filtrate through the formulation module comprises depositing one or more portions of the purified filtrate stream into one or more containers (e.g., bags, vials, syringes, bottles). In some embodiments, the one or more containers are aseptic and/or sterile containers.
Some embodiments described herein relate to systems for producing a single-domain antibody. According to some embodiments, the system comprises a bioreactor (e.g., a perfusion bioreactor), at least one filter, and a purification module. The bioreactor may comprise a reaction chamber, and, as described above, may have any suitable shape and be formed of any suitable material. In some embodiments, the reaction chamber contains a suspension comprising at least one cell culture medium and at least a first type of biological cells configured to express the single-domain antibody. In some embodiments, the biological cells are yeast cells. In certain cases, the yeast cells are Pichia pastoris cells. In some embodiments, for example during a cell growth phase, the at least one cell culture medium comprises chemically defined media comprising a carbon source or buffered glycerol-complex medium (BMGY). In some embodiments, for example during the single-domain antibody production phase, the at least one cell culture medium comprises chemically defined media comprising a carbon source and/or other additive for induction of protein expression or buffered methanol-complex media (BMMY).
In some embodiments, the at least one filter of the system comprises a filter probe, a filtration membrane, and/or a ceramic filter. The at least one filter may, in some embodiments, be fluidically connected (e.g., directly fluidically connected) to the bioreactor. For example, in certain embodiments, the at least one filter comprises at least one filter probe at least partially submerged in the suspension in the bioreactor. In some embodiments, the at least one filter is configured to receive an output of the bioreactor and produce at least one filtrate lean in the first type of biological cells relative to the suspension. In some embodiments, the at least one filtrate comprises a single-domain antibody.
In some embodiments, the system further comprises an adjustment module configured to adjust one or more properties (e.g., pH, conductivity, product stability) of an input stream received by the adjustment module (e.g., the at least one filtrate) to produce an adjusted output stream (e.g., an adjusted filtrate). According to some embodiments, the adjustment module is fluidically connected (e.g., directly fluidically connected) to the bioreactor, the at least one filter, and/or the purification module. In some embodiments, the adjustment module comprises a surge tank. In certain cases, the surge tank has a volume of about 50 mL to about 2 L, about 2 L to about 10 L, or about 10 L to about 100 L. In some cases, the surge tank may advantageously facilitate connection of an upstream process associated with a first flow rate and/or pressure and a downstream process associated with a second, different flow rate and/or pressure.
In certain illustrative embodiments, the adjustment module is configured to increase or decrease the pH of the at least one filtrate to produce a pH-adjusted filtrate. In some embodiments, the pH of the adjusted filtrate is compatible with a first column of the purification module. In certain cases, adjusting the pH of the at least one filtrate may facilitate effective capture of a single-domain antibody on the first column of the purification module. In addition, adjusting the pH of the at least one filtrate may advantageously reduce the amount of product aggregates, increase product yield, increase single-domain antibody stability, and/or maximize product quality. In some embodiments, the pH-adjusted filtrate has a pH of about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, or about 9.0. In certain embodiments, the pH-adjusted filtrate has a pH in the range of about 4.5 to about 7.0 (e.g., about 4.5 and to about 6.0, about 4.5 and to about 5.5).
In some embodiments, the adjustment module is configured to minimize hold time after adjustment. Minimizing hold time after adjustment may, in some cases, advantageously maximize product quality. In certain embodiments, the hold time of the adjusted filtrate is about 12 hours or less, about 6 hours or less, about 1 hour or less, about 30 minutes or less, or about 10 minutes or less. In some embodiments, the hold time of the adjusted filtrate is in the range of about 10 minutes to about 30 minutes, about 10 minutes to about 1 hour, about 10 minutes to about 6 hours, about 10 minutes to about 12 hours, about 10 minutes to about 18 hours, about 10 minutes to about 18 hours, about 30 minutes to about 1 hour, about 30 minutes to about 6 hours, about 30 minutes to about 12 hours, about 30 minutes to about 18 hour, about 30 minutes to about 24 hours, about 1 hour to about 6 hours, about 1 hour to about 12 hours, about 1 hour to about 18 hours, about 1 hour to about 24 hours, about 6 hour to about 12 hours, about 6 hour to about 18 hours, or about 6 hours to about 24 hours.
In some embodiments, the purification module is configured to remove at least a first type of impurity, a second type of impurity, and/or a third type of impurity from an input stream received by the purification module (e.g., the at least one filtrate, the adjusted filtrate) to produce a purified filtrate. In some embodiments, the purification module is fluidically connected (e.g., directly fluidically connected) to the at least one filter and/or the adjustment module.
In some embodiments, the purification module comprises a first partitioning unit configured to remove at least the first type of impurity, a second partitioning unit configured to remove at least the second type of impurity, and optionally a third partitioning unit configured to remove at least the third type of impurity. In certain embodiments, the design framework described above may be used to generate and evaluate candidate sequences of partitioning steps. Using the design framework, for example, a sequence of chromatography columns and associated conditions suitable for capturing and purifying a single-domain antibody may be identified.
In some embodiments, the purification module comprises a first column comprising a multimodal cation exchange resin. In some embodiments, the first column is configured to remove at least the first type of impurity to produce a first partitioned filtrate that comprises a single-domain antibody and is lean in the first type of impurity relative to the first filtrate. According to certain embodiments, the multimodal cation exchange resin comprises Capto MMC, CMM HyperCel, Nuvia cPrime, Toyopearl MX-Trp-650M, Eshmuno HCX, and/or Capto MMC ImpRes. In some embodiments, the multimodal cation exchange resin comprises CMM HyperCel.
In some embodiments, the purification module further comprises a second column comprising an anion exchange resin (e.g., a salt-tolerant anion exchange resin). The second column may be fluidically connected (e.g., directly fluidically connected) to the first column, according to certain embodiments. In some embodiments, the second column is configured to remove at least the second type of impurity to produce a second partitioned filtrate that comprises a single-domain antibody and is lean in the second type of impurity relative to the first partitioned filtrate. In certain cases, the anion exchange resin comprises HyperCel STAR AX, Capto Adhere, and/or PPA HyperCel. In some embodiments, the anion exchange resin comprises HyperCel STAR AX.
In some embodiments, the system further comprises a formulation module configured to produce a formulated product stream. In some embodiments, the formulation module is fluidically connected (e.g., directly fluidically connected) to the purification module. In some embodiments, the formulation module comprises a filtration unit, a viral filtration unit, a dilution adjustment unit, and/or a product packaging unit. According to some embodiments, the filtration unit comprises a tangential flow filtration device.
In some embodiments, the product stream (e.g., the purified filtrate stream from the purification module, the formulated product stream from the formulation module) has a relatively high concentration of the single-domain antibody. In certain embodiments, the product stream has a single-domain antibody concentration of at least about 0.05 mg/mL, at least about 0.1 mg/mL, at least about 0.2 mg/mL, at least about 0.3 mg/mL, at least about 0.4 mg/mL, at least about 0.5 mg/mL, at least about 1 mg/mL, at least about 5 mg/mL, at least about 10 mg/mL, at least about 25 mg/mL, at least about 50 mg/mL, at least about 75 mg/mL, or at least about at least about 90 mg/mL. In some embodiments, the product stream has a single-domain antibody concentration in the range of about 0.05 mg/mL to about 100 mg/mL, about 0.5 mg/mL to about 100 mg/mL, or about 1 mg/mL to about 100 mg/mL. In certain embodiments, the product stream has a single-domain antibody concentration in the range of about 0.05 mg/mL to about 0.2 mg/mL, about 0.05 mg/mL to about 0.3 mg/mL, about 0.05 mg/mL to about 0.4 mg/mL, about 0.05 mg/mL to about 0.5 mg/mL, about 0.1 mg/mL to about 0.3 mg/mL, about 0.1 mg/mL to about 0.4 mg/mL, about 0.1 mg/mL to about 0.5 mg/mL, about 0.2 mg/mL to about 0.3 mg/mL, about 0.2 mg/mL to about 0.4 mg/mL, about 0.2 mg/mL to about 0.5 mg/mL, or about 0.3 mg/mL to about 0.5 mg/mL.
In some embodiments, the product stream has a relatively high product yield. In certain embodiments, the product stream has a product yield of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. In certain embodiments, the product stream has a product yield in the range of about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 99%, about 60% to about 80%, about 60% to about 90%, about 60% to about 95%, about 60% to about 99%, about 70% to about 90%, about 70% to about 95%, about 70% to about 99%, about 80% to about 95%, about 80% to about 99%, or about 90% to about 99%.
In some embodiments, the product stream has a relatively low concentration of impurities. In certain embodiments, for example, the product stream has a relatively low concentration of host cell proteins. In some embodiments, the product stream has a host cell protein concentration of about 100 ng/(mg single-domain antibody) or less, about 50 ng/(mg single-domain antibody) or less, about 20 ng/(mg single-domain antibody) or less, about 10 ng/(mg single-domain antibody) or less, about 8 ng/(mg single-domain antibody) or less, or about 5 ng/(mg single-domain antibody) or less. In some embodiments, the product stream has a host cell protein concentration in the range of about 0 ng/(mg single-domain antibody) to about 5 ng/(mg single-domain antibody), about 0 ng/(mg single-domain antibody) to about 8 ng/(mg single-domain antibody), about 0 ng/(mg single-domain antibody) to about 10 ng/(mg single-domain antibody), about 0 ng/(mg single-domain antibody) to about 20 ng/(mg single-domain antibody), about 0 ng/(mg single-domain antibody) to about 50 ng/(mg single-domain antibody), about 0 ng/(mg single-domain antibody) to about 100 ng/(mg single-domain antibody), about 5 ng/(mg single-domain antibody) to about 8 ng/(mg single-domain antibody), about 5 ng/(mg single-domain antibody) to about 10 ng/(mg single-domain antibody), about 5 ng/(mg single-domain antibody) to about 20 ng/(mg single-domain antibody), about 5 ng/(mg single-domain antibody) to about 50 ng/(mg single-domain antibody), about 5 ng/(mg single-domain antibody) to about 100 ng/(mg single-domain antibody), about 10 ng/(mg single-domain antibody) to about 20 ng/(mg single-domain antibody), about 10 ng/(mg single-domain antibody) to about 50 ng/(mg single-domain antibody), or about 10 ng/(mg single-domain antibody) to about 100 ng/(mg single-domain antibody).
In certain embodiments, the product stream has a relatively low concentration of DNA. For example, in some embodiments, the product stream has a DNA concentration of about 100 ng/(mg single-domain antibody) or less, about 50 ng/(mg single-domain antibody) or less, about 20 ng/(mg single-domain antibody) or less, about 10 ng/(mg single-domain antibody) or less, about 5 ng/(mg single-domain antibody) or less, about 1 ng/(mg single-domain antibody) or less, about 0.5 ng/(mg single-domain antibody) or less, about 0.1 ng/(mg single-domain antibody) or less, about 0.05 ng/(mg single-domain antibody) or less, about 0.01 ng/(mg single-domain antibody) or less, or about 0 ng/(mg single-domain antibody) (e.g., not detectable). In some embodiments, the product stream has a DNA concentration in the range of about 0 ng/(mg single-domain antibody) to about 0.01 ng/(mg single-domain antibody), about 0 ng/(mg single-domain antibody) to about 0.05 ng/(mg single-domain antibody), about 0 ng/(mg single-domain antibody) to about 0.1 ng/(mg single-domain antibody), about 0 ng/(mg single-domain antibody) to about 0.5 ng/(mg single-domain antibody), about 0 ng/(mg single-domain antibody) to about 1 ng/(mg single-domain antibody), about 0 ng/(mg single-domain antibody) to about 5 ng/(mg single-domain antibody), about 0 ng/(mg single-domain antibody) to about 10 ng/(mg single-domain antibody), about 0 ng/(mg single-domain antibody) to about 20 ng/(mg single-domain antibody), about 0 ng/(mg single-domain antibody) to about 50 ng/(mg single-domain antibody), about 0 ng/(mg single-domain antibody) to about 100 ng/(mg single-domain antibody).
In some embodiments, the product stream contains a relatively low amount of aggregates (e.g., aggregates of the product). In some embodiments, the product stream has an aggregate content of about 2% or less, about 1% or less, about 0.90% or less, about 0.80% or less, about 0.70% or less, about 0.60% or less, or about 0.50% or less, about 0.40% or less, about 0.30% or less, about 0.20% or less, or about 0.10% or less. In some embodiments, the product stream has an aggregate content in the range of about 0.10% to about 0.20%, about 0.10% to about 0.30%, about 0.10% to about 0.40%, about 0.10% to about 0.50%, about 0.10% to about 0.60%, about 0.10% to about 0.70%, about 0.10% to about 0.80%, about 0.10% to about 0.90%, about 0.10% to about 1%, or about 0.10% to about 2%.
In some embodiments, the system is configured to be continuously operated. In certain embodiments, for example, the bioreactor is configured to receive at least one feed stream comprising the at least one cell culture medium. In some embodiments, the at least one filtrate is an at least one filtrate stream. In some embodiments, the purified filtrate is a purified filtrate stream.
In some embodiments, the reactor chamber of the bioreactor has a volume of about 1 L or less. In certain of these embodiments, an input stream to the system (e.g., the at least one feed stream) and an output stream of the system (e.g., the purified filtrate stream, the formulated product stream) each have a flow rate of at least about 0.1 mL/min, at least about 0.5 mL/min, at least about 1.0 mL/min, at least about 1.5 mL/min, or at least about 2 mL/min over a period of at least about 1 day. In some embodiments, the input stream and the output stream each have a flow rate in the range of 0.1 mL/min to about 0.5 mL/min, about 0.1 mL/min to about 1.0 mL/min, about 0.1 mL/min to about 1.5 mL/min, about 0.1 mL/min to about 2 mL/min, about 0.5 mL/min to about 1.0 mL/min, about 0.5 mL/min to about 1.5 mL/min, about 0.5 mL/min to about 2 mL/min, or about 1 mL/min to about 2 mL/min over a period of at least about 1 day. In some embodiments, the system is configured to produce at least about 1 mg, at least about 5 mg, at least about 10 mg, at least about 20 mg, at least about 50 mg, at least about 100 mg, at least about 200 mg, at least about 500 mg, at least about 1 g, at least about 2 g, at least about 5 g, at least about 10 g, at least about 15 g, or at least about 18 g of a single-domain antibody per day. In some embodiments, the system is configured to produce an amount of a single-domain antibody in the range of about 1 mg to about 5 mg, about 1 mg to about 10 mg, about 1 mg to about 20 mg, about 1 mg to about 50 mg, about 1 mg to about 100 mg, about 1 mg to about 500 mg, about 1 mg to about 1 g, about 1 mg to about 2 g, about 1 mg to about 5 g, about 1 mg to about 10 g, about 1 mg to about 15 g, about 1 mg to about 20 g, about 10 mg to about 20 mg, about 10 mg to about 50 mg, about 10 mg to about 100 mg, about 10 mg to about 500 mg, about 10 mg to about 1 g, about 10 mg to about 2 g, about 10 mg to about 5 g, about 10 mg to about 10 g, about 10 mg to about 15 g, about 10 mg to about 20 g, about 50 mg to about 100 mg, about 50 mg to about 500 mg, about 50 mg to about 1 g, about 50 mg to about 2 g, about 50 mg to about 5 g, about 50 mg to about 10 g, about 50 mg to about 15 g, about 50 mg to about 20 g, about 100 mg to about 500 mg, about 100 mg to about 1 g, about 100 mg to about 2 g, about 100 mg to about 5 g, about 500 mg to about 1 g, about 500 mg to about 2 g, about 500 mg to about 5 g, about 1 g to about 5 g, about 1 g to about 10 g, about 1 g to about 15 g, or about 1 g to about 20 g per day.
In some embodiments, the reactor chamber of the bioreactor has a volume of about 1 L to about 10 L. In certain of these embodiments, an input stream to the system (e.g., the at least one feed stream) and an output stream of the system (e.g., the purified filtrate stream, the formulated product stream) each have a flow rate of at least about 0.5 mL/min, at least about 1 mL/min, at least about 5 mL/min, at least about 10 mL/min, or at least about 20 mL/min over a period of at least about 1 day. In certain embodiments, the input stream and the output stream each have a flow rate in the range of about 0.5 mL/min to about 1 mL/min, about 0.5 mL/min to about 5 mL/min, about 0.5 mL/min to about 10 mL/min, about 0.5 mL/min to about 20 mL/min, about 1 mL/min to about 5 mL/min, about 1 mL/min to about 10 mL/min, about 1 mL/min to about 20 mL/min, about 5 mL/min to 10 mL/min, about 5 mL/min to about 20 mL/min, or about 10 mL/min to about 20 mL/min over a period of at least about 1 day. In some of these embodiments, the system is configured to produce at least about 50 mg, at least about 100 mg, at least about 200 mg, at least about 500 mg, at least about 1 g, at least about 5 g, at least about 10 g, at least about 50 g, at least about 100 g, at least about 150 g, or at least about 175 g of a single-domain antibody per day. In some embodiments, the system is configured to produce an amount of a single-domain antibody in the range of about 50 mg to about 100 mg, about 50 mg to about 200 mg, about 50 mg to about 500 mg, about 50 mg to about 1 g, about 50 mg to about 5 g, about 50 mg to about 10 g, about 50 mg to about 50 g, about 50 mg to about 100 g, about 50 mg to about 150 g, about 50 mg to about 200 g, about 100 mg to about 500 mg, about 100 mg to about 1 g, about 100 mg to about 5 g, about 100 mg to about 10 g, about 100 mg to about 50 g, about 100 mg to about 100 g, about 100 mg to about 150 g, about 100 mg to about 200 g, about 500 mg to about 1 g, about 500 mg to about 5 g, about 500 mg to about 10 g, about 500 mg to about 50 g, about 1 g to about 10 g, about 1 g to about 50 g, about 10 g to about 50 g per day, about 1 g to about 100 g, about 1 g to about 150 g, or about 1 g to about 200 g.
In some embodiments, the reactor chamber of the bioreactor has a volume of about 10 L to about 50 L. In certain of these embodiments, an input stream to the system (e.g., the at least one feed stream) and an output stream of the system (e.g., the purified filtrate stream, the formulated product stream) each have a flow rate of at least about 5 mL/min, at least about 10 mL/min, at least about 20 mL/min, at least about 50 mL/min, at least about 100 mL/min, at least about 150 mL/min, or at least about 200 mL/min over a period of at least about 1 day. In some embodiments, the input stream and the output stream each have a flow rate in the range of about 5 mL/min to about 10 mL/min, about 5 mL/min to about 20 mL/min, about 5 mL/min to about 50 mL/min, about 5 mL/min to about 100 mL/min, about 5 mL/min to about 150 mL/min, about 5 mL/min to about 200 mL/min, about 10 mL/min to about 20 mL/min, about 10 mL/min to about 50 mL/min, about 10 mL/min to about 100 mL/min, about 10 mL/min to about 150 mL/min, about 10 mL/min to about 200 mL/min, about 50 mL/min to about 100 mL/min, about 50 mL/min to about 150 mL/min, about 50 mL/min to about 200 mL/min, or about 100 mL/min to about 200 mL/min over a period of at least about 1 day. In some of these embodiments, the system is configured to produce at least about 500 mg, at least about 1 g, at least about 2 g, at least about 5 g, at least about 10 g, at least about 50 g, at least about 100 g, at least about 200 g, at least about 500 g, at least about 750 g, or at least about 900 g of a single-domain antibody per day. In some embodiments, the system is configured to produce an amount of a single-domain antibody in the range of about 500 mg to about 1 g, about 500 mg to about 2 g, about 500 mg to about 5 g, about 500 mg to about 10 g, about 500 mg to about 50 g, about 500 mg to about 100 g, about 500 mg to about 200 g, about 500 mg to about 500 g, about 500 mg to about 750 g, about 500 mg to about 1,000 g, about 1 g to about 5 g, about 1 g to about 10 g, about 1 g to about 50 g, about 1 g to about 100 g, about 1 g to about 500 g, about 1 g to about 750 g, about 1 g to about 1,000 g, about 10 g to about 50 g, about 10 g to about 100 g, about 10 g to about 500 g, about 10 g to about 750 g, about 10 g to about 1,000 g, about 100 g to about 500 g, about 100 g to about 750 g, or about 100 g to about 1,000 g per day.
Some embodiments described herein relate to methods for producing a single-domain antibody. In certain embodiments, the method comprises supplying a growth cell medium to a bioreactor (e.g., a perfusion bioreactor). In some embodiments, the method further comprises incubating a first type of biological cells in the growth cell culture medium for a period of at least one day. In some embodiments, the method further comprises at least partially removing the growth cell culture medium from the bioreactor. In some embodiments, the method further comprises supplying at least one cell culture medium (e.g., a production cell culture medium) to the bioreactor. In some embodiments, the method comprises producing, within the bioreactor, a suspension comprising the at least one cell culture medium and at least a first type of biological cells expressing a single-domain antibody.
In some embodiments, the method further comprises causing at least a portion of the suspension to flow through at least one filter to produce at least one filtrate lean in the first type of biological cells. In some embodiments, the at least one filtrate comprises a single-domain antibody.
In some embodiments, the method comprises flowing the at least one filtrate to an adjustment module (e.g., from the at least one filter to the adjustment module). In some embodiments, the method further comprises adjusting, within the adjustment module, one or more properties (e.g., pH, conductivity, product stability) of the at least one filtrate. In certain embodiments, the pH of the at least one filtrate is increased or decreased (e.g., to be compatible with the first column of the purification module). In some embodiments, increasing the pH of the at least one filtrate comprises adding a base to the at least one filtrate. In some embodiments, decreasing the pH of the at least one filtrate comprises adding an acid to the at least one filtrate. In some embodiments, the method further comprises flowing the at least one filtrate and/or the adjusted filtrate through a first column comprising a multimodal cation exchange resin. In certain embodiments, the multimodal cation exchange resin comprises Capto MMC ImpRes, Capto MMC, Nuvia cPrime, Toyopearl MX-Trp-650M, Eshmuno HCX, and/or CMM HyperCel. In certain non-limiting embodiments, the multimodal cation exchange resin comprises a CMM HyperCel resin.
In certain embodiments, the first column is operated in bind-elute mode. In some embodiments, the method further comprises flowing a first mobile phase material through the first column. In some embodiments, the first mobile phase material is configured to promote binding of the single-domain antibody to the multimodal cation exchange resin. In some embodiments, the first mobile phase material comprises sodium citrate, sodium phosphate, sodium chloride, sodium acetate, Tris-HCl, glycine, and/or histidine. According to certain embodiments, the first mobile phase material comprises 20 mM sodium citrate. In some embodiments, the first mobile phase material has a pH in the range of about 4.0 to about 6.0 (e.g., about 4.5 to about 5.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0). In some embodiments, the method further comprises flowing the at least one filtrate and/or the adjusted filtrate through the first column and, subsequently, flowing a second mobile phase material through the first column. In some embodiments, the second mobile phase material is configured to wash one or more impurities from the multimodal cation exchange resin. In some embodiments, the second mobile phase material comprises sodium citrate, sodium phosphate, sodium chloride, sodium acetate, Tris-HCl, glycine, and/or histidine. According to certain embodiments, the second mobile phase material comprises 20 mM sodium phosphate. In some embodiments, the second mobile phase material has a pH in the range of about 5.0 to about 7.0 (e.g., about 5.5 to about 6.5, about 5.0, about 5.5, about 5.8, about 6.0, about 6.2, about 6.5, about 7.0). In some embodiments, the method further comprises subsequently flowing a third mobile phase material through the first column. In some embodiments, the third mobile phase material is configured to elute single-domain antibody from the multimodal cation exchange resin. In some embodiments, the third mobile phase material comprises sodium citrate, sodium phosphate, sodium chloride, sodium acetate, Tris-HCl, glycine, and/or histidine. According to certain embodiments, the third mobile phase material comprises 20 mM sodium phosphate. In some embodiments, the third mobile phase material has a pH in the range of about 6.0 to about 8.0 (e.g., about 6.5 to about 7.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0). In some embodiments, the third mobile phase material has a salt (e.g., sodium chloride) concentration in the range of about 0 mM to about 150 mM (e.g., 50 mM to about 150 mM, about 75 mM to about 125 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM). In some embodiments, the method further comprises collecting one or more first fractions comprising the single-domain antibody from an outflow of the first column. In some embodiments, the one or more first fractions are lean in the first type of impurity relative to the at least one filtrate or the adjusted filtrate. In some embodiments, the one or more first fractions have a concentration of the first type of impurity that is at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 99% less than the concentration of the first type of impurity in the at least one filtrate or the adjusted filtrate.
