Patent Application
20250207149
The present disclosure relates to the field of separation of adeno-associated capsids and is directed to a method for determining elution conditions suitable for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material. Further disclosed are methods for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material, and use of an anion exchange chromatography material for such separations. The present disclosure is applicable to separation of capsids of adeno-associated virus serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10) and variants thereof.
Adeno-associated viruses (AAV) are non-enveloped viruses that have linear single-stranded DNA (ssDNA) genome and that can be engineered to deliver DNA to target cells. Recombinant adeno-associated virus (rAAV) vectors have emerged as one of the most versatile and successful gene therapy delivery vehicles. There is an increasing demand to use viral vectors for gene therapy. To use AAV particles as vectors in therapy it is necessary to purify the virus particles from cell impurities like DNA after transfection. Ultracentrifugation is efficient but not scalable. Normally, several filtration steps and several chromatography steps are used to separate AAV particles from cell cultures (see e.g., Weihong Qu et al).
Therapeutic efficacy of AAV vectors is dependent on high percentage of virus particles fully packaged with genetic material of interest. Upstream expression systems deliver a mixture of fully packaged AAV particles (containing the genetic material of interest), empty AAV particles, and AAV particles which are partially packaged with genetic material of interest), together with impurities. There is thus a need to enrich fully packaged AAV particles in the purification process. However, there are several challenges in relation to achieving an efficient and scalable separation of fully packaged and empty adeno-associated virus capsids, such as:
Methods for separation of fully packaged capsids from not fully packaged capsids have been previously described (see e.g., Hejmowski et al). However, further optimisation of purification strategies is always desired to increase the speed and decrease the cost of downstream processing of different adeno-associated virus serotypes.
The object of the present disclosure is to provide an improved method for separation of fully packaged adeno-associated virus capsids from not fully packaged adeno-associated virus capsids. This is achieved by first performing a method for determining elution conditions suitable for separating fully packaged capsids from not fully packaged capsids. Herein, this method is called a pre-screening method. It is followed by performing a method for separating fully packaged from not fully packaged capsids by eluting not fully packaged capsids at a first conductivity value as determined in the pre-screening method, and by eluting fully packaged capsids at a second conductivity value as determined in the pre-screening method. Thereby, an improved resolution between fully packaged and not fully packaged capsids is obtained, which results in achieving a composition having a higher ratio of fully packaged capsids to not fully packaged capsids. The focus of the disclosure is a pre-screening method for establishing optimal elution conditions for the polishing step of a separation method, also called secondary or final purification.
More particularly, a first aspect of the present disclosure is directed to the so-called pre-screening method, which is a method for determining elution conditions suitable for separating adeno-associated virus (AAV) capsids fully packaged with genetic material from AAV capsids not fully packaged with genetic material, the method comprising:
Even more particularly, the pre-screening method is a method for determining elution conditions suitable for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material, the method comprising the following steps:
The present disclosure further provides a method for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material, the method comprising steps (a)-(d) of the pre-screening method described above, and further comprising the steps:
Additionally, the present disclosure is directed to a method for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material, the method comprising:
The present disclosure also provides use of an anion exchange chromatography material comprising a support, a ligand, and a surface extender connecting the ligand to the support, and being defined by Formula IV:
Preferred aspects of the present disclosure are described below in the detailed description and in the dependent claims.
The present disclosure solves or at least mitigates the problems associated with existing methods for separating fully packaged adeno-associated virus capsids from capsids not fully packaged adeno-associated virus capsids by providing, as illustrated in
Herein, it is shown that the currently disclosed pre-screening method is a universal way of dealing with the weaknesses of previously applied methods, in which differences in ionic capacity, anion exchange ligand density, amount of surface extender, and support material between different lots of chromatography materials, as well as feed variability and variations in buffer preparations, makes it nearly impossible to predict the amount of conductivity that is needed to achieve baseline separation of full and empty capsids.
