Patent Application
20240069001
The present disclosure relates generally to experimental evaluation of drug products, and more particularly, to adaptive perfusions systems and methods for drug release testing.
Since in vitro release testing (IVRT) provides information about the quality and performance of drug products, IVRT has been considered for evaluation of complex drug products, such as nanoemulsions, suspensions, multivesicular liposomes (MVLs), and microspheres. Ideally, an IVRT method would directly correlate changes in a critical quality attribute (CQA) of a drug product formulation to the release characteristics thereof, which correlation could provide valuable information that can be used, for example, to ensure consistent manufacturing quality among different batches of the drug product, to identify post-regulatory-approval changes of a drug product, and/or to allow comparison between different drug products for determination of equivalence. However, developing a fit-for-purpose and robust IVRT method for complex drug products remains a challenge.
Embodiments of the disclosed subject matter provide in vitro release test (IVRT) methods and systems for drug release testing, referred to herein as adaptive perfusion. The adaptive perfusion methods and systems involve a pressure-driven separation technique, for example, a tangential-flow filtration (TFF) process (also known as crossflow filtration). Any species that are smaller than the pores of a hollow fiber filter (HFF) membrane, or any other type of TFF membrane filter, pass therethrough into the permeate. Meanwhile, larger species are retained by the pores of the HFF membrane, and are recirculated to a retentate reservoir. In particular, released drug products can pass through the HFF membrane into the permeate while unreleased drug products are retained in the retentate and recirculated. A continuous supply of fresh media can be provided to the retentate in order to maintain a constant total volume for the sample as well as to maintain sink conditions for the drug release. Drug concentration in the retentate and permeate can be evaluated, for example, by removal of aliquots of the retentate and permeate from the respective reservoirs and subsequent testing via an ultra-performance liquid chromatography (UPLC) method to provide a time history indicative of the release properties of the drug product. Alternatively or additionally, drug concentration can be monitored in situ, for example, using one or more fiber optic sensors. The permeate can also be collected and tested after completion of a particular experiment to provide a measure of the cumulative amount of drug released. In some embodiments, the HFF membrane can be conditioned prior to use to improve the accuracy, precision, or both of the subsequent drug release testing.
In one or more embodiments, a method comprises, using a filter, performing a diafiltration process on a fluid having a drug sample therein. A retentate from the filter can be recirculated to a fluid supply reservoir. A permeate flow from the filter can be collected in a permeate reservoir. The method can further comprise obtaining a first aliquot from the fluid supply reservoir or a flow of the fluid to the filter, and obtaining a second aliquot from the permeate flow. The method can also comprise analyzing the first and second aliquots to determine one or more properties of the drug sample.
In one or more embodiments, a method can condition a filter for use in a diafiltration process involving a drug sample. The method can comprise connecting a first port of a filter to a source of conditioning solution, and connecting a second port of the filter to a third port of the filter. The first port and the second port can connect to a first volume disposed on a first side of a membrane within the filter. The third port and a fourth port can connect to a second volume on an opposite second side of the membrane from the first side. The method can further comprise flowing conditioning solution from the source into the filter via the first port and out through the fourth port, such that the conditioning solution flows over the first and second sides of the membrane via the connection between the second and third ports. The conditioning solution can comprise a surfactant, an emulsifier, or both. In some embodiments, the diafiltration process is an IVRT method for analyzing the drug sample. In other embodiments, the diafiltration process is a purification stage or other stage in the manufacturing of the drug sample.
In one or more embodiments, a method comprises using a filter, performing a diafiltration process on a fluid having a drug sample therein. A retentate from the filter can be recirculated to a fluid supply reservoir. A permeate flow from the filter can be collected in a permeate reservoir. The method can further comprise measuring a first concentration of the drug sample in the fluid supply reservoir, and measuring a second concentration of the drug sample in the permeate flow. The method can also comprise determining one or more properties of the drug sample based on the measured first and second concentrations.
Any of the various innovations of this disclosure can be used in combination or separately. This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. The foregoing and other objects, features, and advantages of the disclosed technology will become more apparent from the following detailed description.
The following description will proceed with reference to the accompanying drawings, which have not necessarily been drawn to scale. Where applicable, some elements may be simplified or otherwise not illustrated in order to assist in the illustration and description of underlying features. Throughout the drawings, like reference numerals denote like elements.
For purposes of this description, certain aspects, advantages, and novel features of the embodiments of this disclosure are described herein. The disclosed methods and systems should not be construed as being limiting in any way. Instead, the present disclosure is directed toward all novel and nonobvious features and aspects of the various disclosed embodiments, alone and in various combinations and sub-combinations with one another. The methods and systems are not limited to any specific aspect or feature or combination thereof, nor do the disclosed embodiments require that any one or more specific advantages be present, or problems be solved. The technologies from any embodiment or example can be combined with the technologies described in any one or more of the other embodiments or examples. In view of the many possible embodiments to which the principles of the disclosed technology may be applied, it should be recognized that the illustrated embodiments are exemplary only and should not be taken as limiting the scope of the disclosed technology.
Although the operations of some of the disclosed methods are described in a particular, sequential order for convenient presentation, it should be understood that this manner of description encompasses rearrangement, unless a particular ordering is required by specific language set forth below. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed methods can be used in conjunction with other methods. Additionally, the description sometimes uses terms like “provide” or “achieve” to describe the disclosed methods. These terms are high-level abstractions of the actual operations that are performed. The actual operations that correspond to these terms may vary depending on the particular implementation and are readily discernible by one of ordinary skill in the art.
The disclosure of numerical ranges should be understood as referring to each discrete point within the range, inclusive of endpoints, unless otherwise noted. Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise implicitly or explicitly indicated, or unless the context is properly understood by a person of ordinary skill in the art to have a more definitive construction, the numerical parameters set forth are approximations that may depend on the desired properties sought and/or limits of detection under standard test conditions/methods, as known to those of ordinary skill in the art. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word “about” is recited. Whenever “substantially,” “approximately,” “about,” or similar language is explicitly used in combination with a specific value, variations up to and including 10% of that value are intended, unless explicitly stated otherwise.
Directions and other relative references may be used to facilitate discussion of the drawings and principles herein, but are not intended to be limiting. For example, certain terms may be used such as “inside,” “outside,”, “top,” “bottom,” “interior,” “exterior,” “left,” right,” “front,” “back,” “rear,” and the like. Such terms are used, where applicable, to provide some clarity of description when dealing with relative relationships, particularly with respect to the illustrated embodiments. Such terms are not, however, intended to imply absolute relationships, positions, and/or orientations. For example, with respect to an object, an “upper” part can become a “lower” part simply by turning the object over. Nevertheless, it is still the same part and the object remains the same.
As used herein, “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.
Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.
Disclosed herein are methods and systems for in vitro release testing (IVRT), for example, to evaluate drug release from complex drug products, such as nanoemulsions, suspensions, multivesicular liposomes (MVLs), microspheres, protein-drug complexes, etc. The disclosed IVRT methods and systems employ a modified tangential flow filtration (TFF) technique, which is referred to herein as adaptive perfusion (AP). In particular, AP involves feeding a diluted drug product sample into a TFF filter, where a pressure differential generated across a filter membrane of the TFF filter drives size-based separation according to a molecular weight cut-off (MWCO) of the filter membrane. Released drug components from the product sample can pass through the filter membrane into the permeate flow, while the remaining product sample is retained by the filter membrane in the retentate flow and recirculated through the TFF filter. Periodic sampling of the retentate (e.g., aliquots from a feed reservoir) and permeate (e.g., aliquots from a permeate flow from the filter) allows for the determination of the rate and extent of drug release from complex drug products. The adaptive aspect of the AP technique arises from feedback-based addition of feed media to maintain a substantially constant dilution volume (e.g., volume of medium on retentate-side of filter membrane, associated fluid circuit, and the feed media reservoir into which the product sample is provided for dilution), as well as from tailoring the AP parameters to correspond to a properties of a particular drug product sample (e.g., size distribution of initial product, size distribution of released product, expected release rate, etc.).
In conventional IVRT methods such as microdialysis, reverse dialysis, or Franz cell diffusion, the testing focuses primarily on quantifying the extent of drug release from the product, with less emphasis on quantifying the rate of drug release. In addition, conventional IVRT methods generally rely on diffusion processes (e.g., via a dialysis membrane) to separate the released drug from the remaining components. Since such processes are necessarily limited by the rate of diffusion, the time required for adequate testing may be prolonged and be unsuitable for testing certain product formulations (e.g., emulsions) or product administration characteristics designed for rapid release (e.g., delivery via ophthalmic route). Conventional IVRT methods may also lack sufficient sensitivity and repeatability to discern minor differences in drug release from complex product formations, for example, differences arising from manufacturing process variations.