In some embodiments, the method further comprises flowing the one or more first fractions through a second column comprising an anion exchange resin. In some embodiments, the anion exchange resin comprises a HyperCel STAR AX resin. In certain embodiments, the second column is operated in flow-through mode. In some embodiments, the method comprises flowing through a first mobile phase material through the second column. In some embodiments, the first mobile phase material comprises sodium citrate, sodium phosphate, sodium chloride, sodium acetate, Tris-HCl, glycine, and/or histidine. According to certain embodiments, the first mobile phase material comprises 20 mM sodium phosphate. In some embodiments, the first mobile phase material has a pH in a range of about 6.0 to about 8.0 (e.g., about 6.5 to about 7.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0). In some embodiments, the first mobile phase material has a salt (e.g., sodium chloride) concentration in a range of about 0 mM to about 150 mM (e.g., about 50 mM to about 150 mM, about 75 mM to about 125 mM, about 100 mM, about 125 mM, about 150 mM). In some embodiments, the method further comprises collecting one or more second fractions comprising the single-domain antibody from an outflow of the second column. In some embodiments, the one or more second fractions are lean in the second type of impurity relative to the first fractions. In some embodiments, the one or more second fractions have a concentration of the second type of impurity that is at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 99% less than the concentration of the second type of impurity in the first fractions.
In some embodiments, the method further comprises flowing the second fractions to a formulation module to produce a formulated product stream. In some embodiments, flowing the purified filtrate through the formulation module comprises flowing the purified filtrate through a tangential flow filtration device. In some embodiments, flowing the purified filtrate through the formulation module comprises flowing the purified filtrate through a viral filtration unit. In some embodiments, the formulated product stream is lean in one or more viruses relative to the purified filtrate stream. In some embodiments, flowing the purified filtrate through the formulation module comprises flowing the purified filtrate through a dilution adjustment unit. In some embodiments, flowing the purified filtrate through the dilution adjustment unit comprises adding a diluent to the purified filtrate. In some embodiments, flowing the purified filtrate through the formulation module comprises depositing one or more portions of the purified filtrate stream into one or more containers (e.g., bags, vials, syringes, bottles). In some embodiments, the one or more containers are aseptic and/or sterile containers.
The term “single-domain antibody” has its ordinary meaning in the art and may refer to a single domain polypeptide comprising (e.g., consisting of) a variable domain (e.g., variable heavy chain domain) that is capable of binding an antigen. In certain embodiments, a single-domain antibody may be a single domain polypeptide comprising (e.g., consisting of) a single variable domain (e.g., variable heavy chain single domain). In other embodiments, a single-domain antibody may be a single domain polypeptide comprising (e.g., consisting of) two or more (e.g., two, three or more, four or more) variable domains (e.g., variable heavy chain single domain) covalently attached. In some embodiments, a single-domain antibody may be derived from an antibody, a heavy chain antibody, an antibody naturally devoid of light chains, an engineered antibody, and/or a single domain scaffold other than those derived from antibodies. In some embodiments, a single-domain antibody may be a nanobody. The term “nanobody” has its ordinary meaning in the art and may include a single-domain antibody derived from a heavy chain antibody of a species such as Camelidae (e.g., camel, llama, dromedary, alpaca, guanaco) and a nurse shark, and humanized variants thereof. For instance, a nanobody may be derived from a heavy chain camelid antibody (e.g., camel antibody, llama antibody, dromedary antibody, alpaca antibody, guanaco antibody).
In general, the systems and methods described herein may be used to manufacture any suitable single-domain antibody. Non-limiting examples of single-domain antibodies that may be manufactured using the systems and methods described herein include camelid antibodies (e.g., 3B2, 2KD1), shark antibodies, and those described in Steeland, S.; Vandenbroucke, R. E.; Libert, C. Nanobodies as Therapeutics: Big Opportunities for Small Antibodies, Drug Discov. Today 2016, 21 (7), 1076-1113, which is incorporated by reference in its entirety. The single-domain antibodies described herein may have a range of indications including, but not limited to, anti-toxin, anti-venom, anti-viral, anti-bacterial, anti-fungal, anti-parasitic, anti-cytokine, anti-hormone, anti-blood factor, anti-tumor, and anti-cancer.
In some embodiments, a single-domain antibody may have an isoelectric point in the range of about 4.0 to about 12.0 (e.g., about 4.0 to about 11.0, about 5.0 to about 10.0, about 5.0 to about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, or about 11.0). For instance, a single-domain antibody may have an isoelectric point in the range of about 5.5 to about 8.0, of about 6.0 to about 8.0, or of about 6.5 to about 8.0. In some embodiments, a single-domain antibody may have a relatively low molecular weight. For instance, in some embodiments, a single-domain antibody may have a molecular weight in the range of about 12 kDa to about 30 kDa (e.g., of about 12 kDa to about 25 kDa, of about 12 kDa to about 20 kDa, of about 12 kDa to about 18 kDa, of about 12 kDa to about 15 kDa).
As used herein, the term “antibody” refers to an immunoglobulin molecule or an immunologically active portion thereof (e.g., antigen-binding portion). The antibody may be naturally produced or wholly or partially synthetically produced. Examples of immunologically active portions of immunoglobulin molecules include F(ab), Fv, and F(ab′) fragments which can be generated by cleaving the antibody with an enzyme such as pepsin. All derivatives thereof which maintain specific binding ability are also included in the term. The term also covers any protein having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. An immunoglobulin molecule may be a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three subdomains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one subdomain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
Exemplary embodiments of the present disclosure are provided below. One exemplary embodiment is generally directed to the following:
1. A biomanufacturing system, comprising a perfusion bioreactor, wherein the perfusion bioreactor comprises:
a reaction chamber configured to receive at least one feed stream comprising at least one cell culture medium;
a suspension comprising the at least one cell culture medium and at least a first type of biological cells configured to express at least one biologically-produced product; at least one filter probe at least partially submerged in the suspension, wherein the at least one filter probe is configured to produce at least one filtrate stream lean in the first type of biological cells relative to the suspension, wherein the at least one filtrate stream comprises the at least one biologically-produced product;
an adjustment module fluidically connected to the perfusion bioreactor, wherein the adjustment module is configured to adjust one or more properties of the at least one filtrate stream to produce an adjusted filtrate stream; and
a purification module fluidically connected to the adjustment module, wherein the purification module is configured to remove at least a first type of impurity and a second type of impurity from the adjusted filtrate stream to produce a purified filtrate stream, wherein the purification module comprises:
a first partitioning unit configured to remove at least the first type of impurity from the adjusted filtrate stream to produce a first partitioned filtrate stream lean in the first type of impurity relative to the adjusted filtrate stream, wherein the first partitioned filtrate stream comprises the at least one biologically-produced product; and
a second partitioning unit configured to remove at least a second type of impurity from the first partitioned filtrate stream to produce a second partitioned filtrate stream lean in the second type of impurity relative to the first partitioned filtrate stream, wherein the second partitioned filtrate stream comprises the at least one biologically-produced product, wherein the system is configured to be continuously operated.
2. The biomanufacturing system of sentence 1, wherein the reactor chamber has a volume of about 1 L or less.
3. The biomanufacturing system of sentence 2, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 0.1 mL/min over a period of at least about 1 day.
4. The biomanufacturing system of any one of sentences 2-3, wherein the system is configured to produce at least about 10 μg of the at least one biologically-produced product per day.
5. The biomanufacturing system of any one of sentences 2-4, wherein the system is configured to produce at least about 50 mg of the at least one biologically-produced product per day.
6. The biomanufacturing system of any one of sentences 2-5, wherein the system is configured to produce at least about 1 g of the at least one biologically-produced product per day.
7. The biomanufacturing system of sentence 1, wherein the reactor chamber has a volume of about 1 L to about 10 L.
8. The biomanufacturing system of sentence 7, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 0.5 mL/min over a period of at least about 1 day.
9. The biomanufacturing system of any one of sentences 7-8, wherein the system is configured to produce at least about 100 μg of the at least one biologically-produced product per day.
10. The biomanufacturing system of any one of sentences 7-9, wherein the system is configured to produce at least about 500 mg of the at least one biologically-produced product per day.
11. The biomanufacturing system of any one of sentences 7-10, wherein the system is configured to produce at least about 10 g of the at least one biologically-produced product per day.
12. The biomanufacturing system of sentence 1, wherein the reactor chamber has a volume of about 10 L to about 50 L.
13. The biomanufacturing system of sentence 12, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 5 mL/min over a period of at least about 1 day.
14. The biomanufacturing system of any one of sentences 12-13, wherein the system is configured to produce at least about 500 μg of the at least one biologically-produced product per day.
15. The biomanufacturing system of any one of sentences 12-14, wherein the system is configured to produce at least about 2.5 g of the at least one biologically-produced product per day.
16. The biomanufacturing system of any one of sentences 12-15, wherein the system is configured to produce at least about 50 g of the at least one biologically-produced product per day.
17. The biomanufacturing system of any one of sentences 1-16, wherein the first type of biological cells are microbial cells, yeast cells, filamentous fungal cells, microalgal cells, or diatom cells.
18. The biomanufacturing system of sentence 17, wherein the yeast cells are Pichia pastoris cells.
19. The biomanufacturing system of any one of sentences 1-18, wherein the at least one biologically-produced product comprises a cytokine, an antibody, an antibody fragment, a nanobody, a hormone, an enzyme, a growth factor, a blood factor, a recombinant immunogen, and/or a fusion protein.
20. The biomanufacturing of sentence 19, wherein the antibody is a single-chain antibody, a bispecific antibody, and/or a monoclonal antibody.
21. The biomanufacturing system of any one of sentences 1-19, wherein the at least one biologically-produced product comprises human growth hormone (hGH).
22. The biomanufacturing system of any one of sentences 1-19, wherein the at least one biologically-produced product comprises granulocyte colony-stimulating factor (G-CSF).
23. The biomanufacturing system of any one of sentences 1-19, wherein the at least one biologically-produced product comprises an interferon.
24. The biomanufacturing system of sentence 23, wherein the interferon is interferon α-2b.
25. The biomanufacturing system of any one of sentences 1-24, wherein the one or more properties comprise pH.
26. The biomanufacturing system of sentence 25, wherein the adjustment module is configured to reduce the pH of the at least one filtrate stream.
27. The biomanufacturing system of sentence 25, wherein the adjustment module is configured to increase the pH of the at least one filtrate stream.
28. The biomanufacturing system of any one of sentences 1-27, wherein the one or more properties comprise conductivity.
29. The biomanufacturing system of sentence 28, wherein the adjustment module is configured to increase or decrease the conductivity of the at least one filtrate stream.
30. The biomanufacturing system of any one of sentences 1-29, wherein the one or more properties comprise biologically-produced product stability.
31. The biomanufacturing system of sentence 30, wherein the adjustment module is configured to increase the stability of the at least one biologically-produced product in the at least one filtrate stream.
32. The biomanufacturing system of any one of sentences 1-31, wherein the first partitioning unit is directly fluidically connected to the second partitioning unit.
33. The biomanufacturing system of any one of sentences 1-32, wherein the first partitioning unit applies a partitioning technique and the second partitioning unit applies the same partitioning technique.
34. The biomanufacturing system of any one of sentences 1-32, wherein the first partitioning unit applies a partitioning technique and the second partitioning unit applies a different partitioning technique.
35. The biomanufacturing system of any one of sentences 1-34, wherein the first partitioning unit comprises a first column comprising at least a first stationary phase material.
36. The biomanufacturing system of sentence 35, wherein the first stationary phase material comprises a cation exchange resin, a multimodal cation exchange resin, an anion exchange resin, a multimodal anion exchange resin, a hydrophobic charge induction chromatography (HCIC) resin, or an affinity chromatography resin.
37. The biomanufacturing system of any one of sentences 1-36, wherein the first partitioning unit comprises a filter.
38. The biomanufacturing system of sentence 37, wherein the filter comprises a filtration membrane and/or a monolith.
39. The biomanufacturing system of any one of sentences 1-38, wherein the first partitioning unit comprises a precipitation apparatus.
40. The biomanufacturing system of any one of sentences 1-39, wherein the first partitioning unit comprises a crystallization apparatus.
41. The biomanufacturing system of any one of sentences 1-40, wherein the second partitioning unit comprises a second column comprising at least a first stationary phase material.
42. The biomanufacturing system of sentence 41, wherein the first stationary phase material comprises a cation exchange resin, a multimodal cation exchange resin, an anion exchange resin, a multimodal anion exchange resin, a hydrophobic charge induction chromatography (HCIC) resin, or an affinity chromatography resin.
43. The biomanufacturing system of any one of sentences 1-42, wherein the second partitioning unit comprises a filter.
44. The biomanufacturing system of sentence 43, wherein the filter comprises a filtration membrane and/or a monolith.
45. The biomanufacturing system of any one of sentences 1-44, wherein the second partitioning unit comprises a precipitation apparatus.
46. The biomanufacturing system of any one of sentences 1-45, wherein the second partitioning unit comprises a crystallization apparatus.
47. The biomanufacturing system of any one of sentences 1-46, wherein the first type of impurity is different from the second type of impurity.
48. The biomanufacturing system of any one of sentences 1-47, wherein the purification module further comprises a third partitioning unit configured to remove at least a third type of impurity from the second partitioned filtrate stream to produce a third partitioned filtrate stream lean in the third type of impurity relative to the second partitioned filtrate stream, wherein the third partitioned filtrate stream comprises the at least one biologically-produced product.
49. The biomanufacturing system of sentence 48, wherein the third partitioning unit comprises a third column comprising at least a first stationary phase material.
50. The biomanufacturing system of sentence 49, wherein the first stationary phase comprises a cation exchange resin, a multimodal cation exchange resin, an anion exchange resin, a multimodal anion exchange resin, a hydrophobic charge induction chromatography (HCIC) resin, or an affinity chromatography resin.
51. The biomanufacturing system of any one of sentences 48-50, wherein the third partitioning unit comprises a filter.
52. The biomanufacturing system of sentence 51, wherein the filter comprises a filtration membrane and/or a monolith.
53. The biomanufacturing system of any one of sentences 48-52, wherein the third partitioning unit comprises a precipitation apparatus.
54. The biomanufacturing system of any one of sentences 48-53, wherein the third partitioning unit comprises a crystallization apparatus.
55. The biomanufacturing system of any one of sentences 48-54, wherein the third type of impurity is different from the first type of impurity and/or the second type of impurity.
56. The biomanufacturing system of any one of sentences 1-55, further comprising a formulation module fluidically connected to the purification module, wherein the formulation module is configured to produce a formulated product stream.
57. The biomanufacturing system of sentence 56, wherein the formulation module comprises a filtration unit.
58. The biomanufacturing system of sentence 57, wherein the filtration unit comprises a tangential flow filtration device.
59. The biomanufacturing system of any one of sentences 56-58, wherein the formulation module comprises a viral filtration unit.
60. The biomanufacturing system of any one of sentences 56-59, wherein the formulation module comprises a packaging unit.
61. The biomanufacturing system of sentence 60, wherein the packaging unit is configured to package one or more doses of the at least one biologically-produced product into one or more bags, one or more vials, one or more syringes, and/or one or more bottles.
62. The biomanufacturing system of any one of sentences 56-61, wherein the formulation module comprises a dilution adjustment unit.
63. The biomanufacturing system of any one of sentences 1-62, further comprising a buffer delivery module fluidically connected to the purification module, wherein the buffer delivery module is configured to deliver at least one buffer to at least one partitioning unit.
64. The biomanufacturing system of any one of sentences 1-63, further comprising a gas concentration device fluidically connected to the bioreactor.
65. The biomanufacturing system of sentence 64, wherein the gas concentration device is an oxygen concentrator.
66. The biomanufacturing system of any one of sentences 1-65, wherein at least one module of the system is disposable.
67. The biomanufacturing system of sentence 66, wherein each module of the system is disposable.
68. The biomanufacturing system of any one of sentences 1-67, wherein at least one module of the system is configured to be cleaned in place.
69. The biomanufacturing system of sentence 68, wherein each module of the system is configured to be cleaned in place.
70. The biomanufacturing system of any one of sentences 1-69, wherein the purified filtrate stream and/or the formulated product stream has a concentration of the pharmaceutical product of at least about 10 μg/mL.
71. The biomanufacturing system of any one of sentences 1-70, wherein the purified filtrate stream and/or the formulated product stream has a concentration of host cell proteins of about 20 ng/(mg product) or less.
72. The biomanufacturing system of any one of sentences 1-71, wherein the purified filtrate stream and/or the formulated product stream has a concentration of DNA of about 100 ng/(mg product) or less.
73. A method of producing at least one biologically-produced product, comprising:
supplying at least one feed stream comprising at least one cell culture medium to a perfusion bioreactor at a first flow rate;
producing, within the perfusion bioreactor, a suspension comprising the at least one cell culture medium and at least a first type of biological cells expressing the at least one biologically-produced product;
causing at least a portion of the suspension to flow through at least one filter probe to produce at least one filtrate stream lean in the first type of biological cells, wherein the at least one filtrate stream comprises the at least one biologically-produced product, wherein the at least one filter probe is at least partially submerged in the suspension;
adjusting one or more properties of the at least one filtrate stream to produce an adjusted filtrate stream; removing, within a purification module, at least a first type of impurity and a second type of impurity from the adjusted filtrate stream to produce a purified filtrate stream flowing at a second flow rate, wherein the purified filtrate stream comprises the at least one biologically-produced product and is lean in the first type of impurity and the second type of impurity relative to the adjusted filtrate stream, wherein producing the purified filtrate stream comprises:
removing, within a first partitioning unit, at least the first type of impurity from the adjusted filtrate stream to produce a first partitioned filtrate stream lean in the first type of impurity relative to the adjusted filtrate stream, wherein the first partitioned filtrate stream comprises the at least one biologically-produced product; and
removing, within a second partitioning unit, at least the second type of impurity from the first partitioned filtrate stream to produce a second partitioned filtrate stream lean in the second type of impurity relative to the first partitioned filtrate stream, wherein the second partitioned filtrate stream comprises the at least one biologically-produced product.
74. The method of sentence 73, further comprising, prior to supplying the at least one feed stream comprising the at least one cell culture medium to the perfusion bioreactor:
supplying a growth cell culture medium to the perfusion bioreactor;
incubating the first type of biological cells in the growth cell culture medium for a period of at least about 1 day; and
at least partially removing the growth cell culture medium from the perfusion bioreactor.
75. The method of any one of sentences 73-74, wherein the at least one feed stream is continuously supplied to the perfusion bioreactor at the first flow rate over a period of at least about 1 day.
76. The method of any one of sentences 73-75, wherein the first flow rate and the second flow rate are substantially the same.
77. The method of any one of sentences 73-76, wherein the perfusion bioreactor comprises a reactor chamber having a volume of about 1 L or less.
78. The method of sentence 77, wherein the first flow rate and/or second flow rate are maintained at about 0.1 mL/min or more over a period of about 1 day or more.
79. The method of any one of sentences 77-78, wherein at least about 10 μg of the at least one biologically-produced product is produced in about 1 day or less.
80. The method of any one of sentences 77-79, wherein at least about 50 mg of the at least one biologically-produced product is produced in about 1 day or less.
81. The method of any one of sentences 77-80, wherein at least about 1 g of the at least one biologically-produced product is produced in about 1 day or less.
82. The method of any one of sentences 73-76, wherein the perfusion bioreactor comprises a reactor chamber having a volume of about 1 L to about 10 L.
83. The method of sentence 82, wherein the first flow rate and/or second flow rate are maintained at about 0.5 mL/min or more over a period of about 1 day or more.
84. The method of any one of sentences 82-83, wherein at least about 100 μg of the at least one biologically-produced product is produced in about 1 day or less.
85. The method of any one of sentences 82-84, wherein at least about 500 mg of the at least one biologically-produced product is produced in about 1 day or less.
86. The method of any one of sentences 82-85, wherein at least about 10 g of the at least one biologically-produced product is produced in about 1 day or less.
87. The method of any one of sentences 73-76, wherein the perfusion bioreactor comprises a reactor chamber having a volume of about 10 L to about 50 L.
88. The method of sentence 87, wherein the first flow rate and/or second flow rate are maintained at about 5 mL/min or more over a period of about 1 day or more.
89. The method of any one of sentences 87-88, wherein at least about 500 μg of the at least one biologically-produced product is produced in about 1 day or less.
90. The method of any one of sentences 87-89, wherein at least about 2.5 g of the at least one biologically-produced product is produced in about 1 day or less.
91. The method of any one of sentences 87-90, wherein at least about 50 g of the at least one biologically-produced product is produced in about 1 day or less.
92. The method of any one of sentences 73-91, wherein the first type of biological cells are yeast cells, filamentous fungal cells, microalgal cells, or diatom cells.
93. The method of sentence 92, wherein the yeast cells are Pichia pastoris cells.
94. The method of any one of sentences 73-93, wherein the at least one biologically-produced product comprises a cytokine, an antibody, an antibody fragment, a nanobody, a hormone, an enzyme, a growth factor, a blood factor, a recombinant immunogen, and/or a fusion protein.
95. The method of sentence 94, wherein the antibody is a single-chain antibody, a bispecific antibody, and/or a monoclonal antibody.
96. The method of any one of sentences 73-95, wherein the at least one biologically-produced product comprises human growth hormone (hGH).
97. The method of any one of sentences 73-96, wherein the at least one biologically-produced product comprises granulocyte colony-stimulating factor (G-CSF).
98. The method of any one of sentences 73-97, wherein the at least one biologically-produced product comprises an interferon.
99. The method of sentence 98, wherein the interferon is interferon α-2b.
100. The method of any one of sentences 73-99, wherein adjusting one or more properties of the at least one filtrate stream comprises decreasing the pH of the at least one filtrate stream.
101. The method of any one of sentences 73-99, wherein adjusting one or more properties of the at least one filtrate stream comprises increasing the pH of the at least one filtrate stream.
102. The method of any one of sentences 73-101, wherein adjusting one or more properties of the at least one filtrate stream comprises increasing the conductivity of the at least one filtrate stream.
103. The method of any one of sentences 73-101, wherein adjusting one or more properties of the at least one filtrate stream comprises decreasing the conductivity of the at least one filtrate stream.
104. The method of any one of sentences 73-103, wherein adjusting one or more properties of the at least one filtrate stream comprises reducing the pH of the at least one filtrate stream.
105. The method of any one of sentences 73-104, wherein adjusting one or more properties of the at least one filtrate stream comprises increasing the stability of the at least one biologically-produced product in the at least one filtrate stream.
106. The method of any one of sentences 73-105, wherein the first partitioning unit and the second partitioning unit are directly fluidically connected.
107. The method of any one of sentences 73-106, wherein the first partitioning unit and the second partitioning unit apply the same partitioning technique.
108. The method of any one of sentences 73-107, wherein the first partitioning unit and the second partitioning unit apply different partitioning techniques.
109. The method of any one of sentences 73-108, wherein removing at least the first type of impurity comprises causing the adjusted filtrate stream to flow through a first column comprising at least a first stationary phase material.
110. The method of any one of sentences 73-109, wherein removing at least the first type of impurity comprises causing the adjusted filtrate stream to flow through a filter.
111. The method of sentence 110, wherein the filter comprises a filtration membrane and/or a monolith.
112. The method of any one of sentences 73-111, wherein removing at least the first type of impurity comprises causing the first type of impurity to precipitate and/or crystallize.
113. The method of any one of sentences 73-112, wherein removing at least the second type of impurity comprises causing the first partitioned filtrate stream to flow through a second column comprising at least a first stationary phase material.
114. The method of any one of sentences 73-113, wherein removing at least the second type of impurity comprises causing the first partitioned filtrate stream to flow through a filter.
115. The method of sentence 114, wherein the filter comprises a filtration membrane and/or a monolith.
116. The method of any one of sentences 73-115, wherein removing at least the second type of impurity comprises causing the second type of impurity to precipitate and/or crystallize.
117. The method of any one of sentences 73-116, wherein the first type of impurity is different from the second type of impurity.
118. The method of any one of sentences 73-117, further comprising removing, within a third partitioning unit, at least a third type of impurity from the second partitioned filtrate stream to produce a third partitioned filtrate stream lean in the third type of impurity relative to the second partitioned filtrate stream, wherein the third partitioned filtrate stream comprises the at least one biologically-produced product.
119. The method of sentence 118, wherein removing at least the third type of impurity comprises causing the second partitioned filtrate stream to flow through a third column comprising at least a first stationary phase material.
120. The method of any one of sentences 118-119, wherein removing at least the third type of impurity comprises causing the second partitioned filtrate stream to flow through a filter.