A “virus particle” is herein used to denote a complete infectious virus particle. It includes a core, comprising the genome of the virus (i.e., the viral genome), either in the form of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and the core is surrounded by a morphologically defined shell. The shell is called a capsid. The capsid and the enclosed viral genome together constitute the so-called nucleocapsid. The nucleocapsid of some viruses is surrounded by a lipoprotein bilayer envelope. In the field of bioprocessing, for the purpose of producing viral vectors for various applications such as therapy, the genome of a virus particle is modified to include a genetic insert, comprising genetic material of interest. Modified virus particles are allowed to infect host cells in a cell culture and the virus particles are propagated in said host cells, after which the virus particles are purified from the cell culture by any means of separation and purification. Herein, a virus particle to be separated from a cell culture by the presently disclosed method may alternatively be referred to as a “target molecule” or “target”. It is to be understood that “a virus particle” is intended to mean a type of virus particle and that the singular form of the term may encompass a large number of individual virus particles. Herein, the term “virus particle” may be used interchangeably with the terms “vector” and “capsid”, respectively, as further defined below.
The term “vector” is herein used to denote a virus particle, normally a recombinant virus particle, which is intended for use to achieve gene transfer to modify specific cell type or tissue. A virus particle can for example be engineered to provide a vector expressing therapeutic genes. Several virus types are currently being investigated for use to deliver genetic material (e.g., genes) to cells to provide either transient or permanent transgene expression. These include adenoviruses, retroviruses (γ-retroviruses and lentiviruses), poxviruses, adeno-associated viruses (AAV), baculoviruses, and herpes simplex viruses. Herein, the term “vector” may be used interchangeably with the terms “virus particle” and “capsid”, respectively.
The term “capsid” means the shell of a virus particle. The capsid surrounds the core of the virus particle, and normally should comprise a viral genome. A modified (recombinant) capsid, as produced in an upstream process of manufacturing, is supposed to comprise a complete viral genome, which genome includes genetic material of interest for one or more applications, for example of interest for various therapeutic applications. However, owing to low packaging efficiency, assembled capsids do not always contain any genetic material or only encapsidate truncated genetic fragments, resulting in so-called empty capsids and partially filled capsids, respectively. These capsids possess no therapeutic function, yet they compete for binding receptors during the cell-mediated processes. This may diminish the overall therapeutic efficacy and trigger undesirable immune responses. As a result, tracking these capsids throughout the production process is crucial to ensure consistent product quality and a proper dosing response (Xiaotong Fu et al). In up to 20-30% of a population of virus particles artificially produced in a cell culture, the capsid is only partially filled with genetic material. Further, in up to as much as 98% of artificially produced virus particles, the capsid does not comprise any part of the viral genome at all, i.e., it is empty. However, generally between 80% to 90% of artificially produced virus particles have empty capsids, and best cases currently achieve as little as 50% empty capsids.
Herein, the term “capsid” may be used interchangeably with the terms “vector” and “virus particle”, respectively. In the context of the present disclosure, a capsid may or may not comprise genetic material.
The term “genetic material of interest” is intended to mean genetic material which in the field of bioprocessing is considered relevant and valuable to get produced by viral replication and to purify such that it can be used in various applications, such as, but not limited to, therapeutic applications. As a non-limiting example, genetic material of interest may comprise a therapeutically relevant genetic material, such as a therapeutically relevant nucleotide sequence.
The term “capsid fully packaged with genetic material” is herein used to denote a capsid which has been correctly produced (by the host cell), or in other words,
The viral genome includes a genetic insert, comprising genetic material of interest, as defined elsewhere herein.
A capsid which comprises a complete viral genome may herein alternatively be called a “full capsid” or a “fully packaged capsid”. The terms “full capsid”, “fully packaged capsid”, and “capsid fully packaged with genetic material” may be used interchangeably throughout this text.
The term “capsid not fully packaged with genetic material” is herein used to denote a capsid which has not been correctly produced (by the host cell), or in other words,
A capsid which is not fully packaged with genetic material is either partially filled with genetic material or is not filled with any genetic material at all.
The term “capsid not fully packaged with genetic material” encompasses the terms “partially filled capsid” and “empty capsid”, as defined below.
A “partially filled capsid” is herein defined as a capsid which comprises parts of its viral genome, such as defective parts of its viral genome, or in other words,
An “empty capsid” is herein defined as a capsid which does not comprise any part of its viral genome, i.e., which comprises 0% of its viral genome, or in other words, a capsid which is not filled with any genetic material at all. Thus, an empty capsid does not comprise any genetic material of interest. Consequently, it is desirable (and sometimes required, e.g., due to clinical regulations) to separate and remove as many as possible of the empty capsids from a population of capsids, before putting the population of capsids to use in its intended application, e.g., a therapeutic application.