Moreover, conventional IVRT methods generally focus on analyzing the released drug, in particular, in order to maintain sink conditions across the membrane (e.g., the use of a larger volume for media on the permeate side of the membrane as compared to retentate side in order to maintain a sufficient concentration gradient to drive diffusion across the membrane). Such methods thus lack an ability to analyze the percentage of drug remaining or to achieve mass-balance in drug transport. Finally, in conventional IVRT methods, the drug release from complex product formulations initiates with passive diffusion of free-drug (e.g., drug not associated with or contained with any product formulation) across the membrane. The process is thus governed by conditions that are not easily adjusted from the standpoint of in vitro drug release testing (e.g., to vary concentration gradient, surface area, etc.), thereby limiting the utility and future avenues for development of conventional IVRT methods.
In contrast, embodiments employing the disclosed AP technique provide a readily-customizable testing platform that does not rely on diffusion for separation and can offer a more complete picture of release characteristics of a drug product by simultaneously measuring components in both the retentate and permeate. In some embodiments, the TFF filter of the AP technique is subject to conditioning with surfactant prior to and after use, which conditioning can reduce variability between experimental runs and/or different filters, thereby improving testing precision, accuracy, or both.
In some embodiments, an AP technique can offer an adjustable rate of removal (e.g., during an experimental run or between experimental runs). The ability to adjust removal rate for each experimental run can allow a user to optimize the feed flow rate and/or the permeate flow based on the type of dosage form under test. For example, higher flow rates can be selected to quickly remove released drug for drug product formulations that exhibit rapid release (e.g., micelle phase within an emulsion that readily releases the drug within a few minutes). Alternatively or additionally, flow rate can be adjusted during an experimental run (e g, manually by a user or automatically by a controller of the AP system) to adjust the rate of drug removal to the permeate, for example, for those drug product formulations that may have multi-phasic release kinetics.
In some embodiments, an AP technique can offer selective retention of components of the drug product formulation. In particular, some preferred components of the drug product formulation can be retained by appropriate selection of the MWCO range of the TFF filter employed. For example, castor oil globules in the nanoemulsions or protein-bound drug containing nanoparticles can be selectively retained by the TFF filter on a retentate-side of the membrane therein and recirculated in a fluid loop connected to the TFF filter, which is otherwise being continuously diluted with fresh media to compensate for any fluid loss via the permeate flow from the TFF filter. This process can assist in effectively differentiating between rapid drug release and extended phase drug release. As noted above, embodiments employing the AP technique allows for performing size-based separation while simultaneously analyzing the drug release from the separated components (e.g., in permeate and retentate).
In some embodiments, an AP technique can provide an environment similar to in vivo conditions. In the AP technique, a continuous flow is provided by the dilution of the drug product sample concurrent with continuous removal of any released drug to a permeate side of the TFF filter, while any remaining drug on a retentate side of the TFF filter is recirculated within the system. Such continuous flow may effectively mimic in vivo conditions experienced by the drug product in actual use, for example, continuous dilution of the drug product at the ocular surface due to tear turnover and continuous absorption of drug after releasing from the complex formulations (e.g., emulsions). In another example, the experience of the drug product within the AP system can be similar to the drug release conditions experienced in parenteral drug delivery, where release of drug into the blood stream occurs during circulation and drug removal occurs due to absorption at the target tissue or organ.
In some embodiments, an AP technique can provide controllable dilution, for example, by altering a rate of permeate flow (e.g., via adjustment of feed flow rate into the TFF filter, adjustment of backpressure applied to flow from an outlet of the TFF filter, or both). Dilution ratio during release testing may be especially critical for certain dosage forms, such as ophthalmic drugs. For example, dilution of nanosuspensions can be give rise to rapid (e.g., near instantaneous) dissolution of the nanoparticles and may thus diminish the potential for differentiating between formulations. Moreover, for nanoemulsions, the initial equilibrium states of the oil/aqueous phases present in the formulation may govern the drug distribution in each phase, with high dilution ratios at initiation of an IVRT testing potentially masking differences in drug release between formulations if the rate of drug removal across the membrane is not rapid enough. Thus, in some embodiments employing the AP technique, a controllable rate of dilution can be achieved by optimizing the rate of permeate flow so that the sample is initially diluted at a lower ratio, and the dilution ratio can be increased to a higher level (e.g., in a linear, step-wise, or any other manner) as the testing progresses. The ability to customize dilution level during an experimental run allows embodiments employing the AP technique to discern minor differences in drug release profiles (e.g., in emulsions with different globule sizes).
In some embodiments, an AP technique can provide selective evaluation of excipient impact on drug release. By appropriate selection of MWCO of the TFF filter used in the AP technique, complex excipients (e.g., polymers composed of hydrophilic and hydrophobic monomers) can be selectively retained on the retentate side of the TFF filter based on their molecular weight and recirculated within the system. The impact of these excipients on drug release can be then be studied. For example, compositional or manufacturing process changes can be made to a particular drug product formulation, and the resulting impact on drug release can be studied using the AP technique.
In some embodiments, an AP technique can minimize, or at least reduce, degradation of the drug during release thereof. As noted above, the AP technique allows for custom tuning of the feed flow rate and the permeate flow rate. Fresh media is supplied as replacement fluid to compensate for volume lost to the permeate flow rate, and thus the fresh media supply rate can also be tuned. The fresh media supply rate governs the rate at which the drug product sample gets diluted with media, as well as the duration for completion of the IVRT experimental run. For drugs that may be susceptible to degradation when exposed to large media volumes for prolonged periods (e.g., as suggested by
An outlet of the feed reservoir 104 is coupled to an inlet of first pump 134 (e.g., a peristaltic pump) via fluid conduits 106, 108. The outlet of the first pump 134 is coupled to an inlet 110 of TFF filter 112, and pressure sensor 136 can be configured to measure a pressure at the inlet 110 and provide a sensor signal responsively thereto. The inlet 110 of TFF filter 112 directs fluid to a first volume 118 (e.g., feed stream or retentate volume) on a retentate-side of each membrane filter 116. Each membrane filter 116 separates the first volume 118 from a second volume 114 (e.g., filtered or permeate volume), wherein fluid and other components therein that have a molecular weight less than a MWCO of membrane filter 116 can pass therethrough from the first volume 118 to the second volume 114. The second volume 114 connects to a first outlet 120, through which permeate in the second volume 114 can flow from the TFF filter 112 to permeate reservoir 144 for collection and/or sample testing. Pressure sensor 137 is coupled to the flow-path between the first outlet 120 and permeate reservoir 144, for example, along fluid conduit 140. Pressure sensor 137 can be configured to measure the pressure at the outlet 120 and provide a sensor signal responsibly thereto. Flux sensor 146 is disposed along the flow-path between the first outlet 120 and permeate reservoir 144, for example, along fluid conduit 142. The flux sensor 146 can be configured to measure a rate of permeate flow (e.g., fluid flow rate or mass flow rate) and provide a sensor signal responsively thereto.
An outlet of fresh media reservoir 148 is coupled to an inlet of second pump 152 (e.g., a peristaltic pump) via fluid conduit 150. The outlet of second pump 152 is coupled to an inlet of feed reservoir 104, such that fresh media from reservoir 148 can be supplied to the feed reservoir 104 by second pump 152 to compensate for any fluid lost to the permeate flow through filter membrane 116, thereby maintaining a substantially constant volume in feed reservoir 104 (and for combination of feed reservoir 104 and the fluid circuit and filter volumes 118 on a retentate side of membrane filter 116).
The second volume 114 can also connect to a third outlet 124, which is disposed at an opposite end of the TFF filter 112 from the second outlet 120 and at a same end of the TFF filter 112 as the inlet 110. As described in further detail below, the third outlet 124 can be selectively opened, for example, via valve 164 (e.g., a multi-position valve or an open-close valve) for pre-testing or post-testing processing of the TFF filter 112. In the illustrated configuration, the third outlet 124 is connected to a reservoir 168 via fluid conduit 166; however, in some configurations, the third outlet 124 can instead be directed to waste (e.g., where fluid conduit 166 serves as drain line for waste) or directly connected to fluid conduit 170 without an intervening reservoir 168.