121. The method of sentence 120, wherein the filter comprises a filtration membrane and/or a monolith.
122. The method of any one of sentences 118-121, wherein removing at least the third type of impurity comprises causing the third type of impurity to precipitate and/or crystallize.
123. The method of any one of sentences 118-122, wherein the third type of impurity is different from the first type of impurity and/or the second type of impurity.
124. The method of any one of sentences 73-123, further comprising flowing the purified filtrate stream through a formulation module configured to produce a formulated product stream.
125. The method of sentence 124, wherein flowing the purified filtrate stream through the formulation module comprises flowing the purified filtrate stream through a tangential flow filtration device.
126. The method of any one of sentences 124-125, wherein flowing the purified filtrate stream through the formulation module comprises flowing the purified filtrate stream through a dilution adjustment unit.
127. The method of sentence 126, further comprising adding a diluent to the purified filtrate stream.
128. The method of any one of sentences 124-127, wherein flowing the purified filtrate stream through the formulation module comprises flowing the purified filtrate stream through a viral filtration unit, wherein the formulated product stream is lean in one or more viruses relative to the purified filtrate stream.
129. The method of any one of sentences 124-128, wherein flowing the purified filtrate stream through the formulation module comprises depositing one or more portions of the purified filtrate stream into one or more containers.
130. The method of sentence 129, wherein the one or more containers are aseptic and/or sterile containers.
131. The method of any one of sentences 129-130, wherein the one or more containers comprise one or more bags, vials, syringes, and/or bottles.
132. The biomanufacturing system of any preceding sentence, further comprising a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
133. The method of any preceding sentence, further comprising monitoring one or more steps of the method using a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
134. The method of any preceding sentence, further comprising implementing one or more corrective action based on information derived from a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
A second exemplary embodiment is generally directed to the following:
1. A biomanufacturing system, comprising a perfusion bioreactor, wherein the perfusion bioreactor comprises:
a reaction chamber configured to receive at least one feed stream comprising at least one cell culture medium;
a suspension comprising the at least one cell culture medium and at least a first type of biological cells configured to express at least one biologically-produced product;
a level sensing system configured to measure a level of the suspension in the reactor chamber of the perfusion bioreactor;
at least one filter probe at least partially submerged in the suspension, wherein the at least one filter probe is configured to produce at least one filtrate stream lean in the first type of biological cells relative to the suspension, wherein the at least one filtrate stream comprises the at least one biologically-produced product;
an adjustment module fluidically connected to the perfusion bioreactor, wherein the adjustment module is configured to adjust one or more properties of the at least one filtrate stream to produce an adjusted filtrate stream; and
a purification module fluidically connected to the adjustment module, wherein the purification module is configured to remove at least a first type of impurity and a second type of impurity from the adjusted filtrate stream to produce a purified filtrate stream, wherein the purification module comprises:
a first partitioning unit configured to remove at least the first type of impurity from the adjusted filtrate stream to produce a first partitioned filtrate stream lean in the first type of impurity relative to the adjusted filtrate stream, wherein the first partitioned filtrate stream comprises the at least one biologically-produced product; and
a second partitioning unit configured to remove at least a second type of impurity from the first partitioned filtrate stream to produce a second partitioned filtrate stream lean in the second type of impurity relative to the first partitioned filtrate stream, wherein the second partitioned filtrate stream comprises the at least one biologically-produced product, wherein the system is configured to be continuously operated.
2. The biomanufacturing system of sentence 1, wherein the level sensing system is a magnetic level sensing system.
3. The biomanufacturing system of sentence 2, wherein the magnetic level sensing system comprises a magnetic float, a non-magnetic shaft, and one or more magnetically-activated switches.
4. The biomanufacturing system of sentence 1, wherein the level sensing system is an optical level sensing system.
5. The biomanufacturing system of sentence 4, wherein the optical level sensing system comprises a colored float and/or a colored probe.
6. The biomanufacturing system of sentence 4, wherein the optical level sensing system comprises a probe comprising a plurality of colored bands.
7. The biomanufacturing system of any one of sentences 1-6, wherein the level sensing system comprises a capacitance-based probe.
8. The biomanufacturing system of any one of sentences 1-7, wherein the reactor chamber has a volume of about 1 L or less.
9. The biomanufacturing system of sentence 8, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 0.1 mL/min over a period of at least about 1 day.
10. The biomanufacturing system of any one of sentences 8-9, wherein the system is configured to produce at least about 10 μg of the at least one biologically-produced product per day.
11. The biomanufacturing system of any one of sentences 8-10, wherein the system is configured to produce at least about 50 mg of the at least one biologically-produced product per day.
12. The biomanufacturing system of any one of sentences 8-11, wherein the system is configured to produce at least about 1 g of the at least one biologically-produced product per day.
13. The biomanufacturing system of any one of sentences 1-7, wherein the reactor chamber has a volume of about 1 L to about 10 L.
14. The biomanufacturing system of sentence 13, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 0.5 mL/min over a period of at least about 1 day.
15. The biomanufacturing system of any one of sentences 13-14, wherein the system is configured to produce at least about 100 μg of the at least one biologically-produced product per day.
16. The biomanufacturing system of any one of sentences 13-15, wherein the system is configured to produce at least about 500 mg of the at least one biologically-produced product per day.
17. The biomanufacturing system of any one of sentences 13-16, wherein the system is configured to produce at least about 10 g of the at least one biologically-produced product per day.
18. The biomanufacturing system of any one of sentences 1-7, wherein the reactor chamber has a volume of about 10 L to about 50 L.
19. The biomanufacturing system of sentence 18, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 5 mL/min over a period of at least about 1 day.
20. The biomanufacturing system of any one of sentences 18-19, wherein the system is configured to produce at least about 500 μg of the at least one biologically-produced product per day.
21. The biomanufacturing system of any one of sentences 18-20, wherein the system is configured to produce at least about 2.5 g of the at least one biologically-produced product per day.
22. The biomanufacturing system of any one of sentences 18-21, wherein the system is configured to produce at least about 50 g of the at least one biologically-produced product per day.
23. The biomanufacturing system of any one of sentences 1-22, wherein the first type of biological cells are yeast cells, filamentous fungal cells, microalgal cells, or diatom cells.
24. The biomanufacturing system of sentence 23, wherein the yeast cells are Pichia pastoris cells.
25. The biomanufacturing system of any one of sentences 1-24, wherein the at least one biologically-produced product comprises a cytokine, an antibody, an antibody fragment, a nanobody, a hormone, an enzyme, a growth factor, a blood factor, a recombinant immunogen, and/or a fusion protein.
26. The biomanufacturing of sentence 25, wherein the antibody is a single-chain antibody, a bispecific antibody, and/or a monoclonal antibody.
27. The biomanufacturing system of any one of sentences 1-26, wherein the at least one biologically-produced product comprises human growth hormone (hGH).
28. The biomanufacturing system of any one of sentences 1-27, wherein the at least one biologically-produced product comprises granulocyte colony-stimulating factor (G-CSF).
29. The biomanufacturing system of any one of sentences 1-28, wherein the at least one biologically-produced product comprises an interferon.
30. The biomanufacturing system of sentence 29, wherein the interferon is interferon α-2b.
31. The biomanufacturing system of any one of sentences 1-40, wherein the one or more properties comprise pH.
32. The biomanufacturing system of sentence 31, wherein the adjustment module is configured to reduce the pH of the at least one filtrate stream.
33. The biomanufacturing system of sentence 31, wherein the adjustment module is configured to increase the pH of the at least one filtrate stream.
34. The biomanufacturing system of any one of sentences 1-33, wherein the one or more properties comprise conductivity.
35. The biomanufacturing system of sentence 34, wherein the adjustment module is configured to increase or decrease the conductivity of the at least one filtrate stream.
36. The biomanufacturing system of any one of sentences 1-35, wherein the one or more properties comprise biologically-produced product stability.
37. The biomanufacturing system of sentence 36, wherein the adjustment module is configured to increase the stability of the at least one biologically-produced product in the at least one filtrate stream.
38. The biomanufacturing system of any one of sentences 1-37, wherein the first partitioning unit is directly fluidically connected to the second partitioning unit.
39. The biomanufacturing system of any one of sentences 1-38, wherein the first partitioning unit applies a partitioning technique and the second partitioning unit applies the same partitioning technique.
40. The biomanufacturing system of any one of sentences 1-39, wherein the first partitioning unit applies a partitioning technique and the second partitioning unit applies a different partitioning technique.
41. The biomanufacturing system of any one of sentences 1-40, wherein the first partitioning unit comprises a first column comprising at least a first stationary phase material.
42. The biomanufacturing system of sentence 41, wherein the first stationary phase material comprises a cation exchange resin, a multimodal cation exchange resin, an anion exchange resin, a multimodal anion exchange resin, a hydrophobic charge induction chromatography (HCIC) resin, or an affinity chromatography resin.
43. The biomanufacturing system of any one of sentences 1-42, wherein the first partitioning unit comprises a filter.
44. The biomanufacturing system of sentence 43, wherein the filter comprises a filtration membrane and/or a monolith.
45. The biomanufacturing system of any one of sentences 1-44, wherein the first partitioning unit comprises a precipitation apparatus.
46. The biomanufacturing system of any one of sentences 1-45, wherein the first partitioning unit comprises a crystallization apparatus.
47. The biomanufacturing system of any one of sentences 1-46, wherein the second partitioning unit comprises a second column comprising at least a first stationary phase material.
48. The biomanufacturing system of sentence 47, wherein the first stationary phase material comprises a cation exchange resin, a multimodal cation exchange resin, an anion exchange resin, a multimodal anion exchange resin, a hydrophobic charge induction chromatography (HCIC) resin, or an affinity chromatography resin.
49. The biomanufacturing system of any one of sentences 1-48, wherein the second partitioning unit comprises a filter.
50. The biomanufacturing system of sentence 49, wherein the filter comprises a filtration membrane and/or a monolith.
51. The biomanufacturing system of any one of sentences 1-50, wherein the second partitioning unit comprises a precipitation apparatus.
52. The biomanufacturing system of any one of sentences 1-51, wherein the second partitioning unit comprises a crystallization apparatus.
53. The biomanufacturing system of any one of sentences 1-52, wherein the first type of impurity is different from the second type of impurity.
54. The biomanufacturing system of any one of sentences 1-53, wherein the purification module further comprises a third partitioning unit configured to remove at least a third type of impurity from the second partitioned filtrate stream to produce a third partitioned filtrate stream lean in the third type of impurity relative to the second partitioned filtrate stream, wherein the third partitioned filtrate stream comprises the at least one biologically-produced product.
55. The biomanufacturing system of sentence 54, wherein the third partitioning unit comprises a third column comprising at least a first stationary phase material.
56. The biomanufacturing system of sentence 55, wherein the first stationary phase comprises a cation exchange resin, a multimodal cation exchange resin, an anion exchange resin, a multimodal anion exchange resin, a hydrophobic charge induction chromatography (HCIC) resin, or an affinity chromatography resin.
57. The biomanufacturing system of any one of sentences 54-56, wherein the third partitioning unit comprises a filter.
58. The biomanufacturing system of sentence 57, wherein the filter comprises a filtration membrane and/or a monolith.
59. The biomanufacturing system of any one of sentences 54-58, wherein the third partitioning unit comprises a precipitation apparatus.
60. The biomanufacturing system of any one of sentences 54-59, wherein the third partitioning unit comprises a crystallization apparatus.
61. The biomanufacturing system of any one of sentences 54-60, wherein the third type of impurity is different from the first type of impurity and/or the second type of impurity.
62. The biomanufacturing system of any one of sentences 1-61, further comprising a formulation module fluidically connected to the purification module, wherein the formulation module is configured to produce a formulated product stream.
63. The biomanufacturing system of sentence 62, wherein the formulation module comprises a filtration unit.
64. The biomanufacturing system of sentence 63, wherein the filtration unit comprises a tangential flow filtration device.
65. The biomanufacturing system of any one of sentences 62-64, wherein the formulation module comprises a viral filtration unit.
66. The biomanufacturing system of any one of sentences 62-65, wherein the formulation module comprises a packaging unit.
67. The biomanufacturing system of sentence 66, wherein the packaging unit is configured to package one or more doses of the at least one biologically-produced product into one or more bags, one or more vials, one or more syringes, and/or one or more bottles.
68. The biomanufacturing system of any one of sentences 62-67, wherein the formulation module comprises a dilution adjustment unit.
69. The biomanufacturing system of any one of sentences 1-68, further comprising a buffer delivery module fluidically connected to the purification module, wherein the buffer delivery module is configured to deliver at least one buffer to at least one partitioning unit.
70. The biomanufacturing system of any one of sentences 1-69, further comprising a gas concentration device fluidically connected to the bioreactor.
71. The biomanufacturing system of sentence 70, wherein the gas concentration device is an oxygen concentrator.
72. The biomanufacturing system of any one of sentences 1-71, wherein at least one module of the system is disposable.
73. The biomanufacturing system of sentence 72, wherein each module of the system is disposable.
74. The biomanufacturing system of any one of sentences 1-73, wherein at least one module of the system is configured to be cleaned in place.
75. The biomanufacturing system of sentence 74, wherein each module of the system is configured to be cleaned in place.
76. The biomanufacturing system of any one of sentences 1-75, wherein the purified filtrate stream and/or the formulated product stream has a concentration of the pharmaceutical product of at least about 10 μg/mL.
77. The biomanufacturing system of any one of sentences 1-76, wherein the purified filtrate stream and/or the formulated product stream has a concentration of host cell proteins of about 20 ng/(mg product) or less.
78. The biomanufacturing system of any one of sentences 1-77, wherein the purified filtrate stream and/or the formulated product stream has a concentration of DNA of about 100 ng/(mg product) or less.
79. A method of producing at least one biologically-produced product, comprising:
supplying at least one feed stream comprising at least one cell culture medium to a perfusion bioreactor at a first flow rate;
producing, within the perfusion bioreactor, a suspension comprising the at least one cell culture medium and at least a first type of biological cells expressing the at least one biologically-produced product;
measuring a level of the suspension in a reaction chamber of the perfusion bioreactor using a level sensing system;
causing at least a portion of the suspension to flow through at least one filter probe to produce at least one filtrate stream lean in the first type of biological cells, wherein the at least one filtrate stream comprises the at least one biologically-produced product, wherein the at least one filter probe is at least partially submerged in the suspension;
adjusting one or more properties of the at least one filtrate stream to produce an adjusted filtrate stream;
removing, within a purification module, at least a first type of impurity and a second type of impurity from the adjusted filtrate stream to produce a purified filtrate stream flowing at a second flow rate, wherein the purified filtrate stream comprises the at least one biologically-produced product and is lean in the first type of impurity and the second type of impurity relative to the adjusted filtrate stream, wherein producing the purified filtrate stream comprises: removing, within a first partitioning unit, at least the first type of impurity from the adjusted filtrate stream to produce a first partitioned filtrate stream lean in the first type of impurity relative to the adjusted filtrate stream, wherein the first partitioned filtrate stream comprises the at least one biologically-produced product; and
removing, within a second partitioning unit, at least the second type of impurity from the first partitioned filtrate stream to produce a second partitioned filtrate stream lean in the second type of impurity relative to the first partitioned filtrate stream, wherein the second partitioned filtrate stream comprises the at least one biologically-produced product.
80. The method of sentence 79, wherein the level sensing system is a magnetic level sensing system.
81. The method of sentence 80, wherein the magnetic level sensing system comprises a magnetic float, a non-magnetic shaft, and one or more magnetically-activated switches.
82. The method of sentence 79, wherein the level sensing system is an optical level sensing system.
83. The method of sentence 82, wherein the optical level sensing system comprises a colored float and/or a colored probe.
84. The method of sentence 82, wherein the optical level sensing system comprises a probe comprising a plurality of colored bands.
85. The method of any one of sentences 79-84, wherein the level sensing system comprises a capacitance-based probe.
86. The method of any one of sentences 79-85, further comprising, prior to supplying the at least one feed stream comprising the at least one cell culture medium to the perfusion bioreactor, supplying a growth cell culture medium to the perfusion bioreactor; incubating the first type of biological cells in the growth cell culture medium for a period of at least about 1 day; and at least partially removing the growth cell culture medium from the perfusion bioreactor.
87. The method of any one of sentences 79-86, wherein the at least one feed stream is continuously supplied to the perfusion bioreactor at the first flow rate over a period of at least about 1 day.
88. The method of any one of sentences 79-87, wherein the first flow rate and the second flow rate are substantially the same.
89. The method of any one of sentences 79-88, wherein the perfusion bioreactor comprises a reactor chamber having a volume of about 1 L or less.
90. The method of sentence 89, wherein the first flow rate and/or second flow rate are maintained at about 0.1 mL/min or more over a period of about 1 day or more.
91. The method of any one of sentences 89-90, wherein at least about 10 μg of the at least one biologically-produced product is produced in about 1 day or less.
92. The method of any one of sentences 89-91, wherein at least about 50 mg of the at least one biologically-produced product is produced in about 1 day or less.
93. The method of any one of sentences 89-92, wherein at least about 1 g of the at least one biologically-produced product is produced in about 1 day or less.
94. The method of any one of sentences 79-88, wherein the perfusion bioreactor comprises a reactor chamber having a volume of about 1 L to about 10 L.
95. The method of sentence 94, wherein the first flow rate and/or second flow rate are maintained at about 0.5 mL/min or more over a period of about 1 day or more.
96. The method of any one of sentences 94-95, wherein at least about 100 μg of the at least one biologically-produced product is produced in about 1 day or less.
97. The method of any one of sentences 94-96, wherein at least about 500 mg of the at least one biologically-produced product is produced in about 1 day or less.
98. The method of any one of sentences 94-97, wherein at least about 10 g of the at least one biologically-produced product is produced in about 1 day or less.
99. The method of any one of sentences 79-88, wherein the perfusion bioreactor comprises a reactor chamber having a volume of about 10 L to about 50 L.
100. The method of sentence 99, wherein the first flow rate and/or second flow rate are maintained at about 5 mL/min or more over a period of about 1 day or more.
101. The method of any one of sentences 99-100, wherein at least about 500 μg of the at least one biologically-produced product is produced in about 1 day or less.
102. The method of any one of sentences 99-101, wherein at least about 2.5 g of the at least one biologically-produced product is produced in about 1 day or less.
103. The method of any one of sentences 99-102, wherein at least about 50 g of the at least one biologically-produced product is produced in about 1 day or less.
104. The method of any one of sentences 79-103, wherein the first type of biological cells are yeast cells, filamentous fungal cells, microalgal cells, or diatom cells.
105. The method of sentence 104, wherein the yeast cells are Pichia pastoris cells.
106. The method of any one of sentences 79-105, wherein the at least one biologically-produced product comprises a cytokine, an antibody, an antibody fragment, a nanobody, a hormone, an enzyme, a growth factor, a blood factor, a recombinant immunogen, and/or a fusion protein.
107. The method of sentence 106, wherein the antibody is a single-chain antibody, a bispecific antibody, and/or a monoclonal antibody.
108. The method of any one of sentences 79-107, wherein the at least one biologically-produced product comprises human growth hormone (hGH).
109. The method of any one of sentences 79-108, wherein the at least one biologically-produced product comprises granulocyte colony-stimulating factor (G-CSF).
110. The method of any one of sentences 79-109, wherein the at least one biologically-produced product comprises an interferon.
111. The method of sentence 110, wherein the interferon is interferon α-2b.
112. The method of any one of sentences 79-111, wherein adjusting one or more properties of the at least one filtrate stream comprises decreasing the pH of the at least one filtrate stream.
113. The method of any one of sentences 79-112, wherein adjusting one or more properties of the at least one filtrate stream comprises increasing the pH of the at least one filtrate stream.
114. The method of any one of sentences 79-113, wherein adjusting one or more properties of the at least one filtrate stream comprises increasing the conductivity of the at least one filtrate stream.
115. The method of any one of sentences 79-114, wherein adjusting one or more properties of the at least one filtrate stream comprises decreasing the conductivity of the at least one filtrate stream.
116. The method of any one of sentences 79-115, wherein adjusting one or more properties of the at least one filtrate stream comprises reducing the pH of the at least one filtrate stream.
117. The method of any one of sentences 79-116, wherein adjusting one or more properties of the at least one filtrate stream comprises increasing the stability of the at least one biologically-produced product in the at least one filtrate stream.
118. The method of any one of sentences 79-117, wherein the first partitioning unit and the second partitioning unit are directly fluidically connected.
119. The method of any one of sentences 79-118, wherein the first partitioning unit and the second partitioning unit apply the same partitioning technique.
120. The method of any one of sentences 79-119, wherein the first partitioning unit and the second partitioning unit apply different partitioning techniques.
121. The method of any one of sentences 79-120, wherein removing at least the first type of impurity comprises causing the adjusted filtrate stream to flow through a first column comprising at least a first stationary phase material.
122. The method of any one of sentences 79-121, wherein removing at least the first type of impurity comprises causing the adjusted filtrate stream to flow through a filter.
123. The method of sentence 122, wherein the filter comprises a filtration membrane and/or a monolith.
124. The method of any one of sentences 79-123, wherein removing at least the first type of impurity comprises causing the first type of impurity to precipitate and/or crystallize.
125. The method of any one of sentences 79-124, wherein removing at least the second type of impurity comprises causing the first partitioned filtrate stream to flow through a second column comprising at least a first stationary phase material.
126. The method of any one of sentences 79-125, wherein removing at least the second type of impurity comprises causing the first partitioned filtrate stream to flow through a filter.
127. The method of sentence 126, wherein the filter comprises a filtration membrane and/or a monolith.
128. The method of any one of sentences 79-127, wherein removing at least the second type of impurity comprises causing the second type of impurity to precipitate and/or crystallize.
129. The method of any one of sentences 79-128, wherein the first type of impurity is different from the second type of impurity.
130. The method of any one of sentences 79-129, further comprising removing, within a third partitioning unit, at least a third type of impurity from the second partitioned filtrate stream to produce a third partitioned filtrate stream lean in the third type of impurity relative to the second partitioned filtrate stream, wherein the third partitioned filtrate stream comprises the at least one biologically-produced product.
131. The method of sentence 130, wherein removing at least the third type of impurity comprises causing the second partitioned filtrate stream to flow through a third column comprising at least a first stationary phase material.
132. The method of any one of sentences 130-131, wherein removing at least the third type of impurity comprises causing the second partitioned filtrate stream to flow through a filter.
133. The method of sentence 132, wherein the filter comprises a filtration membrane and/or a monolith.
134. The method of any one of sentences 130-133, wherein removing at least the third type of impurity comprises causing the third type of impurity to precipitate and/or crystallize.
135. The method of any one of sentences 130-134, wherein the third type of impurity is different from the first type of impurity and/or the second type of impurity.
136. The method of any one of sentences 79-135, further comprising flowing the purified filtrate stream through a formulation module configured to produce a formulated product stream.
137. The method of sentence 136, wherein flowing the purified filtrate stream through the formulation module comprises flowing the purified filtrate stream through a tangential flow filtration device.
138. The method of any one of sentences 136-137, wherein flowing the purified filtrate stream through the formulation module comprises flowing the purified filtrate stream through a dilution adjustment unit.
139. The method of sentence 138, further comprising adding a diluent to the purified filtrate stream.
140. The method of any one of sentences 136-139, wherein flowing the purified filtrate stream through the formulation module comprises flowing the purified filtrate stream through a viral filtration unit, wherein the formulated product stream is lean in one or more viruses relative to the purified filtrate stream.
141. The method of any one of sentences 136-140, wherein flowing the purified filtrate stream through the formulation module comprises depositing one or more portions of the purified filtrate stream into one or more containers.
142. The method of sentence 141, wherein the one or more containers are aseptic and/or sterile containers.
143. The method of any one of sentences 141-142, wherein the one or more containers comprise one or more bags, vials, syringes, and/or bottles.
144. The biomanufacturing system of any preceding sentence, further comprising a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
145. The method of any preceding sentence, further comprising monitoring one or more steps of the method using a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
146. The method of any preceding sentence, further comprising implementing one or more corrective action based on information derived from a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
A third exemplary embodiment is generally directed to the following:
1. A system for producing G-CSF, comprising a bioreactor, wherein the bioreactor comprises a reaction chamber containing a suspension comprising at least one cell culture medium and at least a first type of biological cells configured to express G-CSF;
at least one filter, wherein the at least one filter is configured to receive an output of the bioreactor and produce at least one filtrate lean in the first type of biological cells relative to the suspension, wherein the at least one filtrate comprises G-CSF; and
a purification module, wherein the purification module is configured to remove at least a first type of impurity, a second type of impurity, and a third type of impurity from the first filtrate to produce a purified filtrate, wherein the purification module comprises:
a first column comprising a multimodal cation exchange resin;
a second column comprising an anion exchange resin; and
a third column comprising an HCIC resin.