Before putting a population of virus particles to use in its intended application, e.g., a therapeutic application, it is desirable (sometimes even required, e.g., due to clinical regulations) to enrich the full capsids, i.e., to increase the percentage of full capsids at the expense of the percentage of partially filled capsids and empty capsids.
The percentage of full capsids and empty capsids in a population of capsids can be estimated or analyzed with several methods known in the art. Some of these methods are briefly described below:
1: A260:280 in chromatogram will give an estimation of percentage full capsids present in peaks (ratio 1-1.5 indicate enriched in full capsids, ratio 0.5-0.7 is containing mainly empty capsids).
2. qPCR:ELISA ratio. qPCR quantifies viral genomes and ELISA quantifies total viral particles. A ratio of 2 assays with variation is less accurate and will be uncertain. Requires orthogonal analysis for confirmation (see below, 3,4 or 5).
3. Analytical anion exchange separating full and empty capsids (A260:280 ratio and peak area to calculate the percentage). Accuracy dependent of peak definition.
4. Analytical ultracentrifugation (AUC). Detects and quantifies particles of different density (corresponding to full, partially filled, and empty capsids). This is currently known as the “golden standard” in the art. However, ultracentrifugation is not scalable and thus is not suitable for analysis of large-scale batches of capsids.
5. Transmission electron microscopy (TEM). Image analysis counting particles (full, partially filled, and empty capsids). May introduce artifacts from sample preparation.
Some methods for estimating or analyzing the percentage of full capsids and empty capsids in a population of capsids are described in more detail in Xiaotong Fu et al, which is hereby incorporated by reference herein.
It is to be understood that the term “liquid sample” as used herein encompasses any type of sample obtainable from a cell culture, or from a fluid originating from a cell culture which fluid is at least partly purified, by any means of separation and purification.
The term “separation matrix” is used herein to denote a material comprising a support to which one or more ligands comprising functional groups have been coupled. The functional groups of the ligand(s) bind compounds herein also called analytes, which are to be separated from a liquid sample and/or which are to be separated from other compounds present in the liquid sample. A separation matrix may further comprise a compound which couples the ligand(s) to the support. The terms “linker”, “extender”, and “surface extender” may be used to describe such a compound, as further described below. The term “resin” is sometimes used for a separation matrix in this field. The terms “chromatography material” and “chromatography matrix” are used herein to denote a type of separation matrix.
The term “surface” herein means all external surfaces and includes in the case of a porous support outer surfaces as well as pore surfaces.
Herein, the term “strong anion exchange chromatography material” is intended to mean a chromatography material which comprises a ligand comprising a quaternized amine group. A quaternary amine group is a strong anion exchange group, which is always positively charged irrespective of to which pH it is subjected. For DEAE-based types of chromatography materials, the degree of quaternization of the amine group may vary among the amine groups included in a chromatography material. A degree of quaternization of the amine group of from about 12% to about 100% globally in a chromatography material is generally considered to result in a chromatography material which behaves like a strong, or at least partially strong, anion exchange chromatography material since these at least 12% of all amine groups are always charged. In contrast to quaternized amine groups, almost all other ionic exchange groups are weak, i.e., their charge varies from fully charged to not charged within a reasonable range of pH used (such as pH 2-11) and having a neutral charge (same amount of + and − charges) at pl.
Capto Q (Cytiva, Sweden) is a non-limiting example of a strong anion exchange chromatography material having about 100% quaternized amine groups. Capto DEAE (Cytiva, Sweden) is a non-limiting example of a strong, or partially strong, anion exchange chromatography material having a degree of quaternization of the amine groups of about 15%.
The separation matrix may be contained in any type of separation device, as further defined elsewhere herein. As a non-limiting example, a chromatography material may be packed in a chromatography column, before adding a liquid sample to the chromatography material being contained in the chromatography column. As another example, for the present pre-screening method, the chromatography material may be provided in a multi-well format, such as in the form of a multi-well plate having wells containing the chromatography material (e.g. PreDictor Capto Q plates, Cytiva, Sweden).