At an opposite end of the TFF filter 112 from inlet 110, a second outlet 122 connects to the first volume 118, through which retentate in the first volume 118 can flow from the TFF filter 112. The second outlet 122 is connected back to the feed reservoir 104 via fluid conduits 126, 128, and 132, thereby forming a recirculating fluid circuit or fluid loop by which retentate from TFF filter 112 can be returned to inlet 110 for repeated filtration processing. Pressure sensor 138 can be configured to measure a pressure at the second outlet 122 and provide a sensor signal responsively thereto. Back-pressure valve 130 is disposed along the flow-path between the second outlet 122 and the feed reservoir 104 and is configured to apply a back pressure to the second outlet 122, for example, by reducing a cross-sectional area for fluid flow through conduit 128.
Valves 158, 160, 164, 172 provided at various positions around the fluid circuit with respect to inlet 110 and outlets 120, 122, 124 of the TFF filter 112 can be used to reconfigure the fluid circuit according to different operational modes. One, some, or each of the valves 158, 160, 164, 172 can thus be a multi-position valve (e.g., three or four position valve) capable of directing flows along multiple different paths. During an IVRT operational mode, the valves 158, 160, 164, and 172 are configured as shown in
In some embodiments, the fluid circuit can be provided with additional valves (not shown), for example, to connect the fluid circuit to different fluid sources, such as a de-ionized water source, a source of organic solvent, a source of conditioning fluid, etc. Alternatively, in some embodiments, one or more of valves 158, 160, 164, 172 can be omitted, for example, when the fluid circuit is manually reconfigured by a user according to a desired operational mode.
Operation of the system 100 and its various components can be controlled by a controller 156. As such, controller 156 can be operatively coupled to pumps 134, 152, pressure sensors 136, 137, 138, backpressure valve 130, valves 158, 160, 164, 172, or any combination thereof in order to receive sensor or status signals therefrom and send command or control signals thereto. For example, during an IVRT operational mode, the controller 156 can automatically control pump 152 to provide fresh media to feed reservoir 104 from replacement fluid reservoir 148 at a rate that substantially corresponds to a permeate flow rate measured by flux sensor 146 (e.g., using any type of controller loop feedback mechanism, such as proportional-integral-derivative control).
Controller 156 may also control injection device 102 (e.g., for providing an initial sample of drug product to feed reservoir 104) and/or sampling devices 154, 155 (e.g., for periodically obtaining aliquots for analysis). For example, injection device 102 and/or sampling devices 154, 155 can be a syringe pump, automated pipette, or any other liquid handling device that can deliver fluid to and/or retrieve a portion of fluid from a reservoir. Although shown as interacting with reservoir 144, in some embodiments, sampling device 155 is configured to obtain an aliquot for analysis from the permeate flow from outlet 120 (e.g., within fluid conduit 142) rather than from the permeate collected in reservoir 144. Similarly, although shown as interacting with reservoir 104, in some embodiments, sampling device 154 is configured to obtain an aliquot for analysis from the retentate flow into inlet 110 (e.g., within fluid conduit 106 or 108) or out of outlet 122 (e.g., within fluid conduit 126, 128, or 132).
In some embodiments, weight can be monitored instead of or in addition to permeate flow rate in order to regulate fresh media flow rate. For example, feed reservoir 104 can have a first sensor 174 that monitors a weight thereof, and/or permeate reservoir 144 can have a second sensor 176 that monitors a weight thereof. Controller 156 can control pump 152 to provide fresh media to feed reservoir 104 to maintain a constant weight of feed reservoir 104 based on signals from first sensor 174, and/or to match changes in weight of permeate reservoir 144 based on signals from second sensor 176. In another example, during an IVRT operational mode, the controller 156 can control pump 134 and back-pressure valve 130 responsively to pressure signals from pressure sensors 136, 138, so as to provide a desired transmembrane pressure for TFF filter 112.
The controller 156 can operate system 100 to provide three generally sequential modes of operation—a pre-conditioning mode, an IVRT mode, and a re-conditioning mode. The pre-conditioning mode can involve washing and conditioning of the TFF filter by using a specific medium and with the TFF filter in a particular configuration. For example, the pre-conditioning mode can be performed as described below with respect to
In the IVRT mode, the aspects of the process can be regulated by controller 156 to control drug release. This regulation includes swift initiation of feed flow from feed reservoir 104 upon initial sample dilution (e.g., injection via device 102), supply of fresh media from reservoir 148 to compensate for volume loss due to permeate flow via outlet 120, and monitoring of permeate flow. In addition to providing feedback for replacement fluid, the monitoring of the flow rate through the permeate outlet can also be used to calculate the amount of drug released (or at least provide an approximation thereof based on aliquot analysis), for example, cumulative mass of drug released can be calculated using the following equation: cumulative mass of drug released=Σi=1n(permeate flux)i×(timei−timei-1)× (measured permeate concentration)i
During the IVRT mode, a portion of the sample containing dispersed particles smaller than the pores of the filter membrane 116 pass through into the permeate volume 114, whereas larger particles are recirculated back to the feed reservoir 104 in a continuous loop. The pressure gradient across the filter membrane 116 and the size-based separation can be tailored to the type of drug product formulation being analyzed, for example, by appropriate selection of feed flow rates, backpressure, or the MWCO of the filter membrane. For example, for drug products wherein a rapid rate of drug release is expected, the feed flow can be adjusted to higher flow rates, whereas lower feed flow rates can be used when slow drug release is expected. Even though the sample is circulated in a closed loop in system 100, the simultaneous size based separation and the concurrent dilution of the sample can overcome any media volume restrictions and otherwise provide substantially continuous sink conditions.
During the IVRT mode, samples (e.g., aliquots) are periodically obtained from the feed supply reservoir 104 by sampling device 154 and from the permeate flow from outlet 120 by sampling device 155. The obtained samples can then be analyzed to determine an amount of drug that has been released to the permeate as well as the remaining drug in the retentate that has not yet been released. In some embodiments, the samples can be collected and provided to separate setup 180 for analysis. For example, a batch 184 of samples from an entire IVRT run can be provided to a data analysis system 182 (e.g., ultra-performance liquid chromatography system (UPLC)) for sequential or parallel analysis. For example, the batch 184 of samples can be provided in a microtiter plate, well plate, or any other type of device that can contain multiple aliquots (in an organized arrangement or otherwise) for analysis by liquid chromatography (e.g., high-performance liquid chromatography (HPLC), UPLC, etc.), mass spectrometry, or any other analysis technique (or combination thereof). Alternatively or additionally, each aliquot can be sent for analysis by system 182 without otherwise waiting for other time-sequence samples to compose a batch.
In some embodiments, instead of separate analysis system 182 or in addition thereto, system 100 can be provided with one or more sensors to monitor in situ drug concentration, for example, in real-time or substantially real-time. In such embodiments, the withdrawal of separate aliquots using sampling devices 154, 155 may not be necessary. For example, system 100 can include a first concentration monitoring sensor 186 coupled to the flow-path between the first outlet 120 and permeate reservoir 144, for example, along fluid conduit 142. First concentration monitoring sensor 186 can be configured to measure drug concentration and/or other compositional details of the permeate flowing from outlet 120 to permeate reservoir 144 and to provide a sensor signal responsibly thereto. Alternatively or additionally, system 100 can include a second concentration monitoring sensor 188 coupled to the feed reservoir 104. Second concentration monitoring sensor 188 can be configured to measure drug concentration and/or other compositional details of the retentate within the feed reservoir 104. For example, the first and second concentrating monitoring sensors 186, 188 can be an in situ fiber-optic ultra-violet (UV) sensor, such as or similar to a fiber optic UV-Vis system (Pion μDiss Profiler™, Billerica, MA). It will be appreciated that the configuration, connections, and components of the system 100 are exemplary only, and other configurations, connections, and components can be used to perform AP for IVRT, according to one or more contemplated embodiments. Moreover, operational parameters, configurations, and other options for the IVRT mode and associated system 100 performing the AP technique can be selected to provide optimal results for a particular drug release test and/or particular drug product formulation. For example, the operational parameters, configurations, and other options include feed flow rates, backpressure, initial sample-to-media dilution ratio, tubing lengths, solvents for pre-conditioning, and sequence of pre-conditioning. Selection of an appropriate TFF filter can also depend upon drug properties, dosage form (e.g., emulsions vs. suspensions), filter membrane chemistry, and TFF filter types (length, surface area, internal volume, MWCO and pressure capacity).