2. The system of sentence 1, wherein the bioreactor is a perfusion bioreactor.
3. The system of sentence 2, wherein the bioreactor is configured to receive at least one feed stream comprising the at least one cell culture medium; the at least one filter is fluidically connected to the bioreactor and the first filtrate is an at least one filtrate stream; the purification module is fluidically connected to the at least one filter and the purified filtrate is a purified filtrate stream; and the system is configured to be continuously operated.
4. The system of any one of sentences 1-3, wherein the at least one filter comprises at least one filter probe at least partially submerged in the suspension in the bioreactor.
5. The system of any one of sentences 1-4, wherein the system further comprises a pH adjustment module configured to increase or decrease the pH of the at least one filtrate to produce a pH-adjusted filtrate.
6. The system of sentence 5, wherein the pH adjustment module is fluidically connected to the bioreactor, the at least one filter, and/or the purification module.
7. The system of any one of sentences 5-6, wherein the pH-adjusted filtrate has a pH of about 5.0.
8. The system of any one of sentences 1-7, wherein the first column is configured to remove at least the first type of impurity from the at least one filtrate to produce a first partitioned filtrate lean in the first type of impurity relative to the at least one filtrate, wherein the first partitioned filtrate comprises G-CSF.
9. The system of any one of sentences 1-8, wherein the multimodal cation exchange resin comprises a Capto MMC ImpRes resin.
10. The system of any one of sentences 1-9, wherein the second column is configured to remove at least the second type of impurity from the first partitioned filtrate to produce a second partitioned filtrate lean in the second type of impurity relative to the first partitioned filtrate, wherein the second partitioned filtrate comprises G-CSF.
11. The system of any one of sentences 1-10, wherein the anion exchange resin comprises a HyperCel STAR AX resin.
12. The system of any one of sentences 1-11, wherein the third column is configured to remove at least the third type of impurity from the second partitioned filtrate to produce a third partitioned filtrate lean in the third type of impurity relative to the second partitioned filtrate, wherein the third partitioned filtrate comprises G-CSF.
13. The system of any one of sentences 1-12, wherein the HCIC resin comprises an MEP HyperCel resin.
14. The system of any one of sentences 1-13, wherein the first type of biological cells are yeast cells, filamentous fungal cells, microalgal cells, or diatom cells.
15. The system of sentence 14, wherein the yeast cells are Pichia pastoris cells.
16. The system of any one of sentences 1-15, wherein the cell culture medium comprises chemically defined media comprising a carbon source, chemically defined media comprising an additive, or buffered methanol-complex media (BMMY).
17. The system of any one of sentences 1-16, further comprising a formulation module fluidically connected to the purification module, wherein the formulation module is configured to produce a formulated biologically-produced product stream.
18. The system of sentence 17, wherein the formulation module comprises a filtration unit.
19. The system of sentence 18, wherein the filtration unit comprises a tangential flow filtration device.
20. The system of any one of sentences 17-19, wherein the formulation module comprises a viral filtration unit.
21. The system of any one of sentences 17-20, wherein the formulation module comprises a product packaging unit.
22. The system of sentence 21, wherein the packaging unit is configured to package one or more doses of the at least one biologically-produced product into one or more bags, one or more vials, one or more syringes, and/or one or more bottles.
23. The system of any one of sentences 1-22, wherein the purified filtrate and/or formulated biologically-produced product stream has a host cell protein concentration of about 20 ng/(mg G-CSF) or less.
24. The system of any one of sentences 1-23, wherein the purified filtrate and/or formulated biologically-produced product stream does not have a detectable level of DNA.
25. The system of any one of sentences 1-24, wherein the purified filtrate and/or formulated biologically-produced product stream has an aggregate content of about 1% or less.
26. The system of any of sentences 1-25, wherein the reactor chamber has a volume of about 1 L or less.
27. The system of sentence 26, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 0.1 mL/min over a period of at least about 1 day.
28. The system of any one of sentences 26-27, wherein the system is configured to produce at least about 10 mg of G-CSF per day.
29. The system of any one of sentences 1-26, wherein the reactor chamber has a volume of about 1 L to about 10 L.
30. The system of sentence 29, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 0.5 mL/min over a period of at least about 1 day.
31. The system of any one of sentences 29-30, wherein the system is configured to produce at least about 50 mg of G-CSF per day.
32. The system of any one of sentences 1-31, wherein the reactor chamber has a volume of about 10 L to about 50 L.
33. The system of sentence 32, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 5 mL/min over a period of at least about 1 day.
34. The system of any one of sentences 32-33, wherein the system is configured to produce at least about 500 mg of G-CSF per day.
35. A method of producing G-CSF, comprising:
applying at least one cell culture medium to a bioreactor; producing, within the bioreactor, a suspension comprising the at least one cell culture medium and at least a first type of biological cells expressing G-CSF;
causing at least a portion of the suspension to flow through at least one filter to produce at least one filtrate lean in the first type of biological cells, wherein the at least one filtrate comprises G-CSF;
flowing the at least one filtrate through a purification module to produce a purified filtrate, wherein producing the purified filtrate comprises flowing the at least one filtrate through a first column comprising a multimodal cation exchange resin;
collecting one or more first fractions comprising G-CSF from an outflow of the first column; flowing the one or more first fractions through a second column comprising an anion exchange resin;
collecting one or more second fractions comprising G-CSF from an outflow of the second column;
flowing the one or more second fractions through a third column comprising an HCIC resin; and
collecting one or more third fractions comprising G-CSF from an outflow of the third column.
36. The method of sentence 35, wherein the bioreactor is a perfusion bioreactor.
37. The method of sentence 36, wherein at least one feed stream comprising the at least one cell culture medium is continuously supplied to the perfusion bioreactor at a first flow rate over a period of at least about 1 day; the at least one filter is fluidically connected to the bioreactor and the at least one filtrate is an at least one filtrate stream; and the purified filtrate is a purified filtrate stream flowing at a second flow rate, wherein the purified filtrate stream comprises the one or more third fractions.
38. The method of any one of sentences 35-37, further comprising, prior to supplying the at least one cell culture medium to the bioreactor, supplying a growth cell culture medium to the bioreactor; incubating the first type of biological cells in the growth cell culture medium for a period of at least about 1 day; and at least partially removing the growth cell culture medium from the bioreactor.
39. The method of any one of sentences 35-38, wherein the at least one filter comprises at least one filter probe at least partially submerged in the suspension in the bioreactor.
40. The method of any one of sentences 35-39, further comprising adjusting the pH of the at least one filtrate to produce a pH-adjusted filtrate.
41. The method of sentence 40, wherein the pH-adjusted filtrate has a pH of about 5.0.
42. The method of any one of sentences 40-41, wherein the pH is adjusted in a pH adjustment module that is fluidically connected to the at least one filter and the first column of the purification module.
43. The method of any one of sentences 35-42, wherein the one or more first fractions are lean in a first type of impurity relative to the at least one filtrate.
44. The method of any one of sentences 35-43, wherein the multimodal cation exchange resin comprises a Capto MMC ImpRes resin.
45. The method of any one of sentences 35-44, wherein the first column is operated in bind-elute mode.
46. The method of sentence 45, further comprising flowing a first mobile phase material through the first column prior to flowing the at least one filtrate through the first column, wherein the first mobile phase material is configured to promote binding of G-CSF to the multimodal cation exchange resin.
47. The method of sentence 46, wherein the first mobile phase material has a pH of about 5.0.
48. The method of any one of sentences 46-47, further comprising flowing a second mobile phase material through the first column after flowing the at least one filtrate through the first column.
49. The method of sentence 48, wherein the second mobile phase material has a pH of about 5.8 and a salt concentration of about 150 mM.
50. The method of any one of sentences 46-49, further comprising flowing a third mobile phase material through the first column after flowing the second mobile phase material through the first column, wherein the third mobile phase material is configured to elute G-CSF from the first column.
51. The method of sentence 50, wherein the third mobile phase material has a pH of about 7.0 and a salt concentration of about 150 mM.
52. The method of any one of sentences 35-51, wherein the one or more second fractions are lean in a second type of impurity relative to the first fractions.
53. The method of any one of sentences 35-52, wherein the anion exchange resin comprises a HyperCel STAR AX resin.
54. The method of any one of sentences 35-53, wherein the second column is operated in flow-through mode.
55. The method of any one of sentences 35-54, wherein the one or more third fractions are lean in a third type of impurity relative to the second fractions.
56. The method of any one of sentences 35-55, wherein the HCIC resin comprises an MEP HyperCel resin.
57. The method of any one of sentences 35-56, wherein the third column is operated in bind-elute mode.
58. The method of sentence 57, further comprising flowing a first mobile phase material through the third column prior to flowing the second fractions through the third column, wherein the first mobile phase material is configured to promote binding of G-CSF to the HCIC resin.
59. The method of sentence 58, wherein the first mobile phase material has a pH of about 7.0 and a salt concentration of about 150 mM.
60. The method of any one of sentences 57-59, further comprising flowing a second mobile phase material through the third column after flowing the second fractions through the third column.
61. The method of sentence 60, wherein the second mobile phase material has a pH of about 5.5 and a salt concentration less than about 150 mM.
62. The method of any one of sentences 57-61, further comprising flowing a third mobile phase material through the third column after flowing the second mobile phase material through the third column, wherein the third mobile phase material is configured to elute G-CSF from the third column.
63. The method of sentence 62, wherein the third mobile phase material has a pH of about 3.0 and a salt concentration less than about 150 mM.
64. The method of any one of sentences 35-63, wherein the first type of biological cells are yeast cells, filamentous fungal cells, microalgal cells, or diatom cells.
65. The method of sentence 64, wherein the yeast cells are Pichia pastoris cells.
66. The method of any one of sentences 35-65, wherein the at least one cell culture medium comprises chemically defined media comprising a carbon source, chemically defined media comprising an additive, or buffered methanol-complex media (BMMY).
67. The method of any one of sentences 35-66, further comprising flowing the purified filtrate through a formulation module configured to produce a formulated product stream.
68. The method of sentence 67, wherein flowing the purified filtrate through the formulation module comprises flowing the purified filtrate through a tangential flow filtration device.
69. The method of any one of sentences 67-68, wherein flowing the purified filtrate through the formulation module comprises flowing the purified filtrate stream through a viral filtration unit, wherein the formulated product stream is lean in one or more viruses relative to the purified filtrate stream.
70. The method of any one of sentences 67-69, wherein flowing the purified filtrate through the formulation module comprises depositing one or more portions of the purified filtrate stream into one or more containers.
71. The method of sentence 70, wherein the one or more containers are aseptic and/or sterile containers.
72. The method of any one of sentences 70-71, wherein the one or more containers comprise one or more bags, vials, syringes, and/or bottles.
73. The method of any one of sentences 35-72, wherein the purified filtrate and/or the formulated stream have a host cell protein concentration of about 20 ng/(mg G-CSF) or less.
74. The method of any one of sentences 35-73, wherein the purified filtrate and/or the formulated stream do not have a detectable level of DNA.
75. The method of any one of sentences 35-74, wherein the purified filtrate and/or the formulated stream have an aggregate content of about 1% or less.
76. The method of any one of sentences 36-75, wherein the bioreactor comprises a reactor chamber having a volume of about 1 L or less.
77. The method of sentence 76, wherein the first flow rate and/or the second flow rate are maintained at about 0.1 mL/min or more over a period of about 1 day or more.
78. The method of any one of sentences 76-77, wherein at least about 10 mg of G-CSF is produced in about 1 day or less.
79. The method of any one of sentences 36-75, wherein the bioreactor comprises a reactor chamber having a volume of about 1 L to about 10 L.
80. The method of sentence 79, wherein the first flow rate and/or the second flow rate are maintained at about 0.5 mL/min or more over a period of about 1 day or more.
81. The method of any one of sentences 79-80, wherein at least about 50 mg of G-CSF is produced in about 1 day or less.
82. The method of any one of sentences 35-75, wherein the bioreactor comprises a reactor chamber having a volume of about 10 L to about 50 L.
83. The method of sentence 82, wherein the first flow rate and/or the second flow rate are maintained at about 5 mL/min or more over a period of about 1 day or more.
84. The method of any one of sentences 82-83, wherein at least about 500 mg of G-CSF is produced in about 1 day or less.
85. The method of any one of sentences 67-84, wherein flowing the purified filtrate through the formulation module comprises flowing the purified filtrate through a dilution adjustment unit.
86. The method of sentence 85, wherein flowing the purified filtrate through the dilution adjustment unit comprises adding a diluent to the purified filtrate.
87. The system of any one of sentences 17-34, wherein the formulation module comprises a dilution adjustment unit.
88. The biomanufacturing system of any preceding sentence, further comprising a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
89. The method of any preceding sentence, further comprising monitoring one or more steps of the method using a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
90. The method of any preceding sentence, further comprising implementing one or more corrective action based on information derived from a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
A fourth exemplary embodiment is generally directed to the following:
1. A system for producing interferon-α2b (IFN), comprising:
a bioreactor, wherein the bioreactor comprises a reaction chamber containing a suspension comprising at least one cell culture medium and at least a first type of biological cells configured to express interferon-α2b;
at least one filter, wherein the at least one filter is configured to receive an output of the bioreactor and produce at least one filtrate lean in the first type of biological cells relative to the suspension, wherein the at least one filtrate comprises interferon-α2b; and
a purification module, wherein the purification module is configured to remove at least a first type of impurity, a second type of impurity, and a third type of impurity from the first filtrate to produce a purified filtrate, wherein the purification module comprises:
a first column comprising a multimodal cation exchange resin;
a second column comprising an HCIC resin; and
a third column comprising a cation exchange resin.
2. The system of sentence 1, wherein the bioreactor is a perfusion bioreactor.
3. The system of sentence 2, wherein the bioreactor is configured to receive at least one feed stream comprising the at least one cell culture medium; the at least one filter is fluidically connected to the bioreactor and the first filtrate is an at least one filtrate stream; the purification module is fluidically connected to the at least one filter and the purified filtrate is a purified filtrate stream; and the system is configured to be continuously operated.
4. The system of any one of sentences 1-3, wherein the at least one filter comprises at least one filter probe at least partially submerged in the suspension in the bioreactor.
5. The system of any one of sentences 1-4, wherein the system further comprises a pH adjustment module configured to increase or decrease the pH of the at least one filtrate to produce a pH-adjusted filtrate.
6. The system of sentence 5, wherein the pH adjustment module is fluidically connected to the bioreactor, the at least one filter, and/or the purification module.
7. The system of any one of sentences 5-6, wherein the pH-adjusted filtrate has a pH of about 4.0.
8. The system of any one of sentences 1-7, wherein the first column is configured to remove at least the first type of impurity from the at least one filtrate to produce a first partitioned filtrate lean in the first type of impurity relative to the at least one filtrate, wherein the first partitioned filtrate comprises interferon-α2b.
9. The system of any one of sentences 1-8, wherein the multimodal cation exchange resin comprises a Capto MMC ImpRes resin.
10. The system of any one of sentences 1-9, wherein the second column is configured to remove at least the second type of impurity from the first partitioned filtrate to produce a second partitioned filtrate lean in the second type of impurity relative to the first partitioned filtrate, wherein the second partitioned filtrate comprises interferon-α2b.
11. The system of any one of sentences 1-10, wherein the HCIC resin comprises an MEP HyperCel resin and/or a HEA HyperCel resin.
12. The system of any one of sentences 1-11, wherein the third column is configured to remove at least the third type of impurity from the second partitioned filtrate to produce a third partitioned filtrate lean in the third type of impurity relative to the second partitioned filtrate, wherein the third partitioned filtrate comprises interferon-α2b.
13. The system of any one of sentences 1-12, wherein the cation exchange resin comprises an SP Sepharose HP resin and/or Toyopearl MX-Trp-650M resin.
14. The system of any one of sentences 1-13, wherein the first type of biological cells are yeast cells, filamentous fungal cells, microalgal cells, or diatom cells.
15. The system of sentence 14, wherein the yeast cells are Pichia pastoris cells.
16. The system of any one of sentences 1-15, wherein the cell culture medium comprises chemically defined media comprising a carbon source, chemically defined media comprising an additive, or buffered methanol-complex media (BMMY).
17. The system of any one of sentences 1-16, further comprising a formulation module fluidically connected to the purification module, wherein the formulation module is configured to produce a formulated product stream.
18. The system of sentence 17, wherein the formulation module comprises a filtration unit.
19. The system of sentence 18, wherein the filtration unit comprises a tangential flow filtration device.
20. The system of any one of sentences 17-19, wherein the formulation module comprises a viral filtration unit.
21. The system of any one of sentences 17-20, wherein the formulation module comprises a packaging unit.
22. The system of sentence 21, wherein the packaging unit is configured to package one or more doses of the at least one biologically-produced product into a bag, one or more vials, one or more syringes, and/or one or more bottles.
23. The system of any one of sentences 1-22, wherein the purified filtrate and/or formulated product stream comprises interferon-α2b having a purity of at least about 77%.
24. The system of any one of sentences 1-23, wherein the purified filtrate and/or formulated product stream has a DNA concentration of about 0.51 ng/(mg IFN) or less.
25. The system of any one of sentences 1-24, wherein the purified filtrate and/or formulated product stream has an aggregate content of about 0.5% or less.
26. The system of any of sentences 1-25, wherein the reactor chamber has a volume of about 1 L or less.
27. The system of sentence 26, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 0.1 mL/min over a period of at least about 1 day.
28. The system of any one of sentences 26-27, wherein the system is configured to produce at least about 10 mg of IFN per day.
29. The system of any one of sentences 1-26, wherein the reactor chamber has a volume of about 1 L to about 10 L.
30. The system of sentence 29, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 0.5 mL/min over a period of at least about 1 day.
31. The system of any one of sentences 29-30, wherein the system is configured to produce at least about 50 mg of IFN per day.
32. The system of any one of sentences 1-31, wherein the reactor chamber has a volume of about 10 L to about 50 L.
33. The system of sentence 32, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 5 mL/min over a period of at least about 1 day.
34. The system of any one of sentences 32-33, wherein the system is configured to produce at least about 500 mg of IFN per day.
35. A method of producing interferon-α2b (IFN), comprising:
supplying at least one cell culture medium to a bioreactor; producing, within the bioreactor, a suspension comprising the at least one cell culture medium and at least a first type of biological cells expressing interferon-α2b;
causing at least a portion of the suspension to flow through at least one filter to produce at least one filtrate lean in the first type of biological cells, wherein the at least one filtrate comprises interferon-α2b; and
flowing the at least one filtrate through a purification module to produce a purified filtrate, wherein producing the purified filtrate comprises flowing the at least one filtrate through a first column comprising a multimodal cation exchange resin;
collecting one or more first fractions comprising interferon-α2b from an outflow of the first column;
flowing the one or more first fractions through a second column comprising an HCIC resin;
collecting one or more second fractions comprising interferon-α2b from an outflow of the second column;
flowing the one or more second fractions through a third column comprising a cation exchange resin; and
collecting one or more third fractions comprising interferon-α2b from an outflow of the third column.
36. The method of sentence 35, wherein the bioreactor is a perfusion bioreactor.
37. The method of sentence 36, wherein at least one feed stream comprising the at least one cell culture medium is continuously supplied to the perfusion bioreactor at a first flow rate over a period of at least about 1 day; the at least one filter is fluidically connected to the bioreactor and the at least one filtrate is an at least one first filtrate stream; and the purified filtrate is a purified filtrate stream flowing at a second flow rate, wherein the purified filtrate stream comprises the one or more third fractions.
38. The method of any one of sentences 35-37, further comprising, prior to supplying the at least one cell culture medium to the bioreactor, supplying a growth cell culture medium to the bioreactor; incubating the first type of biological cells in the growth cell culture medium for a period of at least about 1 day; and at least partially removing the growth cell culture medium from the bioreactor.
39. The method of any one of sentences 35-38, wherein the at least one filter comprises at least one filter probe at least partially submerged in the suspension in the bioreactor.
40. The method of any one of sentences 35-39, further comprising adjusting the pH of the at least one filtrate to produce a pH-adjusted filtrate.
41. The method of sentence 40, wherein the pH-adjusted filtrate has a pH of about 4.0.
42. The method of any one of sentences 40-41, wherein the pH is adjusted in a pH adjustment module that is fluidically connected to the at least one filter.
43. The method of any one of sentences 35-42, wherein the one or more first fractions are lean in a first type of impurity relative to the at least one filtrate.
44. The method of any one of sentences 35-43, wherein the multimodal cation exchange resin comprises a Capto MMC ImpRes resin.
45. The method of any one of sentences 35-44, wherein the one or more second fractions are lean in a second type of impurity relative to the first fractions.
46. The method of any one of sentences 35-45, wherein the HCIC resin comprises an MEP HyperCel resin and/or a HEA HyperCel resin.
47. The method of any one of sentences 35-46, wherein the one or more third fractions are lean in a third type of impurity relative to the second fractions.
48. The method of any one of sentences 35-47, wherein the cation exchange resin comprises an SP Sepharose HP resin and/or Toyopearl MX-Trp-650M resin.
49. The method of any one of sentences 35-48, wherein the first type of biological cells are yeast cells, filamentous fungal cells, microalgal cells, or diatom cells.
50. The method of sentence 49, wherein the yeast cells are Pichia pastoris cells.
51. The method of any one of sentences 35-50, wherein the at least one cell culture medium comprises chemically defined media comprising a carbon source, chemically defined media comprising an additive, or buffered methanol-complex media (BMMY).
52. The method of any one of sentences 35-51, further comprising flowing the purified filtrate through a formulation module configured to produce a formulated product stream.
53. The method of sentence 52, wherein flowing the purified filtrate through the formulation module comprises flowing the purified filtrate through a tangential flow filtration device.
54. The method of any one of sentences 52-53, wherein flowing the purified filtrate through the formulation module comprises flowing the purified filtrate stream through a viral filtration unit, wherein the formulated product stream is lean in one or more viruses relative to the purified filtrate stream.
55. The method of any one of sentences 52-54, wherein flowing the purified filtrate through the formulation module comprises depositing one or more portions of the purified filtrate stream into one or more containers.
56. The method of sentence 55, wherein the one or more containers are aseptic and/or sterile containers.
57. The method of any one of sentences 55-56, wherein the one or more containers comprise one or more bags, vials, syringes, and/or bottles.
58. The method of any one of sentences 35-57, wherein the purified filtrate and/or the formulated product stream comprise interferon-α2b having a purity of at least about 77%.
59. The method of any one of sentences 35-58, wherein the purified filtrate and/or the formulated product stream have a DNA concentration of about 0.51 ng/(mg IFN) or less.
60. The method of any one of sentences 35-59, wherein the purified filtrate and/or the formulated product stream have an aggregate content of about 0.5% or less.
61. The method of any one of sentences 35-60, wherein the bioreactor comprises a reactor chamber having a volume of about 1 L or less.
62. The method of sentence 61, wherein the first flow rate and/or the second flow rate are maintained at about 0.1 mL/min or more over a period of about 1 day or more.
63. The method of any one of sentences 61-62, wherein at least about 10 mg of IFN is produced in about 1 day or less.
64. The method of any one of sentences 35-60, wherein the bioreactor comprises a reactor chamber having a volume of about 1 L to about 10 L.
65. The method of sentence 64, wherein the first flow rate and/or the second flow rate are maintained at about 0.5 mL/min or more over a period of about 1 day or more.
66. The method of any one of sentences 64-65, wherein at least about 50 mg of IFN is produced in about 1 day or less.
67. The method of any one of sentences 35-60, wherein the bioreactor comprises a reactor chamber having a volume of about 10 L to about 50 L.
68. The method of sentence 67, wherein the first flow rate and/or the second flow rate are maintained at about 5 mL/min or more over a period of about 1 day or more.
69. The method of any one of sentences 67-68, wherein at least about 500 mg of IFN is produced in about 1 day or less.
70. The method of any one of sentences 52-69, wherein flowing the purified filtrate through the formulation module comprises flowing the purified filtrate stream through a dilution adjustment unit.
71. The method of sentence 70, wherein flowing the purified filtrate stream through the dilution adjustment unit comprises adding a diluent to the purified filtrate stream.