In this context, “ligand” is a molecule that has a known or unknown affinity for a given analyte and includes any functional group, or capturing agent, immobilized on its surface, whereas “analyte” includes any specific binding partner to the ligand. The term “ligand” may herein be used interchangeably with the terms “specific binding molecule”, “specific binding partner”, “capturing molecule” and “capturing agent”. Herein, the molecules in a liquid sample which interact with a ligand are referred to as “analyte”. The analytes of interest according to the present disclosure are adeno-associated virus capsids, more particularly adeno-associated virus capsids either fully packaged or not fully packaged with genetic material. Consequently, herein the terms “analyte”, “adeno-associated virus capsid” and “capsid” may be used interchangeably.
In the herein disclosed method for separating fully packaged capsids from not fully packaged capsids, the chromatography material used comprises a linker connecting the ligand to the support, i.e., the coupling of the ligand to the support is provided by introducing a linker between the support and ligand. The coupling may be carried out following any conventional covalent coupling methodology such as by use of epichlorohydrin; epibromohydrin; allyl-glycidylether; bis-epoxides such as butanedioldiglycidylether; halogen-substituted aliphatic substances such as di-chloro-propanol; and divinyl sulfone. Other non-limiting examples of suitable linkers are: polyethylene glycol (PEG) having 2-6 carbon atoms, carbohydrates having 3-6 carbon atoms, and polyalcohols having 3-6 carbon atoms. These methods are all well known in the art and easily carried out by the skilled person.
The ligand is coupled to the support via a longer linker molecule, also known as a “surface extender”, or simply “extender”. Extenders are well known in this field, and commonly used to sterically increase the distance between ligand and support. Extenders are sometimes denoted tentacles or flexible arms. For a more detailed description of possible chemical structures, see for example U.S. Pat. No. 6,428,707, which is hereby included herein by reference. In brief, the extender may be in the form of a polymer such as a homo- or a copolymer. Hydrophilic polymeric extenders may be of synthetic origin, i.e., with a synthetic skeleton, or of biological origin, i.e., a biopolymer with a naturally occurring skeleton. Typical synthetic polymers are polyvinyl alcohols, polyacryl- and polymethacrylamides, polyvinyl ethers etc. Typical biopolymers are polysaccharides, such as starch, cellulose, dextran, agarose. Extenders may be linear and non-linear (branched) polymers, such as a brush polymer, which is a long linear structure with functional appendices along its length. The results described in the Examples herein surprisingly show that a chromatography material comprising a surface extender provides an improved separation of full AAV capsids from empty AAV capsids compared to the same chromatography material not including a surface extender.
The term “eluent” is used in its conventional meaning in this field, i.e., a buffer of suitable pH and/or ionic strength to release one or more compounds from a separation matrix.
The term “eluate” is used in its conventional meaning in this field, i.e., the part(s) of a liquid sample which are eluted from a chromatography column after having loaded the liquid sample onto the chromatography column.
As mentioned above, in the method for determining elution conditions suitable for separating fully packaged capsids from not fully packaged capsids, the liquid sample which is added to a chromatography material in step (a) comprises adeno-associated virus capsids of a purity of at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and of a concentration of at least 1012, such as 1013, 1014, or 1015, adeno-associated virus capsids/ml, of which at least 5%, such as 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80%, of the adeno-associated virus capsids are adeno-associated virus capsids fully packaged with genetic material. With regard to the purity of adeno-associated capsids in the liquid sample, a purity of at least 90%, such as up to 99%, is intended to mean that at least 90%, such as up to 99%, of the biological material in the liquid sample is represented by adeno-associated capsids (including full, empty, and partially filled capsids) while the remaining up to 10%, such as 1%, is represented by host cell protein and DNA.
As specified in step (b) of the above-described pre-screening method, a step gradient elution is designed in the form of a stepwise slowly increasing conductivity, which starts at from about 0 to about 5 mS/cm, such as at about 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mS/cm. The step gradient increases by approx. 0.5-3 mS/cm increase per step, such as from about 1 to about 2 mS/cm per step, such as from about 1.2 to about 1.5 mS/cm per step, or by about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 mS/cm per step, at least up to and including a conductivity at which the capsids not fully packaged with genetic material and the capsids fully packaged with genetic material have been eluted from the chromatography material, in order to identify which value of conductivity is needed to wash out empty capsids and full capsids, respectively. It is to be understood that the conductivity step gradient may optionally increase above the conductivity at which both empty and full capsids have been eluted.