Available variables or options for conditioning of the filter can include, but are not limited to, surfactant (e.g., type), concentration (e.g., % w/v), and configuration of the fluid circuit connected to the filter during the conditioning process (e.g., open loop, partial-open loop, or closed loop). In some embodiments, the surfactant can be polysorbate-80 at a concentration of 0.07% w/v, and the fluid circuit configuration can be partial-open loop, for example, as described below with respect to
Available variables or options for performing adaptive perfusion can include, but are not limited to, feed flow rate (e.g., ml/min), applied backpressure, and sample dilution (e.g., media-to-sample dilution ratio). The selected feed flow rate and applied backpressure can be a function of the type of drug product to be analyzed (e.g., nanoemulsions versus pure drug solution), for example, to define a removal rate (e.g., permeate flow rate) that substantially corresponds to an anticipated release rate for a particular drug product. Since the pressure difference between the retentate and permeate sides of the membrane filter (e.g., the transmembrane pressure) is a function of both feed flow rate and applied backpressure, the feed flow rate and backpressure can be selected to maximize flux values (or at least achieve a desired minimum permeate flow) through the membrane filter without otherwise exceeding the manufacturer-defined limitations of the filter (e.g., maximum operating pressure and/or maximum operating flow rate). Dilution can also be tailored to the drug product being analyzed, as well as to enhance filter performance In some embodiments, the feed flow rate can be at least 100 ml/min, for example, 200 ml/min, and the sample dilution can be 200:1 on a volume basis. The 200:1 dilution can help reduce the possibility of membrane fouling and can increase overall efficiency of the adaptive perfusion process as compared to lower dilution rates (e.g., 50:1).
Available variables or options for the filter can include, but are not limited to, the configuration of the TFF module (e.g., hollow fibers, spiral wound cartridge, flat plate or cassette, etc.), water affinity (e.g., hydrophilic versus hydrophobic, and the various modifications therefor), size (e.g., diameter, effective length, surface area, etc.), and molecular weight cut-off (MWCO). The MWCO of the filter can be selected based on the drug product to be analyzed (e.g., based on the globule size range of nanoemulsions), in particular, such that released drug particles can pass through the filter into the permeate while unreleased drug particles can be retained on the retentate-side of the filter. In some embodiments, the MWCO can be 100 kD, which may be less susceptible to fouling than higher MWCO values (e.g., 300 kD or 500 kD)
In some embodiments, the water affinity of the filter membrane can be hydrophilic resulting from modified polyethersulfone (mPES), which enable higher flux rates, increase resistance to fouling, and/or lower drug adsorption to the membrane, among other things. Alternative water-affinity modifications can include, but are not limited, to mixed cellulose ester (ME) and polyethersulfone (PES) for hydrophilic and polysulfone (PS) for hydrophobic.
In some embodiments, the TFF filter module can have a hollow fiber configuration, with each fiber having a diameter of 0.5 mm. In some embodiments, the hollow fiber configuration can provide an effective length of 20 cm and a surface area of 20 cm2. The hollow fiber configuration may yield a simpler flow path and lower void volumes as compared to other configurations. For example,
Each fiber 206 has a substantially cylindrical wall surrounding a hollow interior volume (e.g., retentate volume). The cylindrical wall of the fiber is formed of a filter membrane that provides the desired size separation. For example, as shown in the configuration 250 of
In some embodiments, controller 156 of system 100 automate most or all of the AP technique, including adjustment, monitoring, and recording of various operational parameters and coordinating timing of various steps in the AP process. For example,
The controller 156 can have one or more modules or sub-modules that coordinate respective functions associated with performance of an AP process. In the illustrated example of
Pump control sub-module 310 is configured to generate control signals for pump 134 and pump 152. For example, pump control sub-module 310 sends a control signal to pump 134, via I/O interface 302, to provide a user- or system-defined feed flow rate to TFF filter 112 in
Back-pressure control sub-module 312 is configured to generate control signals for back-pressure valve 130. For example, back-pressure control sub-module 312 sends a control signal to valve 130, via I/O interface 302, to provide a user- or system-defined back pressure at the outlet 122 of TFF filter 112 in
Valve control sub-module 314 is configured to generate control signals for valves 160, 164, 172. For example, valve control sub-module 314 sends a control signal to valves 160, 164, 172, via I/O interface 302, to change a configuration of the fluid circuit between open loop, partial open-loop, and closed loop configurations, depending on the mode of operation of system 100 in
Moreover, in some embodiments, functions performed by one of the sub-modules of AP module 304 illustrated in
AP module 304 further includes process flow or recipe details for each operational mode (e.g., as shown in
In some embodiments, the controller 156 can interface with the sample analysis system 182. For example, when system 100 and analysis system 182 are part of a common system, controller 156 can coordinate delivery of aliquots obtained using sampling devices 154, 155 to analysis system 182 for subsequent analysis. Alternatively or additionally, controller 156 can control both systems 100 and 182, and controller 156 can include an analysis module separate from the AP module 304 for controlling operation of sample analysis system 182. In another example, controller 156 can be configured to receive data from sample analysis system 182, for example, to display to a user via user interface 306 and/or store in an appropriate data storage device.
In some embodiments, the user interface 306 can be generated by controller 156, for example, using a display and associated input device (e.g., touch screen, mouse, etc.). The user interface 306 enables a user to monitor performance and specify parameters for operation of system 100. For example,
With reference to
A computing system may have additional features. For example, the computing environment 320 includes storage 360, one or more input devices 370, one or more output devices 380, and one or more communication connections 390. An interconnection mechanism (not shown) such as a bus, controller, or network interconnects the components of the computing environment 320. Typically, operating system software (not shown) provides an operating environment for other software executing in the computing environment 320, and coordinates activities of the components of the computing environment 320.
The tangible storage 360 may be removable or non-removable, and includes magnetic disks, magnetic tapes or cassettes, CD-ROMs, DVDs, or any other medium which can be used to store information in a non-transitory way, and which can be accessed within the computing environment 320. The storage 360 can store instructions for the software 325 implementing one or more innovations described herein.
The input device(s) 370 may be a touch input device such as a keyboard, mouse, pen, or trackball, a voice input device, a scanning device, or another device that provides input to the computing environment 320. The output device(s) 380 may be a display, printer, speaker, CD-writer, or another device that provides output from computing environment 320.
The communication connection(s) 390 enable communication over a communication medium to another computing entity. The communication medium conveys information such as computer-executable instructions, audio or video input or output, or other data in a modulated data signal. A modulated data signal is a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal. By way of example, and not limitation, communication media can use an electrical, optical, RF, or other carrier.
Any of the disclosed methods can be implemented as computer-executable instructions stored on one or more computer-readable storage media (e.g., one or more optical media discs, volatile memory components (such as DRAM or SRAM), or non-volatile memory components (such as flash memory or hard drives)) and executed on a computer (e.g., any commercially available computer, including smart phones or other mobile devices that include computing hardware). The term computer-readable storage media does not include communication connections, such as signals and carrier waves. Any of the computer-executable instructions for implementing the disclosed techniques as well as any data created and used during implementation of the disclosed embodiments can be stored on one or more computer-readable storage media. The computer-executable instructions can be part of, for example, a dedicated software application or a software application that is accessed or downloaded via a web browser or other software application (such as a remote computing application). Such software can be executed, for example, on a single local computer (e.g., any suitable commercially available computer) or in a network environment (e.g., via the Internet, a wide-area network, a local-area network, a client-server network (such as a cloud computing network), or other such network) using one or more network computers.
For clarity, only certain selected aspects of the software-based implementations are described. Other details that are well known in the art are omitted. For example, it should be understood that the disclosed technology is not limited to any specific computer language or program. For instance, aspects of the disclosed technology can be implemented by software written in C++, Java, Perl, any other suitable programming language. Likewise, the disclosed technology is not limited to any particular computer or type of hardware. Certain details of suitable computers and hardware are well known and need not be set forth in detail in this disclosure.
It should also be well understood that any functionality described herein can be performed, at least in part, by one or more hardware logic components, instead of software. For example, and without limitation, illustrative types of hardware logic components that can be used include Field-programmable Gate Arrays (FPGAs), Program-specific Integrated Circuits (ASICs), Program-specific Standard Products (ASSPs), System-on-a-chip systems (SOCs), Complex Programmable Logic Devices (CPLDs), etc.
Furthermore, any of the software-based embodiments (comprising, for example, computer-executable instructions for causing a computer to perform any of the disclosed methods) can be uploaded, downloaded, or remotely accessed through a suitable communication means. Such suitable communication means include, for example, the Internet, the World Wide Web, an intranet, software applications, cable (including fiber optic cable), magnetic communications, electromagnetic communications (including RF, microwave, and infrared communications), electronic communications, or other such communication means. In any of the above described examples and embodiments, provision of a request (e.g., data request), indication (e.g., data signal), instruction (e.g., control signal), or any other communication between systems, components, devices, etc. can be by generation and transmission of an appropriate electrical signal by wired or wireless connections.