72. The system of any one of sentences 17-34, wherein the formulation module comprises a dilution adjustment unit.
73. The biomanufacturing system of any preceding sentence, further comprising a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
74. The method of any preceding sentence, further comprising monitoring one or more steps of the method using a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
75. The method of any preceding sentence, further comprising implementing one or more corrective action based on information derived from a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
A fifth exemplary embodiment is generally directed to the following:
1. A system for producing interferon-α2b (IFN), comprising:
a bioreactor, wherein the bioreactor comprises a reaction chamber containing a suspension comprising at least one cell culture medium and at least a first type of biological cells configured to express interferon-α2b;
at least one filter, wherein the at least one filter is configured to receive an output of the bioreactor and produce at least one filtrate lean in the first type of biological cells relative to the suspension, wherein the at least one filtrate comprises interferon-α2b; and
a purification module, wherein the purification module is configured to remove at least a first type of impurity, a second type of impurity, and a third type of impurity from the first filtrate to produce a purified filtrate, wherein the purification module comprises:
a first column comprising a multimodal cation exchange resin;
a second column comprising a flow-through resin; and
a third column comprising an anion exchange resin.
2. The system of sentence 1, wherein the bioreactor is a perfusion bioreactor.
3. The system of sentence 2, wherein the bioreactor is configured to receive at least one feed stream comprising the at least one cell culture medium; the at least one filter is fluidically connected to the bioreactor and the first filtrate is an at least one filtrate stream; the purification module is fluidically connected to the at least one filter and the purified filtrate is a purified filtrate stream; and the system is configured to be continuously operated.
4. The system of any one of sentences 1-3, wherein the at least one filter comprises at least one filter probe at least partially submerged in the suspension in the bioreactor.
5. The system of any one of sentences 1-4, wherein the system further comprises a pH adjustment module configured to increase or decrease the pH of the at least one filtrate to produce a pH-adjusted filtrate.
6. The system of sentence 5, wherein the pH adjustment module is fluidically connected to the bioreactor, the at least one filter, and/or the purification module.
7. The system of any one of sentences 5-6, wherein the pH-adjusted filtrate has a pH of about 5.0.
8. The system of any one of sentences 1-7, wherein the first column is configured to remove at least the first type of impurity from the at least one filtrate to produce a first partitioned filtrate lean in the first type of impurity relative to the at least one filtrate, wherein the first partitioned filtrate comprises interferon-α2b.
9. The system of any one of sentences 1-8, wherein the multimodal cation exchange resin comprises a Capto MMC ImpRes resin.
10. The system of any one of sentences 1-9, wherein the second column is configured to remove at least the second type of impurity from the first partitioned filtrate to produce a second partitioned filtrate lean in the second type of impurity relative to the first partitioned filtrate, wherein the second partitioned filtrate comprises interferon-α2b.
11. The system of any one of sentences 1-10, wherein the flow-through resin comprises a Q Sepharose HP resin.
12. The system of any one of sentences 1-11, wherein the third column is configured to remove at least the third type of impurity from the second partitioned filtrate to produce a third partitioned filtrate lean in the third type of impurity relative to the second partitioned filtrate, wherein the third partitioned filtrate comprises interferon-α2b.
13. The system of any one of sentences 1-12, wherein the anion exchange resin comprises a Capto Adhere resin.
14. The system of any one of sentences 1-13, wherein the first type of biological cells are yeast cells, filamentous fungal cells, microalgal cells, or diatom cells.
15. The system of sentence 14, wherein the yeast cells are Pichia pastoris cells.
16. The system of any one of sentences 1-15, wherein the cell culture medium comprises chemically defined media comprising a carbon source, chemically defined media comprising an additive, or buffered methanol-complex media (BMMY).
17. The system of any one of sentences 1-16, further comprising a formulation module fluidically connected to the purification module, wherein the formulation module is configured to produce a formulated product stream.
18. The system of sentence 17, wherein the formulation module comprises a filtration unit.
19. The system of sentence 18, wherein the filtration unit comprises a tangential flow filtration device.
20. The system of any one of sentences 17-19, wherein the formulation module comprises a viral filtration unit.
21. The system of any one of sentences 17-20, wherein the formulation module comprises a packaging unit.
22. The system of sentence 21, wherein the packaging unit is configured to package one or more doses of the at least one biologically-produced product into a bag, one or more vials, one or more syringes, and/or one or more bottles.
23. The system of any one of sentences 1-22, wherein the purified filtrate and/or formulated product stream comprises interferon-α2b having a purity of at least about 77%.
24. The system of any one of sentences 1-23, wherein the purified filtrate and/or formulated product stream has a DNA concentration of about 0.51 ng/(mg IFN) or less.
25. The system of any one of sentences 1-24, wherein the purified filtrate and/or formulated product stream has an aggregate content of about 0.5% or less.
26. The system of any of sentences 1-25, wherein the reactor chamber has a volume of about 1 L or less.
27. The system of sentence 26, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 0.1 mL/min over a period of at least about 1 day.
28. The system of any one of sentences 26-27, wherein the system is configured to produce at least about 10 mg of IFN per day.
29. The system of any one of sentences 1-26, wherein the reactor chamber has a volume of about 1 L to about 10 L.
30. The system of sentence 29, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 0.5 mL/min over a period of at least about 1 day.
31. The system of any one of sentences 29-30, wherein the system is configured to produce at least about 50 mg of IFN per day.
32. The system of any one of sentences 1-31, wherein the reactor chamber has a volume of about 10 L to about 50 L.
33. The system of sentence 32, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 5 mL/min over a period of at least about 1 day.
34. The system of any one of sentences 32-33, wherein the system is configured to produce at least about 500 mg of IFN per day.
35. A method of producing interferon-α2b (IFN), comprising:
supplying at least one cell culture medium to a bioreactor; producing, within the bioreactor, a suspension comprising the at least one cell culture medium and at least a first type of biological cells expressing interferon-α2b;
causing at least a portion of the suspension to flow through at least one filter to produce at least one filtrate lean in the first type of biological cells, wherein the at least one filtrate comprises interferon-α2b;
flowing the at least one filtrate through a purification module to produce a purified filtrate, wherein producing the purified filtrate comprises, flowing the at least one filtrate through a first column comprising a multimodal cation exchange resin;
collecting one or more first fractions comprising interferon-α2b from an outflow of the first column;
flowing the one or more first fractions through a second column comprising a flow-through resin;
collecting one or more second fractions comprising interferon-α2b from an outflow of the second column;
flowing the one or more second fractions through a third column comprising an anion exchange resin; and
collecting one or more third fractions comprising interferon-α2b from an outflow of the third column.
36. The method of sentence 35, wherein the bioreactor is a perfusion bioreactor.
37. The method of sentence 36, wherein at least one feed stream comprising the at least one cell culture medium is continuously supplied to the perfusion bioreactor at a first flow rate over a period of at least about 1 day; the at least one filter is fluidically connected to the bioreactor and the at least one filtrate is an at least one first filtrate stream; and the purified filtrate is a purified filtrate stream flowing at a second flow rate, wherein the purified filtrate stream comprises the one or more third fractions.
38. The method of any one of sentences 35-37, further comprising, prior to supplying the at least one cell culture medium to the bioreactor, supplying a growth cell culture medium to the bioreactor; incubating the first type of biological cells in the growth cell culture medium for a period of at least about 1 day; and at least partially removing the growth cell culture medium from the bioreactor.
39. The method of any one of sentences 35-38, wherein the at least one filter comprises at least one filter probe at least partially submerged in the suspension in the bioreactor.
40. The method of any one of sentences 35-39, further comprising adjusting the pH of the at least one filtrate to produce a pH-adjusted filtrate.
41. The method of sentence 40, wherein the pH-adjusted filtrate has a pH of about 5.0.
42. The method of any one of sentences 40-41, wherein the pH is adjusted in a pH adjustment module that is fluidically connected to the at least one filter.
43. The method of any one of sentences 35-42, wherein the one or more first fractions are lean in a first type of impurity relative to the at least one filtrate.
44. The method of any one of sentences 35-43, wherein the multimodal cation exchange resin comprises a Capto MMC ImpRes resin.
45. The method of any one of sentences 35-44, wherein the one or more second fractions are lean in a second type of impurity relative to the first fractions.
46. The method of any one of sentences 35-45, wherein the flow-through resin comprises a Q Sepharose HP resin.
47. The method of any one of sentences 35-46, wherein the one or more third fractions are lean in a third type of impurity relative to the second fractions.
48. The method of any one of sentences 35-47, wherein the anion exchange resin comprises a Capto Adhere resin.
49. The method of any one of sentences 35-48, wherein the first type of biological cells are yeast cells, filamentous fungal cells, microalgal cells, or diatom cells.
50. The method of sentence 49, wherein the yeast cells are Pichia pastoris cells.
51. The method of any one of sentences 35-50, wherein the at least one cell culture medium comprises chemically defined media comprising a carbon source, chemically defined media comprising an additive, or buffered methanol-complex media (BMMY).
52. The method of any one of sentences 35-51, further comprising flowing the purified filtrate through a formulation module configured to produce a formulated product stream.
53. The method of sentence 52, wherein flowing the purified filtrate through the formulation module comprises flowing the purified filtrate through a tangential flow filtration device.
54. The method of any one of sentences 52-53, wherein flowing the purified filtrate through the formulation module comprises flowing the purified filtrate stream through a viral filtration unit, wherein the formulated product stream is lean in one or more viruses relative to the purified filtrate stream.
55. The method of any one of sentences 52-54, wherein flowing the purified filtrate through the formulation module comprises depositing one or more portions of the purified filtrate stream into one or more containers.
56. The method of sentence 55, wherein the one or more containers are aseptic and/or sterile containers.
57. The method of any one of sentences 55-56, wherein the one or more containers comprise one or more bags, vials, syringes, and/or bottles.
58. The method of any one of sentences 35-57, wherein the purified filtrate and/or the formulated product stream comprise interferon-α2b having a purity of at least about 77%.
59. The method of any one of sentences 35-58, wherein the purified filtrate and/or the formulated product stream have a DNA concentration of about 0.51 ng/(mg IFN) or less.
60. The method of any one of sentences 35-59, wherein the purified filtrate and/or the formulated product stream have an aggregate content of about 0.5% or less.
61. The method of any one of sentences 35-60, wherein the bioreactor comprises a reactor chamber having a volume of about 1 L or less.
62. The method of sentence 61, wherein the first flow rate and/or the second flow rate are maintained at about 0.1 mL/min or more over a period of about 1 day or more.
63. The method of any one of sentences 61-62, wherein at least about 10 mg of IFN is produced in about 1 day or less.
64. The method of any one of sentences 35-60, wherein the bioreactor comprises a reactor chamber having a volume of about 1 L to about 10 L.
65. The method of sentence 64, wherein the first flow rate and/or the second flow rate are maintained at about 0.5 mL/min or more over a period of about 1 day or more.
66. The method of any one of sentences 64-65, wherein at least about 50 mg of IFN is produced in about 1 day or less.
67. The method of any one of sentences 35-60, wherein the bioreactor comprises a reactor chamber having a volume of about 10 L to about 50 L.
68. The method of sentence 67, wherein the first flow rate and/or the second flow rate are maintained at about 5 mL/min or more over a period of about 1 day or more.
69. The method of any one of sentences 67-68, wherein at least about 500 mg of IFN is produced in about 1 day or less.
70. The method of any one of sentences 52-69, wherein flowing the purified filtrate through the formulation module comprises flowing the purified filtrate stream through a dilution adjustment unit.
71. The method of sentence 70, wherein flowing the purified filtrate stream through the dilution adjustment unit comprises adding a diluent to the purified filtrate stream.
72. The system of any one of sentences 17-34, wherein the formulation module comprises a dilution adjustment unit.
73. The biomanufacturing system of any preceding sentence, further comprising a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
74. The method of any preceding sentence, further comprising monitoring one or more steps of the method using a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
75. The method of any preceding sentence, further comprising implementing one or more corrective action based on information derived from a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
A sixth exemplary embodiment is generally directed to the following:
1. A system for producing human growth hormone, comprising:
a bioreactor, wherein the bioreactor comprises a reaction chamber containing a suspension comprising at least one cell culture medium and at least a first type of biological cells configured to express human growth hormone;
at least one filter, wherein the at least one filter is configured to receive an output of the bioreactor and produce at least one filtrate lean in the first type of biological cells relative to the suspension, wherein the at least one filtrate comprises human growth hormone; and
a purification module, wherein the purification module is configured to remove at least a first type of impurity and a second type of impurity from the at least one filtrate to produce a purified filtrate, wherein the purification module comprises:
a first column comprising a multimodal cation exchange resin; and a second column comprising an anion exchange resin.
2. The system of sentence 1, wherein the bioreactor is a perfusion bioreactor.
3. The system of sentence 2, wherein the bioreactor is configured to receive at least one feed stream comprising the at least one cell culture medium the at least one filter is fluidically connected to the bioreactor and the at least one filtrate is an at least one filtrate stream; the purification module is fluidically connected to the at least one filter and the purified filtrate is a purified filtrate stream; and the system is configured to be continuously operated.
4. The system of any one of sentences 1-3, wherein the at least one filter comprises at least one filter probe at least partially submerged in the suspension in the bioreactor.
5. The system of any one of sentences 1-4, wherein the system further comprises a pH adjustment module configured to increase or decrease the pH of the at least one filtrate to produce a pH-adjusted filtrate.
6. The system of sentence 5, wherein the pH adjustment module is fluidically connected to the bioreactor, the at least one filter, and/or the purification module.
7. The system of any one of sentences 5-6, wherein the pH-adjusted filtrate has a pH of about 5.0.
8. The system of any one of sentences 1-7, wherein the first column is configured to remove at least the first type of impurity from the at least one filtrate to produce a first partitioned filtrate lean in the first type of impurity relative to the at least one filtrate, wherein the first partitioned filtrate comprises human growth hormone.
9. The system of any one of sentences 1-8, wherein the multimodal cation exchange resin comprises a Capto MMC resin.
10. The system of any one of sentences 1-9, wherein the second column is configured to remove at least the second type of impurity from the first partitioned filtrate to produce a second partitioned filtrate lean in the second type of impurity relative to the first partitioned filtrate, wherein the second partitioned filtrate comprises human growth hormone.
11. The system of any one of sentences 1-10, wherein the anion exchange resin comprises a HyperCel STAR AX resin.
12. The system of any one of sentences 1-11, further comprising a third column comprising a hydrophobic charge induction chromatography (HCIC) resin.
13. The system of sentence 12, wherein the third column is configured to remove at least a third type of impurity from the second partitioned filtrate to produce a third partitioned filtrate lean in the third type of impurity relative to the second partitioned filtrate, wherein the third partitioned filtrate comprises human growth hormone.
14. The system of any one of sentences 12-13, wherein the HCIC resin comprises an MEP HyperCel resin.
15. The system of any one of sentences 1-14, wherein the first type of biological cells are yeast cells, filamentous fungal cells, microalgal cells, or diatom cells.
16. The system of sentence 15, wherein the yeast cells are Pichia pastoris cells.
17. The system of any one of sentences 1-16, wherein the at least one cell culture medium comprises chemically defined media comprising a carbon source, chemically defined media comprising an additive, or buffered methanol-complex media (BMMY).
18. The system of any one of sentences 1-17, further comprising a formulation module fluidically connected to the purification module, wherein the formulation module is configured to produce a formulated pharmaceutical product stream.
19. The system of sentence 18, wherein the formulation module comprises a filtration unit.
20. The system of sentence 19, wherein the filtration unit comprises a tangential flow filtration device.
21. The system of any one of sentences 18-20, wherein the formulation module comprises a viral filtration unit.
22. The system of any one of sentences 18-21, wherein the formulation module comprises a packaging unit.
23. The biomanufacturing system of sentence 22, wherein the packaging unit is configured to package one or more doses of the at least one pharmaceutical product into one or more bags, one or more vials, one or more syringes, and/or one or more bottles.
24. The system of any one of sentences 1-23, wherein the purified filtrate and/or formulated pharmaceutical product stream has a host cell protein concentration of about 50 ng/(mg hGH) or less.
25. The system of any one of sentences 1-24, wherein the purified filtrate and/or formulated pharmaceutical product stream has a DNA concentration of about 100 ng/(mg hGH) or less.
26. The system of any one of sentences 1-25, wherein the purified filtrate and/or formulated pharmaceutical product stream has an aggregate content of about 1% or less.
27. The system of any of sentences 1-26, wherein the reactor chamber has a volume of about 1 L or less.
28. The method of sentence 27, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 0.1 mL/min over a period of at least about 1 day.
29. The method of any one of sentences 27-28, wherein the system is configured to produce at least about 10 mg of hGH per day.
30. The system of any one of sentences 1-26, wherein the reactor chamber has a volume of about 1 L to about 10 L.
31. The system of sentence 30, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 0.5 mL/min over a period of at least about 1 day.
32. The system of any one of sentences 30-31, wherein the system is configured to produce at least about 50 mg of hGH per day.
33. The system of any one of sentences 1-26, wherein the reactor chamber has a volume of about 10 L to about 50 L.
34. The system of sentence 33, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 5 mL/min over a period of at least about 1 day.
35. The system of any one of sentences 33-34, wherein the system is configured to produce at least about 500 mg of hGH per day.
36. A method of producing human growth hormone, comprising:
supplying at least one cell culture medium to a bioreactor;
producing, within the bioreactor, a suspension comprising the at least one cell culture medium and at least a first type of biological cells expressing human growth hormone;
causing at least a portion of the suspension to flow through at least one filter to produce at least one filtrate lean in the first type of biological cells, wherein the at least one filtrate comprises human growth hormone;
flowing the at least one filtrate through a purification module to produce a purified filtrate, wherein producing the purified filtrate comprises flowing the at least one filtrate through a first column comprising a multimodal cation exchange resin;
collecting one or more first fractions comprising human growth hormone from an outflow of the first column;
flowing the one or more first fractions through a second column comprising an anion exchange resin; and
collecting one or more second fractions comprising human growth hormone from an outflow of the second column.
37. The method of sentence 36, wherein the bioreactor is a perfusion bioreactor.
38. The method of sentence 37, wherein at least one feed stream comprising the at least one cell culture medium is continuously supplied to the perfusion bioreactor at a first flow rate over a period of at least about 1 day; the at least one filter is fluidically connected to the bioreactor and the at least one filtrate is an at least one filtrate stream; and the purified filtrate is a purified filtrate stream flowing at a second flow rate.
39. The method of any one of sentences 36-38, further comprising, prior to supplying the at least one cell culture medium to the bioreactor, supplying a growth cell culture medium to the bioreactor; incubating the first type of biological cells in the growth cell culture medium for a period of at least about 1 day; and at least partially removing the growth cell culture medium from the bioreactor.
40. The method of any one of sentences 36-39, wherein the at least one filter comprises at least one filter probe at least partially submerged in the suspension in the bioreactor.
41. The method of any one of sentences 36-40, further comprising adjusting the pH of the at least one filtrate to produce a pH-adjusted filtrate.
42. The method of sentence 41, wherein the pH-adjusted filtrate has a pH of about 5.0.
43. The method of any one of sentences 41-42, wherein the pH is adjusted in a pH adjustment module that is fluidically connected to the at least one filter.
44. The method of any one of sentences 36-43, wherein the one or more first fractions are lean in a first type of impurity relative to the at least one filtrate.
45. The method of any one of sentences 36-44, wherein the multimodal cation exchange resin comprises a Capto MMC resin.
46. The method of any one of sentences 36-45, The method of sentence 46, further comprising flowing a first mobile phase material through the first column prior to flowing the at least one filtrate through the first column, wherein the first mobile phase material is configured to promote binding of human growth hormone to the multimodal cation exchange resin.
48. The method of sentence 47, wherein the first mobile phase material has a pH of about 5.0.
49. The method of any one of sentences 46-48, further comprising flowing a second mobile phase material through the first column after flowing the at least one filtrate through the first column.
50. The method of sentence 49, wherein the second mobile phase material has a pH of about 5.0 and a sodium chloride concentration of about 500 mM.
51. The method of any one of sentences 46-50, further comprising flowing a third mobile phase material through the first column after flowing the second mobile phase material through the first column, wherein the third mobile phase material is configured to elute human growth hormone from the first column.
52. The method of sentence 51, wherein the third mobile phase material has a pH of about 6.0 and a sodium chloride concentration of about 100 mM.
53. The method of any one of sentences 36-52, wherein the one or more second fractions are lean in a second type of impurity relative to the first fractions.
54. The method of any one of sentences 36-53, wherein the anion exchange resin comprises a HyperCel STAR AX resin.
55. The method of any one of sentences 36-54, wherein the second column is operated in flow-through mode.
56. The method of any one of sentences 36-55, further comprising flowing the one or more second fractions through a third column comprising an HCIC resin.
57. The method of sentence 56, further comprising collecting one or more third fractions comprising human growth hormone from an outflow of the third column.
58. The method of sentence 57, wherein the one or more third fractions are lean in a third type of impurity relative to the second fractions.
59. The method of any one of sentences 56-58, wherein the HCIC resin comprises an MEP HyperCel resin.
60. The method of any one of sentences 56-59, wherein the third column is operated in bind-elute mode.
61. The method of sentence 60, further comprising flowing a first mobile phase material through the third column prior to flowing the second fractions through the first column, wherein the first mobile phase material is configured to promote binding of human growth hormone to the HCIC resin.
62. The method of sentence 61, wherein the first mobile phase material has a pH of about 6.0 and a sodium chloride concentration of about 100 mM.
63. The method of any one of sentences 60-62, further comprising flowing a second mobile phase material through the third column after flowing the second fractions through the third column.
64. The method of sentence 63, wherein the second mobile phase material has a pH of about 5.1 and a sodium chloride concentration less than about 100 mM.
65. The method of any one of sentences 60-64, further comprising flowing a third mobile phase material through the third column after flowing the second mobile phase material through the third column, wherein the third mobile phase material is configured to elute human growth hormone from the third column.
66. The method of sentence 65, wherein the third mobile phase material has a pH of about 3.0 and a sodium chloride concentration less than about 100 mM.
67. The method of any one of sentences 36-66, wherein the first type of biological cells are yeast cells, filamentous fungal cells, microalgal cells, or diatom cells.
68. The method of sentence 67, wherein the yeast cells are Pichia pastoris cells.
69. The method of any one of sentences 36-68, wherein the at least one cell culture medium comprises chemically defined media comprising a carbon source, chemically defined media comprising an additive, or buffered methanol-complex media (BMMY).
70. The method of any one of sentences 36-69, further comprising flowing the purified filtrate through a formulation module configured to produce a formulated pharmaceutical product stream.
71. The method of sentence 70, wherein flowing the purified filtrate through the formulation module comprises flowing the purified filtrate through a tangential flow filtration device.
72. The method of any one of sentences 70-71, wherein flowing the purified filtrate through the formulation module comprises flowing the purified filtrate stream through a viral filtration unit, wherein the formulated pharmaceutical product stream is lean in one or more viruses relative to the purified filtrate stream.
73. The method of any one of sentences 70-72, wherein flowing the purified filtrate through the formulation module comprises depositing one or more portions of the purified filtrate stream into one or more containers.
74. The method of sentence 73, wherein the one or more containers are aseptic and/or sterile containers.
75. The method of any one of sentences 73-74, wherein the one or more containers comprise one or more bags, vials, syringes, and/or bottles.
76. The method of any one of sentences 36-75, wherein the purified filtrate and/or the formulated pharmaceutical stream have a host cell protein concentration of about 50 ng/(mg hGH) or less.
77. The method of any one of sentences 36-76, wherein the purified filtrate and/or the formulated pharmaceutical stream have a DNA concentration of about 100 ng/(mg hGH) or less.
78. The method of any one of sentences 36-77, wherein the purified filtrate and/or the formulated pharmaceutical stream have an aggregate content of about 1% or less.
79. The method of any one of sentences 36-78, wherein the bioreactor comprises a reactor chamber having a volume of about 1 L or less.
80. The method of sentence 79, wherein the first flow rate and/or the second flow rate are maintained at about 0.1 mL/min or more over a period of about 1 day or more.
81. The method of any one of sentences 79-80, wherein at least about 10 mg of hGH is produced in about 1 day or less.
82. The method of any one of sentences 36-78, wherein the bioreactor comprises a reactor chamber having a volume of about 1 L to about 10 L.
83. The method of sentence 82, wherein the first flow rate and/or the second flow rate are maintained at about 0.5 mL/min or more over a period of about 1 day or more.
84. The method of any one of sentences 82-83, wherein at least about 50 mg of hGH is produced in about 1 day or less.
85. The method of any one of sentences 36-78, wherein the bioreactor comprises a reactor chamber having a volume of about 10 L to about 50 L.