As specified in steps (c) and (d) of the above-described pre-screening method, a first and second value of conductivity or conductivity-related parameter are determined based on an elution profile obtained in step (b). The elution profile may be in the form of a chromatogram, or a table or a graph comprising elution-related values.
The term “conductivity-related parameter” as used herein is intended to mean a parameter which influences the conductivity of a solution. A conductivity-related parameter may for example be directly correlated or inversely correlated with the conductivity. Non-limiting examples of conductivity-related parameters which may be relevant in this context are salt concentration and pH, as well as presence/concentration of compounds improving the separation between capsids fully packaged with genetic material and capsids not fully packaged with genetic material. Compounds which improve separation may for example be selected from a carbohydrate, a divalent metal ion, and a detergent, as described in detail further below.
The first value of conductivity or conductivity-related parameter determined shall be suitable for eluting the adeno-associated virus capsids not fully packaged with genetic material, and the second value of conductivity or conductivity-related parameter determined shall be suitable for eluting the adeno-associated virus capsids fully packaged with genetic material. In a pre-screening method where empty capsids are eluted before full capsids, the first value of conductivity is normally determined to be the value of conductivity applied when eluting the first peak containing empty and/or full capsids. If the empty capsids do not bind to the column but end up in the flowthrough, the first value of conductivity will be the same as the baseline conductivity value, i.e., the conductivity before the step gradient of conductivity is applied. Accordingly, in some instances the first value of conductivity may be determined to be as low as 0 mS/cm.
In a pre-screening method where empty capsids are eluted before full capsids, the second value of conductivity is normally determined to be a value of conductivity equal to or higher than the conductivity value applied when eluting the last peak containing empty and/or full capsids. For example, if the last peak is eluted at a conductivity value of 5 mS/cm, the second value of conductivity is determined to be ≥5 mS/cm.
The elution buffer applied in step (b) of the pre-screening method may comprise a salt. The step gradient of increasing conductivity in step (b) of the pre-screening method may be a step gradient of increasing salt concentration. In this context, the conductivity-related parameter referred to in steps (c) and (d) of the pre-screening method may be the salt concentration. The salt may be a kosmotropic salt. Salts in water solvent are defined as kosmotropic (order-making) if they contribute to the stability and structure of water-water interactions. In contrast, chaotropic (disorder-making) salts have the opposite effect, disrupting water structure, increasing the solubility of nonpolar solvent particles, and destabilizing solute aggregates. Kosmotropes cause water molecules to favorably interact, which in effect stabilizes intramolecular interactions in macromolecules such as proteins (Moelbert S et al). A scale can be established for example by referring to the Hofmeister series, or lyotropic series, which is a classification of ions in order of their ability to salt out or salt in proteins (Hyde A et al).
More particularly, the salt may comprise (i) an anion selected from a group consisting of CO32−, SO42−, S2O32−, H2PO4−, HPO42−, acetate−, citrate−, and Cl−, and (ii) a cation selected from a group consisting of NH4+, K+, Na+, and Li+. In a currently preferred embodiment, the salt is sodium acetate (NaOAc). Non-limiting examples of suitable concentrations of NaOAc include from about 5 mM to about 500 mM, such as about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mM. However, it is to be understood that other salts consisting of a combination an anion as listed under (i) and a cation as listed under (ii) may alternatively be used to elute the capsids. Non-limiting examples of such other salts are NaCl, LiCl, KCl, or other equivalent metal salt suitable to use for salt elution, as is well known in the art. Non-limiting examples of suitable concentrations of NaCl include from about 5 mM to about 2M, such as about 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, or 2000 mM.
Step (b) of the pre-screening method may comprise adding a volume of the elution buffer corresponding to from about 1 to about 10 volumes, such as from about 2 to about 8 volumes, such as about 5 volumes, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 volumes, of the chromatography material, per step of the step gradient.
The above-described pre-screening method is followed by a two-step elution method, which is designed based on the information that is provided by the elution profile (e.g., chromatogram) obtained in the pre-screening method. More particularly, the present disclosure further provides, as illustrated in
The liquid sample added in step (e) should originate from the same cell culture harvest as the liquid sample of step (a) of the pre-screening method, in order that the elution conditions determined in the pre-screening method are surely applicable also to the liquid sample added in step (e).