The method 500 can initiate at process block 502, where the fluid circuit connected to the TFF filter is set in partial open loop configuration. For example, in AP system 100, valves 158 and 160 can be switched to connect together the outlet 122 (e.g., the retentate outlet) and outlet 120 (e.g., the first permeate outlet), and valve 164 can be switched to connect outlet 124 (e.g., the second permeate outlet) to a waste line or container (e.g., reservoir 168).
Returning to
To perform process block 506 in AP system 100, a water supply can be connected to an inlet of pump 134 (e.g., using valve 172 or another valve, or by filling feed supply 104 with a volume of DI water), and pump 134 can be operated to direct the DI water into inlet 110 of TFF filter 112. Similarly, to perform process block 506 in the partial open loop configuration 520 for HFF 200 in
Returning to
The method 500 can proceed from process block 508 to process block 510, where the TFF filter in the partial open loop configuration is flushed with a third volume of DI water, without any externally applied backpressure. In some embodiments, the flushing of process block 510 may be considered to be a rinsing process, for example, to remove any remaining organic solvent from within the TFF filter. The third volume can be based on a volume of the TFF filter and/or the fluid circuit connected thereto. For example, in some embodiments, the third volume can be at least 5-10 times the volume of the TFF filter. In an exemplary embodiment, the third volume is 1000 mL of DI water for a TFF filter having a volume of 100 mL or less. The performance of process block 510 for AP system 100 in
The method 500 can proceed from process block 510 to process block 512, where the TFF filter in the partial open loop configuration is flushed with a fourth volume of conditioning solution, without any externally applied backpressure. In some embodiments, the flushing of process block 512 may be considered to be a conditioning process to prepare the TFF filter for subsequent use. Flushing with conditioning solution in the partial open loop configuration allows both internal surfaces (e.g., retentate side) and external surfaces (e.g., permeate side) of the membrane filter to be conditioned, which can reduce membrane fouling during subsequent use of the TFF filter as compared to flushing in an open loop configuration (e.g., as shown in
The method 500 can proceed from process block 512 to process block 514, where the TFF filter in the partial open loop configuration is flushed with a fifth volume of DI water, without any externally applied backpressure. In some embodiments, the flushing of process block 514 may be considered to be a rinsing process, for example, to ensure that conditioning solution is fully removed from the TFF filter to avoid adversely impacting IVRT or other subsequent use of the TFF filter. In some embodiments, the flushing of process block 514 may be performed just prior to use of the TFF filter, for example, after calibration of system 100 and before filling the TFF filter with media in performing an IVRT. The fifth volume can be based on a volume of the TFF filter and/or the fluid circuit connected thereto. For example, in some embodiments, the fifth volume can be at least 10-20 times the volume of the TFF filter. In an exemplary embodiment, the fifth volume is 2000 mL of DI water for a TFF filter having a volume of 100 mL or less. The performance of process block 514 for AP system 100 in
Returning to decision block 504, if the TFF filter is a previously used filter that has been subject to re-conditioning (e.g., as described with respect to
Although blocks 502-516 of method 500 have been described as being performed once, in some embodiments, multiple repetitions of a particular process block may be employed before proceeding to the next decision block or process block. Moreover, although
In addition, although
As part of the setup of process block 602, the TFF filter in the open loop configuration can be flushed with a volume of fresh media (e.g., 10 mM pH 7.4 phosphate buffer mixed with ethanol in the ratio of 80:20 v/v), with applied backpressure. In some embodiments, the flushing with fresh media may be considered to be part of a water removal process, for example, to remove any residual DI water remaining from process block 514 of the pre-conditioning method 500. The volume of fresh media can be based on a volume of the TFF filter and/or the fluid circuit connected thereto. For example, in some embodiments, the fresh media volume for flushing can be at least 5 times the volume of the TFF filter. In an exemplary embodiment, the fresh media volume is 500 mL of fresh media for a TFF filter having a volume of 100 mL or less, and the flushing is at a flow rate of 150 mL/min. In some embodiments, rather than returning the fresh media in loop 650 to the feed reservoir 642, the fluid can simply be discarded after back pressure valve 652. Moreover, in some embodiments, after completion of the flushing with fresh media, the fluid conduits connected to the TFF filter can be emptied to remove any residual fluid that may otherwise contribution to sample dilution during subsequent IVRT.
As part of the setup of process block 602, or prior thereto, other components of the system, such as pumps, sensors, etc., can be prepared for use, for example, by initializing (e.g., turning on sensors) and/or performing calibration thereof. For example, tubing calibration can be performed for first pump 134 and second pump 152 to ensure delivery of accurate volumes to TFF filter 112 and feed reservoir 104, respectively. In another example, flux sensor 146 can be calibrated to ensure provision of an accurate permeate flow rate measurement. In some embodiments, the flux sensor calibration can occur at the same time as the fresh media flush.
The method 600 can proceed from process block 602 to process block 604, where a sample of a drug product (e.g., pure drug solution, drug loaded micelles, nanoemulsion, etc.) is provided for IVRT. For example, in AP system 100, a volume of the drug product sample can be spiked into a fixed volume of media in feed reservoir 104 using device 102 or manually. In some embodiments, the feed reservoir can have a stirring mechanism (e.g., a magnetic stirrer) that continuously stirs the contents thereof (e.g., stirred at 1400 rpm). The initial volume of media in the feed reservoir 104 can be selected, for example, based on the dosage form type of the sample, a desired dilution ratio, and/or a desired rate of drug removal (e.g., depending on anticipated drug release characteristics of the sample).
The method 600 can then proceed to process block 606, where an aliquot is obtained to assay for initial drug concentration. In some embodiments, process block 606 may be performed a short time period (e.g., less than 30 seconds, for example, 10 seconds) after introduction of the drug product sample into the media, for example, to allow sufficient time for mixing without the drug product sample otherwise undergoing substantial release. For example, in AP system 100, an aliquot from the feed reservoir 104 can be obtained using sampling device 154 or manually.
The method 600 can then proceed to process block 608, where the AP system is operated to provide IVRT by simultaneous performance of sub-process blocks 610-618. In particular, at sub-process block 610, fluid is pumped from the feed reservoir that initially has the diluted sample therein to a retentate-side inlet of the TFF filter. For example, the feed flow rate to the TFF filter from the feed reservoir can be 200 mL/min. In some embodiments, instead of a continuous feed flow through the filter, a pulsed flow approach could be employed, where the feed flow rate through the filter periodically varies between a maximum value and a minimal or zero value, either in a continuous curve (e.g., sinusoidal, triangular wave, etc.) or step-wise manner, for example, by regulating operation of the peristaltic pumps to increase pulsation effects. Such pulsed flow approaches may further improve filter performance by reducing membrane fouling.
At sub-process block 612, back pressure is applied to the retentate-side outlet of the TFF filter. For example, the back pressure can be adjusted to provide a desired transmembrane pressure for a given feed flow rate. In some embodiments, the feed flow rate and back pressure can be optimized to provide a desired removal rate (e.g., permeate flow rate based on release characteristics of the drug product sample) while ensuring that the inlet pressure to the TFF filter does not exceed specified pressure limits of the TFF filter (e.g., 30 psi). At sub-process block 614, fluid exiting from the retentate-side outlet of the TFF filter is returned to the feed reservoir by way of a recirculating fluid circuit. At sub-process block 616, fluid exiting from the permeate-side outlet of the TFF filter is collected, and a flow rate from the permeate-side outlet can be monitored. At sub-process block 618, fluid is pumped from replacement fluid reservoir to feed reservoir based on the monitored permeate-side flow rate in order maintain a constant volume in the feed reservoir. In some embodiments, fluid can initially be pumped from replacement fluid reservoir to feed reservoir to account for any volume drop in the feed reservoir due to dead volume in the fluid circuit and/or TFF filter. As such, the initial pumping of replacement fluid may be independent of the monitored permeate-side flow rate.
To perform process block 608 in AP system 100, pump 134 can operate to convey media with diluted drug product therein to inlet 110 of TFF filter 112. Back pressure applied via valve 130 to the outlet 122 of TFF filter 112 creates a transmembrane pressure that drives fluid and particles having a molecular weight less than a MWCO of filter membrane from retentate volume 118 to permeate volume 114. The permeate exits TFF filter 112 via outlet 120 to permeate reservoir 144, while flux sensor 146 monitors a rate of flow to the reservoir 144. Pump 152 can operate to convey fresh media from replacement fluid reservoir 148 to feed reservoir 104, using the signal from flux sensor 146 as feedback, to maintain a substantially constant volume in feed reservoir 104.