86. The method of sentence 85, wherein the first flow rate and/or the second flow rate are maintained at about 5 mL/min or more over a period of about 1 day or more.
87. The method of any one of sentences 85-86, wherein at least about 500 mg of hGH is produced in about 1 day or less.
88. The biomanufacturing system of any preceding sentence, further comprising a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
89. The method of any preceding sentence, further comprising monitoring one or more steps of the method using a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
90. The method of any preceding sentence, further comprising implementing one or more corrective action based on information derived from a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
A seventh exemplary embodiment is generally directed to the following:
1. A system for producing a single-domain antibody, comprising, a bioreactor, wherein the bioreactor comprises a reaction chamber containing a suspension comprising at least one cell culture medium and at least a first type of biological cells configured to express a single-domain antibody; at least one filter, wherein the at least one filter is configured to receive an output of the bioreactor and produce at least one filtrate lean in the first type of biological cells relative to the suspension, wherein the at least one filtrate comprises the single-domain antibody; and a purification module, wherein the purification module is configured to remove at least a first type of impurity and a second type of impurity from the at least one filtrate to produce a purified filtrate, wherein the purification module comprises, a first column comprising a multimodal cation exchange resin; and a second column comprising an anion exchange resin.
2. The system of sentence 1, wherein the bioreactor is a perfusion bioreactor.
3. The system of sentence 2, wherein: the bioreactor is configured to receive at least one feed stream comprising the at least one cell culture medium; the at least one filter is fluidically connected to the bioreactor and the at least one filtrate is an at least one filtrate stream; the purification module is fluidically connected to the at least one filter and the purified filtrate is a purified filtrate stream; and the system is configured to be continuously operated.
4. The system of any one of sentences 1-3, wherein the at least one filter comprises at least one filter probe at least partially submerged in the suspension in the bioreactor.
5. The system of any one of sentences 1-4, wherein the system further comprises a pH adjustment module configured to increase or decrease the pH of the at least one filtrate to produce a pH-adjusted filtrate.
6. The system of sentence 5, wherein the pH adjustment module is fluidically connected to the bioreactor, the at least one filter, and/or the purification module.
7. The system of any one of sentences 5-6, wherein the pH-adjusted filtrate has a pH of about 5.0.
8. The system of any one of sentences 1-7, wherein the first column is configured to remove at least the first type of impurity from the at least one filtrate to produce a first partitioned filtrate lean in the first type of impurity relative to the at least one filtrate, wherein the first partitioned filtrate comprises human growth hormone.
9. The system of any one of sentences 1-8, wherein the multimodal cation exchange resin comprises a CMM HyperCel resin.
10. The system of any one of sentences 1-8, wherein the single-domain antibody comprises a single variable domain.
11. The system of any one of sentences 1-10, wherein the second column is configured to remove at least the second type of impurity from the first partitioned filtrate to produce a second partitioned filtrate lean in the second type of impurity relative to the first partitioned filtrate, wherein the second partitioned filtrate comprises the single-domain antibody.
12. The system of any one of sentences 1-11, wherein the anion exchange resin comprises a HyperCel STAR AX resin.
13. The system of any one of sentences 1-11, wherein the anion exchange resin comprises a Capto Adhere resin.
14. The system of any one of sentences 1-11, wherein the single-domain antibody is a camelid single-domain antibody.
15. The system of any one of sentences 1-14, wherein the first type of biological cells are yeast cells, filamentous fungal cells, microalgal cells, or diatom cells.
16. The system of sentence 15, wherein the yeast cells are Pichia pastoris cells.
17. The system of any one of sentences 1-16, wherein the at least one cell culture medium comprises chemically defined media comprising a carbon source, chemically defined media comprising an additive, or buffered methanol-complex media (BMMY).
18. The system of any one of sentences 1-17, further comprising a formulation module fluidically connected to the purification module, wherein the formulation module is configured to produce a formulated pharmaceutical product stream.
19. The system of sentence 18, wherein the formulation module comprises a filtration unit.
20. The system of sentence 19, wherein the filtration unit comprises a tangential flow filtration device.
21. The system of any one of sentences 18-20, wherein the formulation module comprises a viral filtration unit.
22. The system of any one of sentences 18-21, wherein the formulation module comprises a packaging unit.
23. The biomanufacturing system of sentence 22, wherein the packaging unit is configured to package one or more doses of the at least one pharmaceutical product into one or more bags, one or more vials, one or more syringes, and/or one or more bottles.
24. The system of any one of sentences 1-23, wherein the purified filtrate and/or formulated pharmaceutical product stream has a host cell protein concentration of about 50 ng/(mg single-domain antibody) or less.
25. The system of any one of sentences 1-24, wherein the purified filtrate and/or formulated pharmaceutical product stream has a DNA concentration of about 100 ng/(mg single-domain antibody) or less.
26. The system of any one of sentences 1-25, wherein the purified filtrate and/or formulated pharmaceutical product stream has an aggregate content of about 1% or less.
27. The system of any of sentences 1-26, wherein the reactor chamber has a volume of about 1 L or less.
28. The method of sentence 27, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 0.1 mL/min over a period of at least about 1 day.
29. The method of any one of sentences 27-28, wherein the system is configured to produce at least about 10 mg of single-domain antibody per day.
30. The system of any one of sentences 1-26, wherein the reactor chamber has a volume of about 1 L to about 10 L.
31. The system of sentence 30, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 0.5 mL/min over a period of at least about 1 day.
32. The system of any one of sentences 30-31, wherein the system is configured to produce at least about 50 mg of single-domain antibody per day.
33. The system of any one of sentences 1-26, wherein the reactor chamber has a volume of about 10 L to about 50 L.
34. The system of sentence 33, wherein the at least one feed stream and the purified filtrate stream each have a flow rate of at least about 5 mL/min over a period of at least about 1 day.
35. The system of any one of sentences 33-34, wherein the system is configured to produce at least about 500 mg of single-domain antibody per day.
36. A method of producing a single-domain antibody, comprising:
supplying at least one cell culture medium to a bioreactor;
producing, within the bioreactor, a suspension comprising the at least one cell culture medium and at least a first type of biological cells expressing a single-domain antibody;
causing at least a portion of the suspension to flow through at least one filter to produce at least one filtrate lean in the first type of biological cells, wherein the at least one filtrate comprises the single-domain antibody; and
flowing the at least one filtrate through a purification module to produce a purified filtrate, wherein producing the purified filtrate comprises:
flowing the at least one filtrate through a first column comprising a multimodal cation exchange resin;
collecting one or more first fractions comprising the single-domain antibody from an outflow of the first column;
flowing the one or more first fractions through a second column comprising an anion exchange resin; and
collecting one or more second fractions comprising the single-domain antibody from an outflow of the second column.
37. The method of sentence 36, wherein the bioreactor is a perfusion bioreactor.
38. The method of sentence 37, wherein at least one feed stream comprising the at least one cell culture medium is continuously supplied to the perfusion bioreactor at a first flow rate over a period of at least about 1 day;
the at least one filter is fluidically connected to the bioreactor and the at least one filtrate is an at least one filtrate stream; and the purified filtrate is a purified filtrate stream flowing at a second flow rate.
39. The method of any one of sentences 36-38, further comprising, prior to supplying the at least one cell culture medium to the bioreactor, supplying a growth cell culture medium to the bioreactor; incubating the first type of biological cells in the growth cell culture medium for a period of at least about 1 day; and at least partially removing the growth cell culture medium from the bioreactor.
40. The method of any one of sentences 36-39, wherein the at least one filter comprises at least one filter probe at least partially submerged in the suspension in the bioreactor.
41. The method of any one of sentences 36-40, further comprising adjusting the pH of the at least one filtrate to produce a pH-adjusted filtrate.
42. The method of sentence 41, wherein the pH-adjusted filtrate has a pH of about 5.0.
43. The method of any one of sentences 41-42, wherein the pH is adjusted in a pH adjustment module that is fluidically connected to the at least one filter.
44. The method of any one of sentences 36-43, wherein the one or more first fractions are lean in a first type of impurity relative to the at least one filtrate.
45. The method of any one of sentences 36-44, wherein the multimodal cation exchange resin comprises a CMM HyperCel resin.
46. The method of any one of sentences 36-44, wherein the single-domain antibody comprises a single variable domain.
47. The method of any one of sentences 36-46, wherein the first column is operated in bind-elute mode.
48. The method of sentence 47, further comprising flowing a first mobile phase material through the first column prior to flowing the at least one filtrate through the first column, wherein the first mobile phase material is configured to promote binding of the single-domain antibody to the multimodal cation exchange resin.
49. The method of sentence 48, wherein the first mobile phase material has a pH of about 5.0.
50. The method of any one of sentences 47-49, further comprising flowing a second mobile phase material through the first column after flowing the at least one filtrate through the first column.
51. The method of sentence 50, wherein the second mobile phase material has a pH of about 6.0.
52. The method of any one of sentences 47-51, further comprising flowing a third mobile phase material through the first column after flowing the second mobile phase material through the first column, wherein the third mobile phase material is configured to elute the single-domain antibody from the first column.
53. The method of sentence 52, wherein the third mobile phase material has a pH of about 7.0 and a sodium chloride concentration of about 100 mM.
54. The method of any one of sentences 36-53, wherein the one or more second fractions are lean in a second type of impurity relative to the first fractions.
55. The method of any one of sentences 36-54, wherein the anion exchange resin comprises a HyperCel STAR AX resin.
56. The method of any one of sentences 36-54, wherein the anion exchange resin comprises a Capto Adhere resin.
57. The method of any one of sentences 36-56, wherein the second column is operated in flow-through mode.
58. The system of any one of sentences 36-57, wherein the single-domain antibody is a camelid single-domain antibody.
59. The method of any one of sentences 36-58, wherein the first type of biological cells are yeast cells, filamentous fungal cells, microalgal cells, or diatom cells.
60. The method of sentence 59, wherein the yeast cells are Pichia pastoris cells.
61. The method of any one of sentences 36-60, wherein the at least one cell culture medium comprises chemically defined media comprising a carbon source, chemically defined media comprising an additive, or buffered methanol-complex media (BMMY).
62. The method of any one of sentences 36-61, further comprising flowing the purified filtrate through a formulation module configured to produce a formulated pharmaceutical product stream.
63. The method of sentence 62, wherein flowing the purified filtrate through the formulation module comprises flowing the purified filtrate through a tangential flow filtration device.
64. The method of any one of sentences 62-63, wherein flowing the purified filtrate through the formulation module comprises flowing the purified filtrate stream through a viral filtration unit, wherein the formulated pharmaceutical product stream is lean in one or more viruses relative to the purified filtrate stream.
65. The method of any one of sentences 62-64, wherein flowing the purified filtrate through the formulation module comprises depositing one or more portions of the purified filtrate stream into one or more containers.
66. The method of sentence 65, wherein the one or more containers are aseptic and/or sterile containers.
67. The method of any one of sentences 65-66, wherein the one or more containers comprise one or more bags, vials, syringes, and/or bottles.
68. The method of any one of sentences 36-67, wherein the purified filtrate and/or the formulated pharmaceutical stream have a host cell protein concentration of about 50 ng/(mg single-domain antibody) or less.
69. The method of any one of sentences 36-68, wherein the purified filtrate and/or the formulated pharmaceutical stream have a DNA concentration of about 100 ng/(mg single-domain antibody) or less.
70. The method of any one of sentences 36-69, wherein the purified filtrate and/or the formulated pharmaceutical stream have an aggregate content of about 1% or less.
71. The method of any one of sentences 36-70, wherein the bioreactor comprises a reactor chamber having a volume of about 1 L or less.
72. The method of sentence 71, wherein the first flow rate and/or the second flow rate are maintained at about 0.1 mL/min or more over a period of about 1 day or more.
73. The method of any one of sentences 71-72, wherein at least about 10 mg of single-domain antibody is produced in about 1 day or less.
74. The method of any one of sentences 36-70, wherein the bioreactor comprises a reactor chamber having a volume of about 1 L to about 10 L.
75. The method of sentence 74, wherein the first flow rate and/or the second flow rate are maintained at about 0.5 mL/min or more over a period of about 1 day or more.
76. The method of any one of sentences 74-75, wherein at least about 50 mg of single-domain antibody is produced in about 1 day or less.
77. The method of any one of sentences 36-70, wherein the bioreactor comprises a reactor chamber having a volume of about 10 L to about 50 L.
78. The method of sentence 77, wherein the first flow rate and/or the second flow rate are maintained at about 5 mL/min or more over a period of about 1 day or more.
79. The method of any one of sentences 77-78, wherein at least about 500 mg of single-domain antibody is produced in about 1 day or less.
80. The biomanufacturing system of any preceding sentence, further comprising a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
81. The method of any preceding sentence, further comprising monitoring one or more steps of the method using a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
82. The method of any preceding sentence, further comprising implementing one or more corrective action based on information derived from a process and monitoring control system, optionally wherein the process and monitoring control system comprises one or more optical sensors, optionally wherein the one or more optical sensors comprises one or more cameras.
Precision medicine holds the promise of improved treatments for small, well-defined populations of patients or even individuals. This model, however, presents a significant challenge for manufacturing protein biopharmaceuticals, which currently relies on centralized, large-scale facilities to supply global markets. One potential solution for agile and timely delivery of precision biologics to patients is small-scale, on-demand manufacturing within local pharmacies, hospitals, and healthcare clinics. An automated bench-top manufacturing system for end-to-end production of 100's to 1,000's of doses of high-quality, formulated biologic drugs in about 3 days without human intervention was produced. This system is paired with a complementary, integrated approach for accelerating process development of new biologics from sequence to purified drug in as few as 12 weeks. The use of this system with different well-characterized biologic drugs and demonstrate that the biologics have similar identity, purity, and potency as reference products is described below. Process simplification and rapid manufacturing of small volumes of formulated high-quality protein drugs using functionally closed, fully-integrated production systems could provide unique capabilities to supply medicines to patients when and where they need them.
Biologic medicines are an important class of drugs that include recombinant proteins such as cytokines, hormones, replacement enzymes, blood factors, and antibodies. Their therapeutic use in oncology and rare diseases increasingly relies on precise molecular profiles that define diseases of certain, often small, cohorts of patients. This precision in turn reduces the total supply of drug required, but expands the future plurality of medicines needed. The current manufacturing strategy for biologics, however, relies on large-volume, centralized facilities to provide economical production of a few products that support the overall costs of drug development.
Intensification of manufacturing processes with mammalian cells through continuous operations has established alternative cost-effective approaches that can reduce the size of facilities and equipment while retaining large volumetric production of a single drug substance. New technologies to manufacture many different high-quality biopharmaceuticals in small quantities with efficiency and agility are needed to make precision biologic medicines both available and economically feasible. A bench-scale, integrated, and automated manufacturing system that produces, purifies, and formulates high-quality recombinant protein drugs in less than 80 hours without human intervention is described. Additionally, complementary, integrated approach for accelerated process development to make and purify new products on the system in as few as 12 weeks is described.
To accelerate the timing for development and production, these examples utilized as a host the yeast Pichia pastoris, which can grow quickly to high cell densities and secrete recombinant proteins. Other advantages of P. pastoris include low levels of secreted host-cell proteins, little to no risk of viral contamination, documented expression of many classes of proteins including FDA-approved therapeutics, and the capability of human-like post-translational modifications. This host allowed rapid cycles of process development and simplified the architecture of the production system.
The benchtop system described in the certain examples included fluidically connected elements for fermentation, multi-stage chromatography, and ultrafiltration/diafiltration as well as integrated sensors and system controllers for programmed operations.
Fractionation Experiments for Cell Culture Fluid Spiked with G-CSF
Formulated drug substance Filgrastim, similar to naturally occurring granulocyte-colony stimulating factor (G-CSF) and considered for the purposes of these experiments to be equivalent to G-CSF and hereafter referred to interchangeably, was directly spiked into null strain Pichia pastoris cell culture fluid (CCF) at a concentration of 1.18 mg/mL and the resulting solution was titrated to pH 3.0, 5.0, 6.0, or 7.0 as needed from a starting pH of approximately 6.5. Titrated CCF was diluted with deionized (DI) water to a total CCF dilution of three times (3×) and filtered using a filter having a pore size of 0.2 μm. CCF was titrated to lower pH using 100 mM citric acid. CCF was titrated to higher pH using 100 mM Tris base because, by contrast, the addition of strong bases such as sodium hydroxide induced the irreversible local precipitation of some CCF components. CCF was prepared no greater than 24 hours in advance of its use.
Fractionation experiments on the null Pichia CCF spiked with G-CSF were carried out using an ÄKTA Explorer 10 system (GE Healthcare) equipped with a Frac-950 fraction collector and a P-960 sample pump and controlled by Unicorn 5.1 software. The resins listed in Table 3 were packed in GE Tricorn 5/20 columns at approximately 0.5 mL column volumes (CVs), and gradient elution experiments were performed using the gradient conditions listed in Table 3.
Table 3 lists the chromatographic resins used in product and impurity characterization screens and the corresponding gradients performed on them. In Table 3, “salt” denotes salt gradients (0-1.5 M NaCl) at pH 5.0, 6.0, and 7.0, while pH gradients were run from pH 3.0 to 7.0 or 7.0 to 3.0 using a 20 mM citrate buffer. This set of resins was selected for the ability to bind material at elevated conductivities and for maximum operational flexibility across a small set of resins.
Columns were equilibrated with 10 CV of buffer A, loaded with 120 CV of conditioned null Pichia CCF spiked with G-CSF, washed with 20 CV of buffer A, eluted with a 40 CV gradient to buffer B, and washed with 10 CV of buffer B. Table 4 lists the compositions of the buffers used, each of which contained 0.02% azide.
All solvents used 20 mM sodium citrate as a buffer species. Multimodal cation exchange resins (MMC) were stripped with 20 CV of 0.1 M sodium hydroxide. Multimodal anion exchange resins (MMA), hydrophobic charge-induction chromatography resins (HCIC), and salt-tolerant anion exchange resins (AEX) were stripped with 20 CV of 0.1 M citric acid.
For null Pichia CCF spiked with G-CSF, the flow rate was 0.5 CV/min during the load stage and 1 CV/min for all other stages. CCF flow-through was collected in two 60 CV fractions. Gradient elutions were collected in 2 CV fractions, which were combined into 4 CV fractions for analysis. The first 10 CVs of the strip were collected for analysis.
Reversed phase-ultra high pressure liquid chromatography (RP-UPLC) analysis of samples from the Pichia CCF partitioning fractionations was performed on a Waters Acquity ultra high pressure liquid chromatography (UPLC) H-class system equipped with a photodiode array (PDA) detector and controlled by Empower 3 software. Samples were run on an Acquity UPLC Protein bridged ethylsiloxane/silica hybrid (BEH) C4 column (300 angstrom, 1.7 μm, 2.1 mm×100 mm) with an Acquity UPLC Protein BEH VanGuard Pre-Column (300 angstrom, 1.7 μm, 2.1 mm×5 mm). Column temperature was set to 60° C. Sample temperature was set to 8° C. Buffer A was 0.1% formic acid in water, and buffer B was 0.1% formic acid in acetonitrile (ACN) (v/v basis). System flow rate was held constant at 0.5 mL/min. The gradient method used was a 1 minute hold at 0% B, followed by a 7 minute linear gradient to 100% with a 2 minute hold prior to re-equilibration. Total method time was 12.5 min. Sample injection volumes were 50 μL. UV absorbance was collected as a wavelength scan from 200 to 400 nm at 2.4 nm resolution and 40 Hz frequency.
RP-UPLC chromatograms were taken as raw data .ARW files at A210, A260, and A280 with 40 Hz resolution. A MATLAB script was written to convert all raw RP-UPLC data from .ARW files to a single .MAT file. Chromatograms corresponding to blank deionized water (DI) injections were used to baseline subtract from RP-UPLC chromatograms. These DI injections were performed each day, and fractionated Pichia CCF RP-UPLC chromatograms were baseline-subtracted using the appropriate DI injection run.
Data reduction was performed by integrating each chromatogram over 0.5 second time intervals. Only data from 1.5 to 10.0 minutes were considered in order to eliminate the effects of t0 baseline disturbances. The process-related impurity retention data set was then stored as a 5-dimensional array with the first dimension corresponding to the wavelength of the data collected, the second dimension corresponding to the resin, the third dimension corresponding to the gradient type, the fourth dimension corresponding to the ÄKTA fraction number, and the fifth dimension corresponding to the integration window from RP-UPLC data.
The downstream process (DSP) generation tool consisted of subroutines for total process generation and characterization, process constraint implementation, process ranking, table generation and graphical process output generation.
Downstream Process Generation: Process Generation with Implementation of Process Constraints
Data from the RP-UPLC regarding host cell proteins (HCP) and G-CSF was loaded into the program. Each resin or step was categorized as bind-elute, explicit flow-through, or implicit flow-through. Explicit flow-through steps were identified as such when the product was experimentally observed to flow through at the selected solution conditions of the load solution for the column. Implicit flow-through steps were identified as such when the load solution for the column was at a condition later in the salt or pH gradient than the condition at which the product eluted. Steps in which the product eluted in the strip were not considered.
The data was used as inputs to generate a list of all candidate 3-step process sequences, of both resin types and operating conditions, which recover the product, wherein each step corresponded to running the Pichia CCF spiked with G-CSF through a column with a resin from the screening process (Table 3) using a pH or salt gradient (Table 4). A number of constraints were implemented to reject undesirable processes. Capture steps were required to operate in bind-elute mode. The elution condition from one column was required to be the load condition for the subsequent column without adjustment to the salt and pH of the elution pool, so as to allow for integrated manufacturing. For sequences containing MEP HyperCel, only pH transitions were considered since salt gradient elution experiments were not performed. HCIC resins were not permitted to be used as a capture step. Each process was only permitted to use a given resin once. Since the first step of each candidate process was bind-elute, the second and third steps were classified as bind-elute, flow-through (explicit or implicit), or non-allowable transitions. The list of processes was further trimmed by imposing the constraint of no more than one (implicit or explicit) flow-through step.
Process ranking for predicted process-related impurity clearance was performed using the following equation, for which lower scores indicated better (more orthogonal) processes:
In Equation 1, . . . , K is the number of UPLC integration fractions; P is the number of purification steps in a given process; F is a set of steps which are flow-through steps; B is a set of steps which are bind-elute steps; ai,j is the area under the RP-UPLC chromatogram for a given wavelength, resin, gradient type, ÄKTA fraction number, and RP-UPLC integration window; RElute is the elution fraction number of the current column (corresponding to a specific pH and salt content); and N is the number of fractions collected in the ÄKTA gradient. Assumptions were made of optimized washing and relatively sharp elution peaks, so that in bind-elute operations, process-related impurities were considered to co-elute with the product in only a single gradient elution fraction. For flow-through steps, impurity clearance was determined using the sum of the RPLC chromatograms of all gradient elution fractions subsequent to the column inlet condition.
By use of the equation, the resins and conditions that offered the highest degree of orthogonal selectivity for process-related impurities were determined. By this method, scores were assigned and each set was rank-ordered (Table 5). Scores were assigned for data collected at a wavelength of 210 nm. This wavelength was chosen because it offered high sensitivity of detection and non-specificity so as to measure as many impurities as possible at a single wavelength. Profiles of process-related impurity removal were generated to visualize step orthogonality for each sequence.
Table 5 shows the top 20 process sequences selected by the process selection tool for the purification of G-CSF. Sequences are presented with their scores calculated by Equation 1 along with a score normalized to that of the top-ranked process. Individual steps are presented in the format of “resin, operating mode/pH, product elution condition”, where an operating pH is given when using a salt gradient elution and “flow-through” is used to indicate that the product elution condition is the same as the load condition.
Once potential sequences were identified using the process generation tool, a decision process was implemented to select the specific potential sequence for process development. This was important since the sequences were generated based on orthogonal selectivity while not including additional important considerations such as binding capacity and yield constraints. One constraint that was used to eliminate potential sequences was that for bind/elute steps, the loading and elution condition should be sufficiently different, because this generally resulted in higher binding capacities. For example, in sequence number 4 (Table 5), for the first step using Toyopearl MX-Trp-650M, the elution salt concentration was 300 mM, which was close to the conductivity of the Pichia CCF load. Thus, this sequence was not selected. This heuristic was employed to select “straw man” processes which were then used to initiate process development as described below.