The aim of steps (f) and (g) of the above-disclosed method is to obtain fully packaged capsids of a purity which is as high as possible. A person skilled in the art readily understands that this may be achieved by applying various different separation conditions. Non-limiting examples of separation conditions to obtain fully packaged capsids of a purity as high as possible include separation conditions which allow binding of not fully packaged capsids to the chromatography material, while:
As mentioned above, there are small differences between fully packaged capsids and not fully packaged capsids in relation to several parameters relevant for purification, e.g., their isoelectric point. This often leads to (at least partial) co-elution of fully packaged and not fully packaged capsids. Accordingly, realistically, the adeno-associated virus capsids eluted in step (b) of the above-disclosed method will not be completely separated into full, empty, and partially filled capsids. However, there will be eluate fractions which comprise a substantially higher percentage of full capsids than in the liquid sample added to the chromatography material in step (e). More particularly, the adeno-associated virus capsids eluted in step (g), i.e., adeno-associated virus capsids fully packaged with genetic material, may be eluted into eluate fractions, which eluate fractions combined comprise at least 50%, such as 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, of the adeno-associated virus capsids of the liquid sample added in step (e), of which at least 60%, such as 65%, 70%, 75%, 80%, 85%, or 90%, of the adeno-associated virus capsids are fully packaged with genetic material. Non-limiting examples of recovery and purification of full capsids achieved by the presently disclosed method are a recovery of at least 50% of the capsids of the liquid sample added in step (e), of which at least 60% are full capsids, such as a recovery of at least 70% of the capsids of the liquid sample added in step (a), of which at least 80% are full capsids. In Example 1 described further below, the results show a recovery of at least 80% of viral genomes from harvest, of which at least 70% are full capsids.
It has been found advantageous to perform step (f) for a duration of time which is at least 3 times, such as 4 times, 5 times or more, compared to the duration of step (g). Without wishing to be bound by theory, it is believed that the relatively longer duration of step (f) is beneficial or even crucial for eluting substantially all, or nearly all, of the empty capsids present in the liquid sample.
An alternative to performing step (f) for a duration at least 3 times the duration of step (g), the method may comprise applying an additional step (f′) between step (f) and step (g), wherein the duration of steps (f) and (f′) is at least 3 times, such as 4 times, 5 times or more, compared to the duration of step (g). Step (f′) may for example comprise:
The difference in duration between step (f) and step (g) may be accomplished by adding a volume of the elution buffer in step (f), which is at least 3 times, such as 4 times or more, higher than the volume of elution buffer added in step (g). As a non-limiting example, in step (f) a volume of elution buffer corresponding to from about 3 to about 30 volumes, such as from about 6 to about 24 volumes, such as about 15 volumes, or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 volumes of the chromatography material is added, while in step (g), a volume of elution buffer corresponding to from about 1 to about 10 volumes, such as from about 2 to about 8 volumes, such as about 5 volumes, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 volumes, of the chromatography material is added. Similarly, if the method comprises a step (f′), the difference in duration between steps (f)+(f′) compared to step (g) may be accomplished by adding a volume of elution buffer in steps (f)+(f′) which in total is at least 3 times, such as 4 times or more, the volume of elution buffer added in step (g).
The elution buffer applied in steps (b), (f), optionally (f′), and (g) of the above-described method for separating capsids (and as illustrated in
More particularly, the salt may comprise (i) an anion selected from a group consisting of CO32−, O42−, S2O32−, H2PO4−, HPO42−, acetate−, citrate−, and Cl−, and (ii) a cation selected from a group consisting of NH4+, K+, Na+, and Li+. In a currently preferred embodiment, the salt is sodium acetate. However, it is to be understood that other salts consisting of a combination an anion as listed under (i) and a cation as listed under (ii) may alternatively be used to elute the capsids.
Step (b) of the method may comprise adding a volume of the elution buffer corresponding to from about 1 to about 10 volumes, such as from about 2 to about 8 volumes, such as about 5 volumes, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 volumes, of the chromatography material, per step of the step gradient.
As illustrated in
The method as illustrated in
It is to be understood that the embodiments and details described above for step (e), (f), optionally (f′), and (g), respectively, of the method of
As mentioned above, the chromatography material applied in any of the presently disclosed methods, as described in detail above and as illustrated in
The strong anion exchange chromatography material may be defined by the following Formula I:
As a non-limiting example, each of R1, R2, and R3 is CH3.