Similarly, to perform process block 608 in the open loop configuration 640 for HFF 200 in
Returning to
If a time interval has been reached, the method 600 can proceed from decision block 620 to process block 622, where aliquots are obtained from both the feed reservoir and the permeate flow. For example, in AP system 100, an aliquot from the feed reservoir 104 can be obtained using sampling device 154 or manually, an aliquot from the permeate flow in outlet conduit 142 can be obtained using sampling device 155 or manually. In some embodiments, the obtained aliquots can be sent for immediate analysis, for example, by separate analysis system 182. Alternatively, in some embodiments, the aliquots from different time intervals are accumulated and sent for analysis at the conclusion of the IVRT run. For example, the obtained aliquots can be stored in a microtiter plate, well plate, or any other type of device that can contain multiple aliquots (in an organized arrangement or otherwise) for analysis by liquid chromatography (e.g., high-performance liquid chromatography (HPLC), UPLC, etc.), mass spectrometry, or any other analysis technique (or combination thereof). Alternatively or additionally, in some embodiments, concentrations within fluid in the system can be periodically or continuously monitored in situ. For example, in AP system 100, concentration monitoring sensors 186, 188 can interrogate fluid in the feed reservoir 104 and permeate flow in outlet conduit 142, respectively, in order to determine a concentration of drug therein (or other compositional details). In such embodiments, the obtaining of separate aliquots of process block 622 may be optional.
Once initiated, process block 608 may be performed in a substantially continuous manner until conclusion of the IVRT run, for example, due to expiration of a predetermined time limit or based on a user command. At decision block 620, it can be further evaluated if the time limit for the IVRT run has been reached. If so, the method 600 can proceed from decision block 620 to process block 624, where performance of process block 608 is stopped and final aliquots and measurements are obtained. In some embodiments, both feed pump and replacement fluid pump are stopped at the same time, and the monitoring systems (e.g., pressure and flux sensors) can be turned off or otherwise idled. The fluid conduits connecting the TFF filter to the feed reservoir can be detached from the TFF filter, and fluid therein can be allowed to completely drain into the feed reservoir. In some embodiments, at the end of the IVRT run, an aliquot can be taken from the permeate reservoir and the total volume of permeate can be measured in order to determine the amount of cumulative drug release. The aliquot can be collected for analysis with the other aliquots from performance of process block 622 or can otherwise be separately sent for analysis. The method 600 can thus conclude at 630, subject to any system de-commissioning and/or TFF filter re-conditioning in preparation for subsequent IVRT.
Although blocks 602-624 of method 600 have been described as being performed once, in some embodiments, multiple repetitions of a particular process block may be employed before proceeding to the next decision block or process block. Moreover, although
The method 700 can initiate at process block 702, where the fluid circuit connected to the TFF filter is set in the partial open loop configuration. For example, in AP system 100, valves 158 and 160 can be switched to connect together the outlet 122 (e.g., the retentate outlet) and outlet 120 (e.g., the first permeate outlet), and valve 164 can be switched to connect outlet 124 (e.g., the second permeate outlet) to a waste line or container (e.g., reservoir 168).
Returning to
To perform process block 704 in AP system 100, an organic solvent supply can be connected to an inlet of pump 134 (e.g., using valve 172 or another valve, or by replacing the contents feed supply 104 with organic solvent), and pump 134 can be operated to direct the organic solvent into inlet 110 of TFF filter 112. Similarly, to perform process block 704 in the partial open loop configuration 520 for HFF 200 in
Returning to
The method 700 can then proceed from either process block 708 or process block 710 to process block 712, where the fluid circuit connected to the TFF filter is set in a closed loop configuration. For example, in AP system 100, valves 158 and 160 can maintain their configuration that connect the outlet 122 (e.g., the retentate outlet) and outlet 120 (e.g., the first permeate outlet) together, and valves 164 and 170 can be switched to connect inlet 110 (e.g., the feed inlet) and outlet 124 (e.g., the second permeate outlet) to a common reservoir 168.
Returning to
To perform process block 714 in AP system 100, solvent reservoir 168 can be filled with the desired volume of organic solvent, which reservoir 168 is connected to an inlet of pump 134 via valve 172. Pump 134 can then be operated to direct the organic solvent from reservoir into inlet 110 of TFF filter 112. Similarly, to perform process block 714 in the closed loop configuration 740 for HFF 200 in
Returning to
Returning to decision block 730, if the TFF filter will not be used within 24 hours, the method 700 can proceed to process block 724, where the TFF filter in the partial open loop configuration is flushed with diluted organic solvent, for example, in a manner similar to that described above for process block 508 in
Although blocks 702-730 of method 700 have been described as being performed once, in some embodiments, multiple repetitions of a particular process block may be employed before proceeding to the next decision block or process block. Moreover, although
In addition, although
Materials: Difluprednate (>97%) was purchased from RIA International LLC (East Hanover, NJ). Castor oil was purchased from Fisher (Pittsburgh, PA). Polysorbate-80 was purchased from Acros Organics (Morris Plains, NJ). Glycerin, sodium acetate, boric acid, edetate disodium and sodium dodecyl sulfate were purchased from Fisher Scientific (Waltham, MA). Sorbic acid was obtained from MP Biomedicals (Solon, OH). Sodium dihydrogen phosphate and sodium hydroxide (1N and 2N) was purchased from Sigma Aldrich (St. Louis, MO). Phosphoric acid was obtained from EMD Millipore Corporation (Burlington, MA). Acetonitrile was obtained from Fisher Scientific (Waltham, MA). Ethanol was procured from Decon Labs (King of Prussia, PA). Deionized water was obtained from Milli-Q Ultrapure Water Systems, EMD Millipore Corporation (Burlington, MA). Unless otherwise specified, all materials were of analytical grade.
Preparation of drug loaded micelles: Difluprednate micelles were prepared by dispersing and sonicating difluprednate powder in 3.6% w/v polysorbate-80 solution in deionized water at room temperature. The final concentration of difluprednate in the polysorbate-80 micelles was determined to be about 100 μg/mL, by ultra-performance liquid chromatography (UPLC) method (Method 1).
Preparation of nanoemulsions: Three nanoemulsions with different globule size distributions were manufactured by microfluidization process, while keeping their composition qualitatively (Q1) and quantitatively (Q2) the same as the reference listed drug, Durezol®. Briefly, primary emulsion was formed by high-shear mixing of the aqueous phase-I (containing a mixture of glycerin, polysorbate-80, and deionized water) with the oil phase (castor oil containing difluprednate) by maintaining the temperature at 65°−70° C. Primary emulsion was further mixed with the aqueous phase-II (containing sorbic acid, sodium acetate, boric acid and edetate disodium dissolved in deionized water) maintained at 65°−70° C. to obtain a coarse emulsion. pH of the coarse emulsion was then adjusted to 5.2 to 5.8 at room temperature by using 1N sodium hydroxide. Thereafter, the coarse emulsion was subjected to microfluidization process, with precise control over the variation of critical process parameters (CPPs) such as pressure and temperature. These variations in CPPs of microfluidization process produced nanoemulsions with small (approximately 80 nm mean size), medium (approximately 120 nm mean size) and large (approximately 150 nm mean size) globule size distributions. The globule size distribution of the nanoemulsions was determined by dynamic light scattering method using Zetasizer Nano ZSP instrument (Malvern Panalytical Inc., Westborough, MA). The results of Z-average, polydispersity index (PdI) and intensity weighed distribution (D10, D50 and D90) for each formulation is provided in Table 1.
Ultra performance liquid chromatography: The UPLC system included a Waters Acquity UPLC I-Class (Waters Corporation, Milford, MA) equipped with degasser, binary solvent pump, thermostatted autosampler, thermostatted column compartment, and a photo diode array detector. Acquity UPLC BEH C18, 2.1 mm×150 mm (1.7 μm packing) column (Waters Corporation, Milford, MA) was used along with an Acquity UPLC BEH C18, 2.1 mm×5 mm (1.7 μm packing) vanguard precolumn (Waters Corporation, Milford, MA). Column temperature was maintained at 50° C. whereas autosampler temperature was kept at 8° C. A 100 μL extension loop was used. Two separate gradient elution methods (Method 1 in Table 2 and Method 2 in Table 3) were used to analyze difluprednate in different media, e.g., Method 1 for retentate samples, and Method 2 for permeate samples. Injection volume was 3 μL and 80 μL for Method 1 and Method 2, respectively. The eluted difluprednate was detected at 240 nm. Data collection and analysis were performed using Empower 3 software.