The development of a process to purify G-CSF from Pichia CCF was initiated using the output from the process selection tool shown in Table 5. The specified purification targets were host cell proteins (HCP) less than 100 ppm and DNA less than limit of detection (LOD) of a Quant-iT™ PicoGreen® dsDNA Assay Kit (ThermoFisher) kit. Sequences 1-4 and 6 were discounted due to the use of Toyopearl MX-Trp-650M as the capture step since the elution salt concentration was 300 mM, which was close to the conductivity of the Pichia CCF load. For G-CSF sequence 5, this particular combination of Capto MMC and CMM HyperCel was highly sensitive to the pH of the Capto MMC elution buffer/CMM HyperCel binding buffer. In order to circumvent this problem, sequence 7 was selected as the starting point for G-CSF process development.
Upon experimental process optimization, the Capto MMC ImpRes and MEP HyperCel steps were developed, and it was found that recovery for Capto Adhere was poor. The next highly ranked process, which contained Capto MMC ImpRes and MEP HyperCel, was selected, only requiring development of the HyperCel STAR AX step.
The final process (shown in
PicoGreen DNA analysis was performed using the Quant-iT™ PicoGreen® dsDNA Assay Kit from ThermoFisher Scientific. To generate a dsDNA calibration curve, samples were prepared from lambda DNA standard included with the kit diluted to concentrations of 500, 100, 50, 25, 10, 5, and 1 pg/μL in TE buffer included with the kit. Pure TE buffer was also included in the calibration as a null concentration point. Samples for analysis were diluted with TE buffer if needed. 20 μL of each sample to be analyzed were added to a Costar black 96-well plate in triplicate. In darkness, PicoGreen reagent was prepared by mixing with TE buffer, and then 200 μL was added to each well for analysis and mixed by pipetting. The plate was then analyzed on a plate reader, which included a 2 minute agitation period to further mix the wells followed by excitation at 480 nm and measurement of emission at 520 nm.
The results presented in Table 6 show that overall product recovery was approximately 80% with total HCP clearance (5.4 logs) and total DNA clearance (greater than 4.1 logs) exceeding the purification targets. The final product concentration was 0.232 mg/mL, and the final aggregate content was 0.60%. For DNA, error bars represent one standard deviation of triplicate measurements. For HCP ELISA, error bars denote the 95% confidence interval.
66 ± 17.9
Chromatographic Process Design for Purification of Human Growth Hormone (hGH)
Null strain Pichia pastoris cell culture fluid (CCF) fractionation experiments were carried out as described in Example 1.
Pure human growth hormone (hGH) was dissolved from lyophilized powder form into pH 3.0 citrate buffer, pH 4.0 citrate buffer, pH 5.0 citrate buffer, pH 6.0 citrate buffer, or pH 7.0 Tris buffer as needed at approximately 0.5 mg/mL and 0.2 μm filtered. The final concentration of hGH in the solution was 640 μg/mL. The hGH solution was prepared less than 24 hours in advance of its use. hGH fractionation experiments were carried out according to nearly the same protocols and using the same chromatographic resins and buffers as the CCF G-CSF fractionation experiments described in Example 1. The difference was that in this case system flow rate was kept constant at 1 CV/min throughout the method. Column load challenge was kept constant at 0.3 mg/mL, and product elution pH or salt concentration was determined by peak maximum at 280 nm. Pure component hGH retention data was obtained directly from the ÄKTA chromatogram, shown in
RP-UPLC analysis of samples from the CCF partitioning fractionations was performed according to the protocol described in Example 1. The process-related impurity retention data set was constructed according to the protocols described in Example 1, and the downstream process (DSP) generation tool described in Example 1 was used to design a downstream process appropriate for hGH.
Initially, hGH product retention data was loaded into the program. As described in Example 1, each resin or step was categorized as bind-elute, explicit flow-through, or implicit flow-through. The hGH product retention data was used as inputs to generate a list of all candidate 3-step process sequences, of both resin types and operating conditions, which recover the product, wherein each step corresponded to running the hGH product solution through a column with a resin from the screening process using a pH or salt gradient. The constraints described above in Example 1 were implemented to reject undesirable processes.
Then, using both the hGH product retention data and host cell protein (HCP) data from the null cell culture fluid (CCF) fractionation experiments, the processes were ranked using Equation 1 as described in Example 1. Using this equation, scores were assigned and each set was rank-ordered. The top 20 process sequences selected by the process selection tool for the purification of hGH are shown in Table 7.
Profiles of process-related impurity removal were generated to visualize step orthogonality for each sequence.
Once potential sequences were identified using the process generation tool, a decision process was implemented to select the specific potential sequence for process development according to the protocols described in Example 1.
The development of a process to purify hGH from Pichia CCF was initiated using the output from the process selection tool shown in Table 7. The specified purification targets were host cell proteins (HCP) less than 100 ppm and DNA less than the limit of detection (LOD) of a Quant-iT™ PicoGreen® dsDNA Assay Kit (ThermoFisher) kit. Sequence 1 was selected as the starting point for hGH process development.
The initial top-scoring process (
The final process shown in
The results presented in Table 8 show that overall product recovery was approximately 86%, with total HCP clearance (4.5 logs) exceeding the purification target and total DNA clearance (2.7 logs) falling short of the purification target. The final product concentration was 0.631 mg/mL, and the final aggregate content was 0.48%. In Table 8, for DNA, error bars represent one standard deviation of triplicate measurements, and for HCP ELISA, error bars denote the 95% confidence interval.
Interferon α-2β (IFN) was partially purified from a cell culture fluid from a Pichia pastoris culture expressing IFN in order to concentrate the problematic product variants being produced. A multimodal cation exchange (MMC) CMM HyperCel column was used to capture the IFN and related products and separate them from many of the other host impurities. Partially purified IFN was fractionated by reversed phase-ultra high pressure liquid chromatography (RP-UPLC) using a linear gradient elution and analyzed by direct injection electrospray ionization mass spectrometry (ESI-MS).
To identify variants, ten fractions were collected from the RP-UPLC fractionation of IFN, each enriched with one of the dominant peaks observed by RP-UPLC.
Charge state isoforms of the proteins in each injection allowed for identification of charge state and molecular weight. Molecular weights were matched with a pool of possible identities based upon protein and signal peptide sequence, cleavages and excisions therein, and common post-translational modifications (PTM). Species identified consisted primarily of incomplete signal peptide cleavage or C-terminal cleavage variants (Table 9). A singularly oxidized variant was also observed (Table 9). There was no evidence of glycosylated variants from MS. This was confirmed with an enzymatic deglycosylation assay, using Jack Bean mannosidase specific to Pichia glycosylation patterns. Problematic product variants were identified and assigned corresponding RPLC peaks for tracking through a downstream purification process selection screening process that will be described next.
Null strain Pichia pastoris cell culture fluid (CCF) fractionation experiments were performed as described in Example 1.
Partially purified IFN was diluted to approximately 0.5 mg/mL in an equilibration buffer 20 mM sodium citrate at pH 4.0 and 0.2 μm filtered, and the solution was titrated to pH 3.0, 5.0, 6.0, or 7.0 as needed. The solution was titrated to lower pH using 100 mM citric acid. The solution was titrated to higher pH using 100 mM Tris base. The final concentration of IFN in the solution ranged from 100 to 500 μg/mL. The IFN solution was prepared less than 24 hours in advance of its use. IFN fractionation experiments were carried out according to the protocol described for G-CSF fractionation experiments in Example 1.
Reversed phase-ultra high pressure liquid chromatography (RP-UPLC) analysis of samples from the CCF partitioning fractionations and IFN solution fractionations was performed according to the protocol described in Example 1.
RP-UPLC chromatograms were taken as raw data .ARW files at A210, A260, and A280 with 40 Hz resolution. A MATLAB script was written to convert all raw RP-UPLC data from .ARW files to a single .MAT file. Chromatograms corresponding to blank deionized water (DI) injections were used to baseline subtract from RP-UPLC chromatograms. These DI injections were performed each day, and fractionated CCF and IFN RP-UPLC chromatograms were baseline-subtracted using the appropriate DI injection run.
Data reduction was performed by integrating each chromatogram over 0.5 second time intervals. Only data from 1.5 to 10.0 minutes were considered in order to eliminate the effects of t0 baseline disturbances. The process-related impurity and product-related impurity retention data set was then stored as a 5-dimensional array with the first dimension corresponding to the wavelength of the data collected, the second dimension corresponding to the resin, the third dimension corresponding to the gradient type, the fourth dimension corresponding to the ÄKTA fraction number, and the fifth dimension corresponding to the integration window from RP-UPLC data.
A partial selection of results from IFN purification using different resins with characterizing language is presented in Table 10.
indicates data missing or illegible when filed
The development of a process to purify IFN from Pichia CCF and product variants was initiated using the product-related impurity retention data set. The specified purification targets were host cell proteins (HCP) less than 100 ppm and DNA less than limit of detection (LOD) of a Quant-iT™ PicoGreen® dsDNA Assay Kit (ThermoFisher) kit.
An example of a chromatogram analyzing the purity of IFN after a downstream process purification is shown in
Only one resin—HEA HyperCel, an MMA/HCIC (aliphatic)—showed a high degree of selectivity for the N-terminal variant (HEA, Table 10). Process development of the purification step demonstrated that the step was highly sensitive to pH. Exploration of additional resins for N-terminal selectivity identified SP Sepharose HP as being capable of resolving the variant using a salt elution.
The final process, which employed MMC ImpRes as a capture step, MEP HyperCel as a first polishing step, and SP Sepharose HP as a second polishing step, was used to purify IFN from Pichia CCF and problematic IFN variants, and the resulting solution was characterized for product recovery and clearance of HCP, DNA, product variants, and product aggregates. For MMC ImpRes, the bind condition was 20 mM sodium citrate, pH 5.0; the wash condition was 20 mM sodium phosphate, pH 6.8; and the elute condition was 200 mM sodium citrate, 100 mM sodium chloride, 20 mM sodium phosphate, pH 7.6. For MEP HyperCel, the bind condition was 200 mM sodium citrate, 100 mM sodium chloride, 20 mM sodium phosphate, pH 7.6; the wash condition was 2 0 mM sodium citrate, pH 5; and the elute condition was 20 mM sodium citrate, pH 4.0. For SP Sepharose HP, the bind condition was 10 mM sodium citrate, pH 4.3; the wash condition was 180 mM sodium chloride, 10 mM sodium citrate, pH 4.3; and the elute condition was 250 mM sodium chloride, 10 mM sodium citrate, pH 4.3. The load challenge for capture step was approximately 10 mg IFN/mL resin. An SDS-PAGE gel was run to test product quality. Enzyme-linked immunosorbent assay (ELISA) (Cygnus Technologies) was carried out on Pichia to determine information on process contaminants including host cell proteins (HCPs). A Quant-iT™ PicoGreen® dsDNA Assay Kit (ThermoFisher) was carried out to determine information on DNA process contaminants. Size-exclusion chromatography (SEC) was used to quantify aggregates. Reverse phase liquid chromatography (RPLC) was used to determine charge variants and product titer. Capillary electrophoresis (Perkin Elmer GXII) was also used to determine product titer. A cell-based proliferation assay was conducted to determine product activity.
The results presented in Table 11 show that overall product purity was 94.80%. The final product concentration (conc.) was 0.058 mg/mL, and the final aggregate content was 0.63%.
In the three-column process, the most problematic product variants were greatly reduced. By using the selected process, N-terminal variant was reduced to 4.79%. A composition profile was determined by RP-HPLC for IFN and N-terminal variants during the isocratic elution condition identified for the final process.
To ensure that glycosylation was not a lingering issue in the final product pool, enzymatic deglycosylation was conducted. The C-terminal deletion peak was the only one exhibiting an increase over the incubation period, which did not suggest glycosylation. This can be seen from
The downstream purification process determined as in Example 1 was used to purify granulocyte-colony stimulating factor under similar operating conditions to those used in Example 1. The product quality obtained contained host cell proteins (HCP) in an amount less than 70 PPM as determined by ELISA (Cygnus Technologies), DNA in an amount less than 65 PPB as determined by qPCR (resDNASEQ Quantitative Pichia DNA Kit—Life Technologies), and an overall purity of greater than 95% as determined by RPLC, as in
The pharmacokinetic profile as well as pharmacodynamic effect (neutrophil stimulation) of the experimentally purified G-CSF was tested in an animal model (Sprague Dawley rats) compared to Neupogen (predicate control) following a single subcutaneous administration. Briefly, thirty-nine male Sprague Dawley rats were assigned to four groups: 12 animals in each of Groups 2-4 and three animals in untreated control Group 1. Animals in Groups 2 and 3 received experimentally purified G-CSF at concentrations of 115 μg/kg and 575 μg/kg respectively and animals in Group 4 received predicate control (Neupogen) at a concentration of 115 μg/kg. Blood samples were collected for pharmacokinetic assessment (four time points from three rats per group) at pre-dose, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, and 120 hours post-dose.
Blood samples were also collected from three rats per group for neutrophil analysis 24 hours post dose. A statistically significant increase in relative neutrophil counts was observed in the peripheral blood 24-hours post dose in both experimentally purified G-CSF and Neupogen® treated groups compared to vehicle control group. Increased relative neutrophil counts observed were considered to be treatment related and an expected pharmacological or pharmacodynamic effect of both experimentally purified G-CSF and Neupogen®. The pharmacokinetic profile and the pharmacodynamic effect (neutrophil stimulation) of the experimentally purified G-CSF and Neupogen were similar.
As shown in
Chromatographic Process Performance for Human Growth Hormone (hGH)
Another downstream purification process was used to purify human growth hormone (hGH). The downstream purification process used in this example included a first chromatography column with Pall CMM HyperCel (multimodal cation exchange) resin operated in bind-elute mode. The bind conditions were pH 5.5 (from 5.0 to 6.8 possible), conductivity in the range 0 to 50 mS/cm. The wash conditions were pH 6.0 (from 5.8 to 6.5 possible), conductivity 4 mS/cm (from 0 to 10 mS/cm possible). The elution conditions were pH 8.0 (from 7.5 to 8.5 possible), conductivity 4 mS/cm (from 0 to 10 mS/cm possible). The next step in the downstream purification process was a second chromatography column with Pall HyperCel STAR AX (anion exchange) resin operated in flow-through mode. In this example, there were no further purification modules.
The purification of hGH was found to be consistent across multiple cycles and multiple runs, as shown by the chromatograms in
SDS-PAGE was used to analyze the purified product, as shown in
The downstream purification process determined as in Example 3 was used to purify interferon α-2β (IFN) under similar operating conditions to those used in Example 3. The product quality obtained contained host cell proteins (HCP) in an amount less than 75 PPM, DNA in an amount less than 300 PPB, and an overall purity of greater than 90%, as in
This Example describes a system comprising a magnetic level sensor and a perfusion bioreactor.
Sensing the level of liquids in fluid-holding systems such as bioreactors, hold tanks, surge tanks, etc. can provide one measure for enabling process control since the rate of perfusion and feeding can be regulated as required by the operator to create the optimized production conditions in the vessel. Current approaches for perfusion bioreactors use in-vessel level probes for this purpose. Non-invasive sensing can offer multiple benefits, including reduced risk of contamination, reduced geometric complexity, and potential cost savings. For disposable bioreactors, non-invasive sensors could allow for reuse of the sensor, facilitating rapid turnaround of process equipment without necessitating sterilization.
The few non-invasive technologies for sensing liquid level that exist in other industries do not typically lend themselves to applications in bioreactor vessels. For example, external capacitive level sensors work by measuring the change in dielectric constant through the vessel wall. The ionic conductivity of the fluid in fermentation typically changes during the course of a run, however, and can interfere with these measurements.
In this Example, a non-invasive liquid level sensing technique using a magnetic float and externally-mounted reed switches is described. This technique does not depend on fluid properties, but instead leverages the fluid level itself to allow direct measures of the fluid level in the reactor.
A magnetic liquid level sensor using reed switches and a magnetic float was designed and prototyped. The magnetic liquid level sensor was a discrete sensor suitable for use in applications where the process control is based upon point-level control. With high-resolution, multi-level modifications, it can also be used in a pseudo-continuous manner if the required liquid level resolution is sufficiently high.
A magnetic float was suspended around a non-magnetic shaft placed in a vessel. The proximity to the edge of the vessel ensured that the magnetic field penetrated the outer surface of the vessel. To achieve a specific gravity less than that of the reactor fluid (approximately that of water), a foam polystyrene floatation ring was used as the float. Four nickel-coated neodymium [NdFeB] magnets were equidistantly spaced and embedded in the float. The magnets were of a ½″ disc diameter and ¼″ thickness, with an individual pull force of 6.1 pounds. An array of reed switches were placed external to the vessel. Reed switches generally refer to electric switches that turn on in the presence of a magnetic field. Depending on the specific reed switch and magnet strength, the distance and orientation required to turn them on may vary. In this Example, the reed switches used were SPST-NO, with a magnetic sensitivity of 12-18 Ampere-turns. The probe holding the magnetic float was a plastic shaft. The vessel was a glass beaker with a diameter of 4.25 inches (10.80 cm) and a wall thickness of 0.15 inches (0.38 cm). The vessel geometry can affect sensor performance, since level sensing resolution is based on height, and a smaller diameter vessel can lead to increased resolution for the same volume change.
One configuration tested included an array of 5 reed switches. To provide visual feedback on the system for the active switch, each reed switch was placed in series with a different colored light-emitting diode (LED). In a setup for process control, other means of communications such as current sensors or a direct voltage or current signal can be sent to the processor, instead or in addition to LEDs. In this Example, the spacing of the reed switches was intended to turn on either one or two LEDs.
The liquid level was varied between 0 mL and 1000 mL. To minimize the effect of LEDs flickering on and off due to the magnetic field only temporarily reaching the reed switches, the reading was allowed to stabilize for 10 seconds before the LED was said to be ON. This mimicked a process control step that verifies a positive signal for 10 seconds before processing the level. The results corresponded very closely to the designed logic. The discrepancies occurred due to lack of robustness of the experimental setup and the fluctuations and instabilities introduced due to testing.
In a stable bioreactor environment, with a commercially prototyped array of reed switches, and knowing the exact magnetic field environment, as well as the interference from surrounding objects, a precise sensor can easily be developed to follow its assigned logic perfectly. Additionally, the discrete level sensor can transform into a pseudo-continuous sensor by adding reed switches and increasing the amount of steps (n). As n goes to infinity, the sensor will overlap the actual level exactly, and the sensor will become continuous. Depending on the resolution of liquid level sensing required, n will need not go to infinity, but instead be high enough to achieve sensing to the accuracy desired. If the logic dictates that each step is evenly spaced (which is not the case in the current setup due to leveraging combinations of ON states to achieve more steps), then n equals the number of reed switches, and the number of different level states that can be predicted. As n increases, the resolution becomes fine enough to resemble a continuous sensor. For example, at n=100, the measured liquid level overlaps with the true level.
An improved version of the Magnetic Float Liquid Level Sensor would comprise a custom-made adhesive strip of reed switches that attaches externally to the vessel. All the necessary information regarding which switch is sensing current would be parsed through to one end of the strip and output to the processor. The strip would be carefully manufactured so that the switches are equidistant but also with the knowledge of the rest of the system (such as the magnetic float, vessel wall properties, etc.) so as to ensure perfect relay of logic as the magnetic float changes with level. The magnetic float itself would have a uniform distribution of magnetic material so that rotation of the float does not change the magnetic field. The binary nature of reed switches makes them reliable and robust, but Hall Effect sensors are an alternative to reed switches that can also be used. Hall Effect sensors are similar to reed switches in that they react to a magnetic field, but they can act as a continuous sensor due to their ability to linearly increase output voltage (until saturation) with increasing magnetic field density.
A liquid level sensor using a magnetic float and an array of reed switches was successfully tested. This non-invasive option to liquid level sensing may have a wide range of applications in biotechnology as well as other industries in which non-invasive fluid sensing would be beneficial. While the magnetic level sensor is primarily a discrete sensor that lends itself to point-level alarm control, with increased resolution (or implementation of Hall Effect sensors), it can also be used for process control when continuous level sensing is required. Using an external magnetic level sensor allows for reduced reactor complexity, reduced risk of contamination, increased cost savings from reuse, and other benefits when used for process control in a single-use, continuous perfusion bioreactor.
This Example describes a system comprising an optical level sensor and a perfusion bioreactor.
To enable perfusion in small-scale bioreactors, hold tanks, surge tanks or other fluid-holding tanks, there is an opportunity for advancing probes for real-time, online monitoring of various process parameters such as temperature, dissolved oxygen, pH, and cell growth, for process control. Liquid level in the reactor is another parameter that can be monitored for controlling the rate of perfusion, the rate of nutrient feed, or in any step that requires volume control. Depending on the nature and requirements for process control, the liquid level sensor could sense a single point, multiple discrete points, or continuous levels.
Stirred single-use bioreactors configured for perfusion currently use invasive liquid level sensors such as conductive level probes. Having non-invasive disposable alternatives to sense liquid level could offer several advantages, including reduced vessel and headplate complexity, reduced risk of contamination, and increased cost savings.
A few non-invasive level sensing methods exist, but are typically unsuitable for use in bioreactors. Externally-mounted capacitive level sensors that sense the changing dielectric constant of the fluid are generally sensitive to changes in ionic conductivity of the reactor fluid during a bioreactor run. Methods such as load cells and pressure transducers are often subject to variations in the density of the reactor fluid, as well as altered by vibrational noise. Time of flight methods such as ultrasonic or lasers typically require very high resolution due to the small geometries, and are susceptible to change due to a wide range of parameters, thus needing to be fairly expensive in order to work in this scenario, negating the benefits of the non-invasive method.
In this Example, three different optical-based level sensors that provide low-cost and accurate means of discrete as well as continuous level monitoring were designed and demonstrated.
A clear plastic probe was constructed with an outer diameter of 8 mm, a wall thickness of 1 mm, and a height of 13 inches. At a distance of 3.5″ from the bottom of the probe, bright blue tape (VWR) with a width of ½″ was wrapped around the length of the plastic probe once, to cover exactly ½″ of height. After a spacing of ⅛″ below the end of the blue tape, orange tape (VWR) with a width of ½″ was wrapped the length of the plastic probe once, to cover exactly ½″ of height. Similarly, keeping the spacing and length consistent, green tape (VWR), bright red tape (VWR), and lavender tape (VWR) were respectively wrapped around the probe. The plastic probe was tape-free for the last half-inch at the bottom.
A clear plastic probe was constructed with an outer diameter of 8 mm, a wall thickness of 1 mm, and a height of 13 inches. A float was constructed by using an annular, semi-hollow polypropylene piece having an outer diameter 27.5 mm, an inner diameter of 11 mm (thickness of 8.25 mm), a height of 12 mm, and a weight of 4.19 g (density of 175 kg/m3). Bright red tape (VWR) with a width of ½″ was wrapped around the float so as to cover it completely. After the addition of the tape, the float weighed 4.52 g. The float was then placed around the plastic probe.
A clear plastic probe was constructed with an outer diameter of 8 mm, a wall thickness of 1 mm, and a height of 13 inches. Bright red tape (VWR) with a width of ½″ was wrapped around the length of the plastic probe to cover 10 inches of height. The tape was overlapped such that the plastic was not visible along the 10 inches. Above 10 inches, the clear plastic was visible.
A laptop running MATLAB R2015b (Windows 8.1), with an Intel® Core™ i7-3630QM CPU @ 2.40 GHz processor with 8.00 GB of installed memory RAM, was used for its built-in webcam for image acquisition, and to run the MATLAB image processing algorithms online, in real-time. The liquid level was then varied as required, while the process computer's webcam acquired images. For all three methods described here, the algorithms developed as described were then run continuously using the acquired images, and the level was predicted.
A CBB algorithm was developed to create a binary (black and white) image from the original image by selecting for the colors of interest. The acquired image was first converted from the RGB (red, green, blue) space to the HSV (hue, saturation, value) space. The H-value generally provides a truer representation of the color of the object and is less sensitive to environmental lighting conditions, unlike the RGB values in which color and luminance information are coupled. The HSV image was then filtered using thresholding of the different hue, saturation, and value data against the known ranges of the colors of interest. To remove optical and physical noise, the holes in the binary image (for example, a couple of black pixels amongst many white ones) were then filled by using the “imfill” function in order to ensure objects remain together and are not compromised.
The binary image generated from the CBB algorithm was then converted into discrete objects. The COD algorithm applied a Gaussian blur and filter to the binary image to smooth erroneous pixels, physical, and optical imperfections and to reduce vibrational noise. This transformation was done by clustering binary data and creating “blobs” that represented objects of the specific color in the original image. To avoid flecks of the specific color in the image, and other minor objects of the same color that may interfere with the process, an area-based filter was then applied to only retain objects within a certain pixel area range. For the geometric setup and camera resolution (640×800 pixels) implemented, the allowed range of area was set to filter objects smaller than 40 pixels when searching for the colored float or the painted bands, and 500 pixels when searching for the colored shaft. This range was determined by knowing the positioning of the camera in relation to the setup, as well as the camera resolution, which allowed correlations to the range of sizes of the binary objects expected.