Release of difluprednate from pure drug solution was studied using a reverse-dialysis setup, where drug was transferred from outside a dialysis device to the inside. As compared to a dialysis configuration, the reverse-dialysis configuration provides an advantage in terms of allowing for better control of dilution and maximum drug concentration gradient across oil/water interface to drive the drug release. Diffusion of difluprednate through dialysis membrane was determined in a USP 2 apparatus setup (Vision Elite 8, Teledyne Hanson Research, with mini-vessels 800) using commercially available dialysis tubes 808 (Float-A-Lyzer G2, 100 kD MWCO, regenerated cellulose, Spectrum Labs, CA, USA), as shown in
Within dialysis tube 808, difluprednate concentration (e.g., drug that had diffused through the membrane filter of dialysis device 808) was monitored in real time using an in-situ fiber optic probe 804, in particular a fiber optic UV-Vis system (Pion μDiss Profiler™, Billerica, MA). The scanning frequency was once every minute for the first 6 hours, then it switched to once every 5 minutes for the next 18 hours. After first 24 hours, the scanning frequency was changed to once every 10 minutes for the next 40 hours. For the last 104 hours, the scanning frequency was once every 30 min. The total experimental time was 168 hours (7 days). Pure difluprednate solution diluted with release medium at the same concentration of 5 μg/mL was also monitored in parallel as a control to determine any possible change in drug concentration, e.g., due to degradation. Prior to the start of the experiment, UV signals were calibrated using various concentrations of difluprednate standard solutions (0.05, 0.10, 0.50, 1.00, 2.00, 3.00, 4.00, and 6.00 μg/mL). Zero Intercept Method (ZIM) analysis was performed to remove polysorbate-80 interference by using wavelength within the range of 271-274 nm (exact ZIM wavelength varied between probes). Ethanol was used as a co-solvent to prevent the precipitation of difluprednate in the release media.
Release of difluprednate from nanoemulsions was studied using a reverse-dialysis setup, where drug was transferred from outside a dialysis device to the inside. As compared to a dialysis configuration, the reverse-dialysis configuration provides an advantage in terms of allowing for better control of dilution and maximum drug concentration gradient across oil/water interface to drive the drug release. The IVRT study was performed by using a reverse dialysis setup inside a USP 2 apparatus equipped with mini-vessels 900, as shown in
Into mini-vessel 900, 2 mL of nanoemulsion was added directly to 200 mL release media 902 in order to achieve an initial concentration of 5 μg/mL for difluprednate. Sampling was performed from alternating dialysis tubes 906a, 906b. At each pre-determined sampling time point, 0.2 mL of media was taken from both inside the dialysis tubes (e.g., using respective conduits 904a, 904b) and the outside media 902. Due to the interference of other formulation components, in-situ UV analysis was not possible, and accordingly difluprednate concentration was determined by UPLC method (e.g., Method 2). An equivalent volume of fresh media was replenished inside the dialysis tubes 906a, 906b after each sampling.
As shown in
In summary, despite the high dilution ratio (100 time dilution by release medium), aggressive agitation (100 rpm), and elevated temperature (34° C.), all of the conditions favoring the drug release from emulsions, the rate and extent of drug release was still slow and low, rendering reverse-dialysis method incapable of discerning differences in the drug release profiles of the small and large globule size nanoemulsions.
AP setup 1000 provided a unique platform for in vitro release testing of various complex dosage forms. The adaptive nature of the process allows the user to optimize the feed flow rate based on the type of dosage form. For example, higher flow rate may be selected to quickly remove the released drug in case of rapid release from formulations (e.g., micelle phase within emulsions which readily releases the drug within few minutes). Likewise, for formulations with multiphasic release kinetics, flow rate may be adjusted during the experiment to adjust the rate of drug removal. Even though the sample is circulated in a closed loop (e.g., from reservoir 1008 to first port 1022 of HFF 1020 and from second port 1024 of HFF 1020 back to the reservoir 1008), the simultaneous size-based separation (e.g., via HFF 1020) and the concurrent dilution of sample (e.g., via fluid from reservoir 1002 into feed reservoir 1008) helps in overcoming the limitation due to small medium volume in maintaining continuous sink conditions.
Formulation components can be selectively retained based on the MWCO range of the membrane filter of the HFF 1020. For example, castor oil globules in the nanoemulsions or protein-bound drug containing nanoparticles can be selectively retained and made to continuously circulate in fluid circuit 1030, which is being continuously diluted with fresh medium (e.g., via fluid from reservoir 1002 into feed reservoir 1008).
Furthermore, there is an increasing need to analyze the role of critical excipients and their impact on drug release. Complex excipients, such as polymers composed of hydrophilic and hydrophobic monomers, can be selectively retained based on their molecular weight by using the AP method. The impact of these polymers on drug release could then be studied, for example, by inducing compositional or manufacturing process changes and evaluating their effect on drug release of resulting product.
In addition, the continuous processes in the AP method (i.e., concomitant dilution of the sample and removal of the released drug from the membrane and recirculation of the remaining drug within the AP system) mimics in-vivo conditions, for example, continuous dilution of a drug on an ocular surface due to tear turnover and continuous absorption of drug after release from the complex formulation. Similarly, drug release testing using the AP method also closely resembles the drug release condition in parenteral drug delivery, where release of drug into the blood stream occurs during circulation and drug removal occurs due to absorption at the target tissue or organ.
Dilution ratio during release testing, which may be highly critical for some dosage forms such as ophthalmics, can be controlled using the AP method. Such control may be especially useful for nanosuspensions, where dilution can give rise to rapid (e.g., substantially instantaneous) dissolution of the nanoparticles and thereby diminish the possibility of differentiating formulations. Also, for nanoemulsions the initial equilibrium between the oil/aqueous phases present in the formulation governs the drug distribution in each phase. Thus, for very high dilution at the beginning of an IVRT may disturb this equilibrium and potentially mask the difference in drug distribution between formulations, especially if the rate of drug removal across the membrane is slow.
In the AP method, dilution rate can be controlled by adjusting the rate of permeate flow (e.g., by controlling feed flow rate via pump 1014 and/or back pressure via valve 1028) so that the sample could be diluted at a lower ratio initially, followed by higher dilution as the test progresses. This unique feature enables the AP method to potentially discern minor differences in drug release profiles of emulsions with different globule sizes. The AP method also provides the flexibility of tuning the rates of feed flow and fresh medium supply, which in turn governs the rate at which the drug sample gets diluted as well as the time necessary to complete an IVRT. The AP method can adjust these flow rates to avoid, or at least reduce, degradation of drug products that may degrade when otherwise exposed to large volume of media for a prolonged period (e.g., as occurred with the drug samples in comparative examples 1-2,
Pure difluprednate solution and difluprednate polysorbate-80 micelles was evaluated using the setup 1000 of
In addition, to demonstrate the discriminatory capability of the AP method, IVRT was conducted for nanoemulsions having small, medium and large globule size distribution using the setup 1000 of
Drug transfer from the pure difluprednate solution and the difluprednate polysorbate-80 micelles was evaluated using setup 1000.
Similar to the reverse-dialysis setup, 20% ethanol was used in the release media for testing of pure difluprednate solutions, in order to provide a comparison with the media used in reverse-dialysis method as well as to prevent drug precipitation due to the higher rate of drug transfer achieved with the AP method. In the AP method, diffusion of drug across the membrane may still occur, but it is otherwise overshadowed by the rapid drug transfer from the pressure driven-filtration process. In other words, the overall rate of drug transfer across the TFF membrane is primarily a function of the filtration process rather than diffusion. As shown in
With respect to nanoemulsions,
As shown in Table 4 above, the AP method is capable of discriminating between samples of different dosage forms, e.g., pure drug solution, micelles, and nanoemulsions. A statistically significant difference was observed when the rates of drug transfer were compared for pure drug solution and drug loaded micelles with the nanoemulsions. The rates of drug transfer were reduced by almost 50% for nanoemulsions as compared to the pure drug solution and drug in micelles, which reduction was expected considering the additional barrier of drug transfer from oil globules to the release medium. The difference in terms of release media used for pure drug solution (i.e., 10 mM pH 7.4 phosphate buffer mixed with ethanol in the ratio of 80:20 v/v) from that used for drug loaded micelles and nanoemulsions (i.e., 10 mM pH 7.4 phosphate buffer) may also have contributed to the reduced drug transfer rate for nanoemulsions. However, the different release processes between solution, micelles, emulsions are expected to be the primary driver of the differences in drug transfer rates.
The AP method was able to provide a clear discrimination between the release profiles of different globule size nanoemulsions. The most rapid extent of release came from smaller globules, followed by medium and larger globule size nanoemulsions. It should be further noted that the globule size difference between the large and small nanoemulsions was about 2.4 times higher when compared with that of the medium size nanoemulsions (see Table 1). This difference in the globule size distribution between the different nanoemulsions is evident by the difference observed in the extent of drug release from each of these formulations, as shown in
The above described IVRT data for complex nanoemulsions using the AP method demonstrate that the rate and extent of drug release can be a product of surfactant distribution in the nanoemulsions and the flux during the AP process. In particular, surfactant distribution exhibited a direct correlation with the size of the globules, with larger globules (smaller surface area) requiring smaller amount of surfactant for stabilizing the interface. Accordingly, a higher percentage of surfactant was available in the bulk aqueous phase to form micelles. As suggested by
To evaluate the effect of pre-conditioning on filter performance, IVRT was conducted for nanoemulsions having small GSD using the AP method in the setup of
To evaluate the effect of pre-conditioning using partial open loop configuration of
In view of the above described implementations of the disclosed subject matter, this application discloses the additional examples in the clauses enumerated below. It should be noted that one feature of a clause in isolation, or more than one feature of the clause taken in combination, and, optionally, in combination with one or more features of one or more further clauses are further examples also falling within the disclosure of this application.
Clause 1. A method, comprising:
Clause 2. The method of clause 1, wherein the first aliquot and the second aliquot are obtained at a same time during the performance of the diafiltration process or after the performance of the diafiltration process.
Clause 3. The method of any one of clauses 1-2, wherein the first aliquot is obtained prior to the performance of the diafiltration process.
Clause 4. The method of any one of clauses 1-3, wherein the obtaining the first aliquot, the obtaining the second aliquot, and the analyzing the first and second aliquots are repeated periodically during the performance of the diafiltration process.
Clause 5. The method of any one of clauses 1-4, wherein the determined one or more properties of the drug sample comprises a percentage of the drug sample released over time.
Clause 6. The method of any one of clauses 1-5, wherein the analyzing comprises ultra-performance liquid chromatography (UPLC).
Clause 7. The method of any one of clauses 1-6, wherein the drug sample comprises a drug solution or suspension, drug-loaded micelles, nanoemulsions comprising drug molecules, or any combination of the above.
Clause 8. The method of any one of clauses 1-7, further comprising, prior to performing the diafiltration process, conditioning the filter by flowing a conditioning solution over retentate and permeate sides of each membrane in the filter.
Clause 9. The method of any one of clauses 1-8, further comprising, after the performing the diafiltration process, flowing a conditioning solution over retentate and permeate sides of each membrane in the filter, and storing the filter within the conditioning solution.
Clause 10. The method of any one of clauses 8-9, wherein the conditioning solution comprises a surfactant, an emulsifier, or both.
Clause 11. The method of clause 10, wherein the conditioning solution comprises polysorbate-80.
Clause 12. The method of any one of clauses 1-11, further comprising, after the performing the diafiltration process, flushing at least a retentate side of each membrane in the filter with a solvent, and recovering any drug sample retained in the solvent exiting the filter from the flushing.
Clause 13. The method of any one of clauses 1-12, wherein:
Clause 14. The method of clause 13, further comprising measuring a second flow rate of the permeate flow, wherein the adding fluid to the fluid supply reservoir is responsive to the measured second flow rate.
Clause 15. The method of any one of clauses 13-14, further comprising measuring volume, weight, or both of the fluid supply reservoir, the permeate reservoir, or both, wherein the adding fluid to the fluid supply reservoir is responsive to the measuring.
Clause 16. The method of any one of clauses 13-15, further comprising:
Clause 17. A method for conditioning a filter for use in a diafiltration process involving a drug sample, the method comprising:
Clause 18. The method of clause 17, wherein the conditioning solution comprises polysorbate-80.
Clause 19. The method of any one of clauses 17-18, wherein the first side is a retentate side of the membrane, the second side is a permeate side of the membrane, the first and fourth ports are disposed at one end of the filter, and the second and third ports are disposed at an opposite end of the filter from the first and fourth ports.
Clause 20. The method of any one of clauses 17-19, further comprising, prior to or after the flowing conditioning solution, flowing deionized water into the filter via the first port and out through the fourth port, such that the deionized water flows over the first and second sides of the membrane via the connection between the second and third ports.
Clause 21. The method of any one of clauses 17-20, further comprising, after the flowing conditioning solution, storing the filter within the conditioning solution.
Clause 22. The method of any one of clauses 1-21, wherein the filter is a tangential flow filter.
Clause 23. The method of any one of clauses 1-22, wherein the filter is a hollow fiber filter.
Clause 24. A method, comprising:
Clause 25. The method of clause 24, wherein:
Clause 26. The method of any one of clauses 24-25, wherein:
Clause 27. The method of clause 26, wherein the first concentration monitoring sensor, the second concentration monitoring sensor, or both comprise an in situ fiber optic ultra-violet (UV) sensor.
Clause 28. The method of any one of clauses 24-27, wherein:
Clause 29. The method of clause 28, wherein the analyzing the first aliquot, the analyzing the second aliquot, or both comprises ultra-performance liquid chromatography (UPLC).
Clause 30. The method of any one of clauses 24-29, wherein the determined one or more properties of the drug sample comprises a percentage of the drug sample released over time.
Clause 31. The method of any one of clauses 24-30, wherein the drug sample comprises a drug solution or suspension, drug-loaded micelles, nanoemulsions comprising drug molecules, or any combination of the above.
32. The method of any one of clauses 24-31, wherein:
Clause 33. The method of clause 32, further comprising measuring a second flow rate of the permeate flow, wherein the adding fluid to the fluid supply reservoir is responsive to the measured second flow rate.
Clause 34. The method of any one of clauses 32-33, further comprising measuring volume, weight, or both of the fluid supply reservoir, the permeate reservoir, or both, wherein the adding fluid to the fluid supply reservoir is responsive to the measuring.
Clause 35. The method of any one of clauses 32-34, further comprising:
Clause 36. The method of any one of clauses 24-35, further comprising, prior to performing the diafiltration process, conditioning the filter by flowing a conditioning solution over retentate and permeate sides of each membrane in the filter.
Clause 37. The method of any one of clauses 24-36, further comprising, after the performing the diafiltration process, flowing a conditioning solution over retentate and permeate sides of each membrane in the filter, and storing the filter within the conditioning solution.
Clause 38. The method of any one of clauses 36-37, wherein the conditioning solution comprises a surfactant, an emulsifier, or both.
Clause 39. The method of any one of clauses 36-38, wherein the conditioning solution comprises polysorbate-80.
Clause 40. The method of any one of clauses 24-39, further comprising, after the performing the diafiltration process, flushing at least a retentate side of each membrane in the filter with a solvent, and recovering any drug sample retained in the solvent exiting the filter from the flushing.
Clause 41. The method of any one of clauses 24-40, wherein the filter is a tangential flow filter.
Clause 42. The method of clause 41, wherein the filter is a hollow fiber filter.
Clause 43. A control system, comprising:
Clause 44. A method of conditioning a TFF filter with surfactant, the TFF filter being connected in a partial-open loop configuration, so as to minimize run-to-run and device-to-device variability, as otherwise described herein above.
Clause 45. A method of conducting drug release testing using a TFF filter with analysis of drug components in both the permeate and retentate, as otherwise described herein above.
Clause 46. A method of using an AP technique for size-based separation of complex drug product to study release characteristics thereof, as otherwise described herein above.
Clause 47. A method of using an AP technique to assess quality and performance differences in a complex drug product, as otherwise described herein above.
Clause 48. The method of any one of clauses 1-42 and 44-47, wherein the drug sample or the complex drug product comprises an emulsion, suspension, liposome, protein-drug complex, or any combination thereof.
Clause 49. An adaptive perfusion system, comprising:
Clause 50. The adaptive perfusion system of clause 49, further comprising one or more in situ sensors configured to measure drug concentration, composition of a fluid, or both.
Clause 51. The adaptive perfusion system of clause 50, wherein the one or more in situ sensors comprises an in situ fiber optic ultra-violet (UV) sensor.
Any of the features illustrated or described with respect to
Although the operations of some of the disclosed methods are described in a particular, sequential order for convenient presentation, it should be understood that this manner of description encompasses rearrangement, unless a particular ordering is required by specific language set forth below. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed methods can be used in conjunction with other methods.
In view of the many possible embodiments to which the principles of the disclosed technology may be applied, it should be recognized that the illustrated embodiments are only preferred examples and should not be taken as limiting the scope of the disclosed technology. Rather, the scope is defined by the following claims. We therefore claim all that comes within the scope of these claims.
This application claims the benefit of U.S. Provisional Application 63/128,505, filed Dec. 21, 2020, which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/064387 | 12/20/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63128505 | Dec 2020 | US |