The retained objects of the specific color were then counted. Within the specified range, only one object should remain for each color of interest, since the choice of apparatus and geometry were designed to leave one remaining object per color. These visualized objects were leveraged in different ways for each sensor method described.
The PB algorithm used the COD algorithm to extract information from the binary images it created. The objects for each band color were counted in the COD algorithm and fed to the PB algorithm. The PB algorithm used these counts to detect the presence or absence of the specific painted band in the image and correlated that to the point-level being above or below certain values associated with the bands at those levels.
The RCF algorithm used the COD algorithm to extract information from the binary image it created. The algorithm detected the location of the red object and determined its centroid. By pre-determined geometrical calculations and knowledge of camera-acquired image specifications, the centroid of the red float that was detected was then used to correlate to liquid level. An average of five level readings were conducted at each point, and the calculated mean value was the resulting predicted level value. In case of an erroneous zero reading, the code disregarded this value in calculating the mean.
The RCS algorithm used the COD algorithm to extract information from the binary image it created. This algorithm detected the residual size of the red probe. By pre-determined geometrical calculations and knowledge of camera-acquired image specifications, the area was then used to determine the liquid level. An average of five level readings were conducted at each point, and the calculated mean value was the resulting predicted level value. In there was an erroneous zero reading, the code disregarded this value in calculating the mean.
A 1000 mL glass beaker with wall thickness of 0.15 inches (0.38 cm) was used to model the bioreactor. The beaker was filled with a spent cell culture of Pichia pastoris post-run that was stirred at 600 rpm using a VWR mini-magnetic stirrer and plate, thus simulating an agitated bioreactor. Along with the image-capturing camera and process control computer, the elements constructed for each level sensing method were then introduced into the assembly for each experiment.
Process control in bioreactors generally requires continuous monitoring of liquid level. Due to the drawbacks of in-vessel level sensors, three non-invasive optical methods were developed.
In lieu of an in-vessel point level sensor, an optical method comprising painted bands along the impeller shaft was designed. Taking advantage of the typical opacity of the reactor fluid, the camera could only see the colored bands above the surface. To sense certain pre-defined measurement points, the system read the visible colors to determine the current level. To ensure the image processing algorithms worked robustly, the colors were chosen to be different enough from each other and from the reactor fluid. In this Example, five colors were used—blue, orange, green, red, lavender. To test the Discrete Optical Level Sensor, the vessel volume was varied from 0 mL to 1000 mL. The camera and computer sensed the presence or absence of the five colors and predicted the point level. As a safety margin, the process control set the level above the associated level when a color was detected. This setting could be set at the lower bound as well.
The number of cells generally varies during the course of fermentation, and the opacity of the reactor fluid also generally changes. To determine if the level sensor could accurately report the fluid level throughout the process, the optical densities (OD) of reactor fluid were varied from 0 to 0.600, equivalent to a cell concentration of 0 to 3.6×107 cells/mL. To do this, the OD600 measurement of BMGY media without cells was used as a blank reference in the UV spectrophotometer. The cell optical density was then varied from an OD600 value of 0 to 0.600, while the level sensor was tested.
The lighting conditions where the reactor is housed could also fluctuate during long fermentations. To test the robustness of the optical level sensors with changing light surroundings, the experiment was repeated by changing the light intensity incident on the setup, in a room with only one source of light, from 1,000 to 100,000 lux, while the percentage of failed readings (no detection of desired objects, or a zero reading) was measured. The optical density was set to a constant OD600 of 0.300 (cell concentration of 1.8×107 cells/mL) for this experiment.
In the experiment, the sensor triggered the level above “X” to which it was assigned accurately for each level, with the highest absolute mean error being 4 mL and the highest percentage mean error being 0.67%. Each individual calculation took an average of 1.4 seconds to complete. The results show that the sensor worked at optical densities above 0.171 OD600 (1.03×107 cells/mL) and had no failed readings once the illuminance was above 29100 lux on the apparatus, at an optical density of 0.300 OD600.
While a point-level sensor may be appropriate in some applications, a continuous level sensor that tracked liquid level directly by using a float was developed to provide more control in the process while maintaining the benefits of a non-invasive optical level sensor. As the float varied directly with the level of liquid in the vessel, capturing the co-ordinates of the float enabled prediction of the liquid level. To ensure the float was easily detectable by the algorithms, its color was different from that of the liquid. In this Example, a red float was used, and the experiment was set up and built as detailed above. To test the Continuous Coordinate-based Optical Level Sensor, the vessel volume was varied from 400 mL to 1000 mL while the camera and computer sensed the position of the red float and used it to predict the liquid level. Similar to the case of the Discrete Optical Level Sensor, the OD and light intensity experiments were also conducted to determine the range in which the sensor works.
The results show that there were no erroneous zero readings in the primary experiment conducted due to good lighting conditions. The average time taken for each individual level calculation using the current setup was 1.6 seconds, with 7.9 seconds required to predict a level using five averaged readings. To assess the accuracy of the predicted values, a range of liquid level values (between 400 mL and 1000 mL) was measured. The highest absolute error was 10.8 mL, with a highest percentage error of 2.38%. The standard deviation for the individual level readings had a highest value of 8.7 mL and a highest standard error value of 2.6 mL. The sensor worked at all tested optical densities between 0 to 0.600 OD600 and had no failed readings once the illuminance was above 1800 lux on the apparatus, at an optical density of 0.300 OD600.
The benefits of a non-invasive, continuous liquid level monitoring system were achieved using the previous setup. However, the use of an additional part (the float) was necessary to achieve the desired functionality. In order to try and achieve the same functionality without the addition of added parts, a different continuous level sensor was created using the agitator shaft that was already in the vessel. By detecting the amount of visible shaft area using a camera, the computer predicted the level by assuming the liquid in the vessel blocked the rest of the agitator shaft. To ensure the shaft was easily distinguishable so the algorithms worked appropriately, the agitator shaft of the bioreactor was a contrasting color to the reactor fluid—bright red was used in this Example.
In order to test the Continuous Area-based Optical Level Sensor, the experiment was built and set up as detailed above. The vessel volume was varied from 400 mL to 1000 mL, while the camera and computer sensed the amount of visible shaft area and correlated it to liquid level. Similar to the previous optical level sensors, the OD and light intensity experiments were also conducted to determine the range in which the sensor works.
The results show that there were no erroneous zero readings in the primary experiment conducted due to good lighting conditions. The average time taken for each individual level calculation using the current setup was 1.5 seconds, with 7.7 seconds required to predict a level using five averaged readings. To assess the accuracy of the predicted values, a range of liquid level values (between 400 mL and 1000 mL) was measured. The highest absolute error was 15.0 mL, with a highest percentage error of 2.34%. The standard deviation had a highest standard deviation value of 5.0 mL and a highest standard error value of 1.5 mL. The results also show that the sensor worked at optical densities above 0.133 OD600 (7.98×106 cells/mL) and had no failed readings once the illuminance was above 2900 lux on the apparatus, at an optical density of 0.300 OD600.
The successful testing of the optical level sensors validated a novel solution to process control using level sensing in bioreactors. There is no longer a requirement for an invasive sensor that inconveniences the setup as well as introduces a potential sterility concern. An external sensor may allow for reusability even in the case of disposable bioreactors and may be cost effective in the long run. Two of methods created were continuous to offer complete process control, whereas one method was discrete, lending itself to point-level applications in biotechnology.
As seen in the results, the sensors worked on the order of seconds. The reactor fluid volume typically changes at a rate of 15-30 mL/hour, which means the sensors developed are adequate for process control. Typically, the rate of fluid flux incoming or outgoing in perfusion bioreactors is not fast enough to alter the liquid level faster than the sensor calculates the level. The results also demonstrate that the sensors work in a large range of lighting conditions and a large range of optical densities. The Continuous Coordinate-based Optical Level Sensor worked at all densities, whereas the other two designs began to work at very low cell concentrations as well. With regards to illuminance, the Continuous Coordinate-based Optical Level Sensor worked perfectly even at 1800 lux, whereas the Continuous Area-based Optical Level Sensor required marginally more illuminance at 2900 lux to ensure no zero readings. The Discrete Optical Level Sensor required the most light to work all the time, with a minimum illuminance of 29100 lux. The Continuous Coordinate-based Optical Level Sensor required the addition of the float as a piece of equipment, whereas the other two methods used an existing part of the setup (the shaft) to achieve functionality. Since the Discrete Optical Level Sensor only gauged the presence or absence of a specific color band, it was more robust for its application, as opposed to the continuous level sensors. However, the continuous sensors offered more control due to the ability to predict level to a closer degree.
Since all methods used optical sensing and chrominance filtering to achieve their respective results, after a certain point, the drastically changing light conditions made the optical level sensors vulnerable to stop working. This problem may be circumvented in future designs by using a more sophisticated setup with a controlled and consistent light source inside a closed environment. Alternatively, a slightly less robust but cheaper modification to the current setup would be to use a “gray card” approach, where the code uses the known gray card HSV values as a reference and corrects the image for changing lighting before implementing the algorithms. The formation of a vortex at certain stirrer speeds could potentially slightly skew the level sensors since the liquid surface becomes concave and has different heights at different points. If the formation of a vortex is a concern to proper process control, and very precise level sensing is required, the two continuous techniques can be used in tandem to correct for the vortex. With the float level at the wall, and the agitator shaft level at the center, the two methods will experience opposite effects of the vortex level change, and can be combined to correct to the true level (volume) of the vessel. Alternatively, knowledge of the vortex formed with stirrer speed can be used to correct and recalibrate the level sensor.
Overall, the methods and algorithms developed worked effectively to gauge liquid level for the purpose of process control in running a single-use, continuous perfusion bioreactor, and offer plenty of advantages over traditional level sensors. The optical methods offer a non-invasive alternative that do not cause sterility concerns, avoid added reactor complexity and cost, and are reusable even in a single-use bioreactor setup.
This example describes an alternative process for the downstream purification of IFN. The chromatographic process design was the same as in Example 3, except an additional constraint was placed on the downstream process generation tool. The 3-step process was required to have a single flow-through step. The downstream purification process having a single flow-through step had a higher percent product recovery than the process in Example 3 and a similar host cell proteins (HCP) concentration and total variant content.
Table 13 shows the top 20 process sequences selected by the process selection tool for the purification of IFN. Sequences are presented with their scores calculated by Equation 1 along with a host-related impurity rank, variant removal rank, and a summed rank. Individual steps are presented in the format of “resin, operating mode/pH, product elution condition”, where an operating pH is given when using a salt gradient elution and “flow-through” is used to indicate that the product elution condition is the same as the load condition.
The development of a process to purify IFN from Pichia CCF was initiated using the output from the process selection tool shown in Table 13. The specified purification targets were host cell proteins (HCP) less than 100 ppm and DNA less than limit of detection (LOD) of a Quant-iT™ PicoGreen® dsDNA Assay Kit (ThermoFisher) kit. The initial top-ranked process (i.e., sequence 1) was discounted due to the use of Nuvia cPrime as the capture step, because the wash conditions for Nuvia cPrime resulted in a significant loss of the product. Sequences 3 and 4 were discounted due to the use of Toyopearl MX-Trp-650M as the capture step since the elution salt concentration was 300 mM, which was close to the conductivity of the Pichia CCF load. Sequence 19 was selected, because step 1 was the same as the process utilized in Example 3 and steps 2 and 3 were the same as sequence 1.
The final process, which employed Capto MMC ImpRes as a capture step, Q Sepharose HP as a flow-through step, and Capto Adhere as a polishing step, was used to purify IFN from Pichia CCF and problematic IFN variants, and the resulting solution was characterized for product recovery and clearance of HCP, DNA, product variants, and product aggregates. For Capto MMC ImpRes, the bind condition was 20 mM sodium citrate, pH 5.0; the wash condition was 20 mM sodium phosphate, pH 6.8; and the elute condition was 100 mM sodium chloride, 20 mM sodium phosphate, pH 7.6. For Q Sepharose HP, the flow-through condition was 100 mM sodium chloride, 20 mM sodium phosphate, pH 7.6. For Capto Adhere, the bind condition was 100 mM sodium chloride, 20 mM sodium phosphate, pH 7.6; the wash condition was 350 mM sodium chloride, 20 mM sodium phosphate, pH 7.0; and the elute condition was 610 mM sodium chloride, 20 mM sodium phosphate, pH 7.0. The load challenge for capture step was approximately 9.2 mg IFN/mL resin. An SDS-PAGE gel was run to test product quality. Enzyme-linked immunosorbent assay (ELISA) (Cygnus Technologies) was carried out on Pichia to determine information on process contaminants including host cell proteins (HCPs). A Quant-iT™ PicoGreen® dsDNA Assay Kit (ThermoFisher) was carried out to determine information on DNA process contaminants. Size-exclusion chromatography (SEC) was used to quantify aggregates. Reverse phase liquid chromatography (RPLC) was used to determine charge variants and product titer. Capillary electrophoresis (Perkin Elmer GXII) was also used to determine product titer. A cell-based proliferation assay was conducted to determine product activity.
The results presented in Table 14 show that overall product yield was 71.1%. The final product concentration was 0.172 mg/mL and the final total variant content was 7.9%.
Null strain Pichia pastoris cell culture fluid (CCF) fractionation experiments were carried out as described in Example 1.
In order to generate the product retention database, single-domain antibody 3B2 (i.e., SEQ. ID. No. 1), which is specific for the VP6 protein of rotavirus A, was produced using an upstream component, as described herein, and partially purified on a multimodal cation exchange resin to concentrate the product and reduce host cell proteins.
The partially purified 3B2 at a concentration of about 4 mg/ml was diluted 20× into a load buffer for the various gradient screens (i.e., 20 mM citrate, pH 5 for the pH 5 salt gradient screen; 20 mM sodium phosphate, pH 6 for the pH 6 salt gradient screen; or 20 mM sodium phosphate, pH 7 for the pH 7 salt gradient screen). The 3B2 solution was prepared less than 24 hours in advance of its use. 3B2 fractionation experiments were carried out according to nearly the same protocols and using the same chromatographic resins and buffers as the CCF G-CSF fractionation experiments described in Example 1. The difference was that in this case system flow rate was kept constant at 1 CV/min throughout the method. Column load challenge was kept constant at 2.5 mg/mL, and product elution pH or salt concentration was determined by peak maximum at 280 nm. Pure component 3B2 retention data was obtained directly from the ÄKTA chromatogram.
RP-UPLC analysis of samples from the CCF partitioning fractionations was performed according to the protocol described in Example 1. The process-related impurity retention data set was constructed according to the protocols described in Example 1. The downstream process (DSP) generation tool was used as described in Example 1, except an additional constraint was placed on the downstream process generation tool. The process was required to be two steps.
Initially, 3B2 product retention data was loaded into the program. As described in Example 1, each resin or step was categorized as bind-elute, explicit flow-through, or implicit flow-through. The 3B2 product retention data was used as inputs to generate a list of all candidate 2-step process sequences, of both resin types and operating conditions, which recover the product, wherein each step corresponded to running the 3B2 product solution through a column with a resin from the screening process using a pH or salt gradient. The constraints described above in Example 1 were implemented to reject undesirable processes.
Then, using both the 3B2 product retention data and host cell protein (HCP) data from the null cell culture fluid (CCF) fractionation experiments, the processes were ranked using Equation 1 as described in Example 1. Using this equation, scores were assigned and each set was rank-ordered. The top 20 process sequences selected by the process selection tool for the purification of 3B2 are shown in Table 15.
Once potential sequences were identified using the process generation tool, a decision process was implemented to select the specific potential sequence for process development according to the protocols described in Example 1.
The development of a process to purify 3B2 from Pichia CCF was initiated using the output from the process selection tool shown in Table 15. The specified purification targets were host cell proteins (HCP) less than 100 ppm and DNA less than the limit of detection (LOD) of a Quant-iT™ PicoGreen® dsDNA Assay Kit (ThermoFisher) kit. Sequence 1 was selected as the starting point for 3B2 process development.
The initial top-scoring process utilized Capto MMC as the capture step and Capto Adhere as the polishing step. Upon experimental validation, capacity and process robustness were improved by replacing the capture step with CMM HyperCel and the polishing step with HyperCel STAR AX. Capto MMC was found to have poor product recovery and was replaced with CMM HyperCel, which appeared in other top ranking processes. Capto Adhere resulted in substantial dilution of the final product. HyperCel STAR AX, which appeared in other top ranking processes, was found to provide comparable performance with respect to impurity clearance and resulted in less dilution. This revised process corresponds to process 25.
The results presented in Table 16 show that overall product recovery was approximately 94.8%. The final product concentration was 0.244 mg/mL.
This example describes the purification of camelid single-domain antibody 2KD1 using the downstream purification process for camelid single-domain antibody 3B2 described in Example 10. Though single-domain antibodies 3B2 and 2KD1 differ in both sequence and isoelectric point (i.e., 6.71 and 7.75, respectively), the process developed in Example 10 effectively purified the single domain antibody 2KD1 (i.e., protein SEQ. ID. No. 2). The final process described in Example 10 was used to purify 2KD1 from Pichia CCF, and the resulting solution was characterized for product recovery and clearance of HCP, DNA, and product aggregates. The load challenge for capture step was approximately 10 mg 2KD1/mL resin. An SDS-PAGE gel was run to test product quality.
The results presented in Table 17 show that overall product recovery was approximately 60%. The final product concentration was 0.398 mg/mL.
IFN was partially purified from cell culture fluid from a Pichia pastoris culture expressing IFN as described in Example 3. Partially purified IFN was fractionated by RP-UPLC.
Null strain Pichia pastoris cell culture fluid (CCF) fractionation experiments were performed as described in Example 1, and IFN fractionation experiments were carried out as described in Example 3. Reversed phase-ultra high pressure liquid chromatography (RP-UPLC) analysis of samples from the CCF partitioning fractionations and IFN solution fractionations was performed according to the protocol described in Example 1. Based on this analysis, a process-related impurity retention data set and a product-related impurity retention data set were constructed according to the protocol described in Example 3.
Using the process-related and product-related impurity retention data sets, each candidate sequence of partitioning steps was assigned a rank for expected removal of process-related impurities and, independently, a rank for expected removal of product-related impurities. To illustrate,
Based on a combination of the process-related impurity removal rank and the product-related impurity removal rank, the top five candidate sequences (out of 655 possible sequences) were identified, as shown in
This refined purification process was used to purify IFN from Pichia CCF and IFN variants, and the resulting solution was characterized for product recovery, HCP concentration, DNA concentration, and total product variant content.
The process described in Example 12 was used to identify another IFN purification process. A schematic representation of this sequence is shown in
The process described in Example 12 was used to identify another IFN purification process. A schematic representation of this sequence is shown in
While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.
Having thus described several aspects of at least one embodiment, it is to be appreciated that various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be within the spirit and scope of the present disclosure. Accordingly, the foregoing description and drawings are by way of example only.
The above-described embodiments of the present disclosure can be implemented in any of numerous ways. For example, the embodiments may be implemented using hardware, software or a combination thereof. When implemented in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers.
Also, the various methods or processes outlined herein may be coded as software that is executable on one or more processors that employ any one of a variety of operating systems or platforms. Additionally, such software may be written using any of a number of suitable programming languages and/or programming or scripting tools, and also may be compiled as executable machine language code or intermediate code that is executed on a framework or virtual machine.
In this respect, the concepts disclosed herein may be embodied as a non-transitory computer-readable medium (or multiple computer-readable media) (e.g., a computer memory, one or more floppy discs, compact discs, optical discs, magnetic tapes, flash memories, circuit configurations in Field Programmable Gate Arrays or other semiconductor devices, or other non-transitory, tangible computer storage medium) encoded with one or more programs that, when executed on one or more computers or other processors, perform methods that implement the various embodiments of the present disclosure discussed above. The computer-readable medium or media can be transportable, such that the program or programs stored thereon can be loaded onto one or more different computers or other processors to implement various aspects of the present disclosure as discussed above.
The terms “program” or “software” are used herein to refer to any type of computer code or set of computer-executable instructions that can be employed to program a computer or other processor to implement various aspects of the present disclosure as discussed above. Additionally, it should be appreciated that according to one aspect of this embodiment, one or more computer programs that when executed perform methods of the present disclosure need not reside on a single computer or processor, but may be distributed in a modular fashion amongst a number of different computers or processors to implement various aspects of the present disclosure.
Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically the functionality of the program modules may be combined or distributed as desired in various embodiments.
Also, data structures may be stored in computer-readable media in any suitable form. For simplicity of illustration, data structures may be shown to have fields that are related through location in the data structure. Such relationships may likewise be achieved by assigning storage for the fields with locations in a computer-readable medium that conveys relationship between the fields. However, any suitable mechanism may be used to establish a relationship between information in fields of a data structure, including through the use of pointers, tags or other mechanisms that establish relationship between data elements.
Various features and aspects of the present disclosure may be used alone, in any combination of two or more, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is therefore not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.
Also, the concepts disclosed herein may be embodied as a method, of which an example has been provided. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.
Use of ordinal terms such as “first,” “second,” “third,” etc. in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.
Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Ser. No. 62/480,418, filed Apr. 1, 2017, and entitled “Using Chromatography Behavior Characteristics of Impurities and Target Products to Design Downstream Processes,” U.S. Provisional Patent Application Ser. No. 62/480,422, filed Apr. 1, 2017, and entitled “Using Chromatography Screens to Characterize Impurities and Target Products,” U.S. Provisional Patent Application Ser. No. 62/480,436, filed Apr. 1, 2017, and entitled “Using Chromatography Screens to Characterize Impurities and Target Products,” U.S. Provisional Patent Application Ser. No. 62/480,438, filed Apr. 1, 2017, and entitled “Using Chromatography Screens to Characterize Impurities and Target Products to Design Downstream Processes,” U.S. Provisional Patent Application Ser. No. 62/480,426, filed Apr. 1, 2017, and entitled “Characterizing Behavior of Impurities and Target Products with Respect to Various Partitioning Techniques,” U.S. Provisional Patent Application Ser. No. 62/480,427, filed Apr. 1, 2017, and entitled “Using Behavior Data of Impurities and Target Proteins to Design Downstream Processes,” U.S. Provisional Patent Application Ser. No. 62/480,428, filed Apr. 1, 2017, and entitled “Process/Equipment for High Concentration/Throughput Rapid Perfusion based Production of Biotherapeutics,” U.S. Provisional Patent Application Ser. No. 62/480,430, filed Apr. 1, 2017, and entitled “Process/Equipment for Producing G-CSF,” U.S. Provisional Patent Application Ser. No. 62/480,432, filed Apr. 1, 2017, and entitled “Process/Equipment for Producing IFN,” U.S. Provisional Patent Application Ser. No. 62/480,435, filed Apr. 1, 2017, and entitled “Process/Equipment for Producing HGH,” U.S. Provisional Patent Application Ser. No. 62/533,765, filed Jul. 18, 2017, and entitled “Systems and Methods for Manufacturing Biologically-Produced Products,” U.S. Provisional Patent Application Ser. No. 62/553,105, filed Aug. 31, 2017, and entitled “Level Sensing Systems for Perfusion-Based Systems and Methods for Manufacturing Biologically-Produced Products,” U.S. Provisional Patent Application Ser. No. 62/644,285, filed Mar. 16, 2018, and entitled “Process/Equipment for Producing IFN,” and U.S. Provisional Patent Application Ser. No. 62/646,236, filed Mar. 21, 2018, and entitled “Process/Equipment for Producing Single-Domain Antibodies,” each of which is incorporated herein by reference in its entirety for all purposes.
This invention was made with Government support under Contract No. N66001-13-C-4025 awarded by the Space and Naval Warfare Systems Center. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
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| PCT/US2018/025583 | 3/30/2018 | WO | 00 |
| Number | Date | Country | |
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| 62646236 | Mar 2018 | US | |
| 62644285 | Mar 2018 | US | |
| 62553105 | Aug 2017 | US | |
| 62533765 | Jul 2017 | US | |
| 62480418 | Apr 2017 | US | |
| 62480422 | Apr 2017 | US | |
| 62480436 | Apr 2017 | US | |
| 62480438 | Apr 2017 | US | |
| 62480426 | Apr 2017 | US | |
| 62480427 | Apr 2017 | US | |
| 62480428 | Apr 2017 | US | |
| 62480430 | Apr 2017 | US | |
| 62480432 | Apr 2017 | US | |
| 62480435 | Apr 2017 | US |