There are currently available chromatography materials comprising a ligand defined by Formula I, wherein each of R1, R2, and R3 is CH3; e.g., a chromatography material made available under the name Capto Q, provided by Cytiva, Sweden (www.cytivalifesciences.com). Capto Q further comprises dextran as surface extender and is a chromatography medium for high-resolution polishing steps in industrial purification processes, e.g., for purification of monoclonal antibodies.
According to another non-limiting example, R1 and R2 are ethyl, and R3 is methyl.
According to yet another non-limiting example, R1 and R2 are methyl, and R3 is CH2CHOHCH3.
The density of ligand defined by Formula I may be from about 60 to about 500 μmol, such as from about 160 to about 350 μmol, such as from about 160 to about 220 μmol, of ligand per ml of the strong anion exchange chromatography material.
Alternatively, the strong, or partially strong, anion exchange chromatography material may be defined by the following Formula II:
As a non-limiting example, the ligand is defined by Formula III and comprises a combination of two or more of the following structures (i)-(iv):
One currently available chromatography material comprising a ligand defined by Formula III and comprising a combination of the above-mentioned structures (i)-(iv) is the chromatography resin called Capto DEAE (Cytiva, Sweden). Capto DEAE further comprises dextran as surface extender and is a chromatography medium for high-resolution polishing steps in industrial purification processes, e.g., for purification of monoclonal antibodies.
According to another non-limiting example, the ligand is defined by Formula III, wherein m is 1; n is 1, 2, or 3; each R1, R2, R3, and R4 is methyl; and R5 is hydrogen.
According to yet another non-limiting example, the ligand is defined by Formula III, wherein m is 1; n is 1, 2, or 3; each R1, R2, R3, and R4 is methyl; and R5 is CH2CHOHCH3.
According to another non-limiting example, the ligand is defined by Formula III, wherein m is 1 and the ligand comprises a combination of two or more of the following structures (i)-(iv):
The density of ligand defined by Formula II or Formula III may be from about 60 to about 500 μmol, such as from about 160 to about 350 μmol, such as from about 290 to about 350 μmol, of ligand per ml of the strong anion exchange chromatography material.
As described above, the chromatography material comprises a surface extender connecting the ligand to the support, wherein the surface extender is a polymer, wherein the polymer is selected from:
As a non-limiting example, the surface extender is dextran. The dextran may have a molecular weight of from about 10 to about 2000 kDa, such as about 10, 40, 70, 250, 750, or 2000 kDa, such as 40 kDa. The density of dextran may be from about 5 to about 30 mg dextran per ml of the chromatography material. It is to be understood that the amount of dextran immobilized on the chromatography material may vary, for example depending on the molecular weight of the dextran immobilized. Normally, decreasing amounts are required for increasing molecular weights of dextran.
Steps (a) and (b) of the above-disclosed pre-screening method (
Said buffer is suitably selected from buffers generally recommended for anion exchange chromatography and may for example comprise tris(hydroxymethyl)amino-methane (i.e., Tris), 1,3-bis(tris(hydroxymethyl)methylamino) propane (i.e., bis-Tris propane), triethanolamine, N-methyldiethanolamine, Diethanolamine, 1,3-diaminopropane, or ethanolamine. A person skilled in the art is able to choose a suitable concentration for any one of the above-listed buffers.
In the above-disclosed pre-screening method, step (b) may comprise applying a buffer, optionally one of the buffers mentioned above, wherein the buffer comprises a compound which improves separation between capsids fully packaged with genetic material and capsids not fully packaged with genetic material. If so, in the separation method (
Where said compound which improves separation is a carbohydrate, it may for example be selected from sucrose, sorbitol, and a polysaccharide.
Where said compound which improves separation is a divalent metal ion, it may for example be selected from Mg2+, Fe2+, and Mn2+. The metal ion may be present in the form of a salt, optionally in combination with for example chloride ions or sulphate ions. A non-limiting example of a suitable metal salt to include in the buffer of step (b) is MgCl2. Non-limiting examples of suitable concentrations of MgCl2 include from about 0.5 to about 30 mM of MgCl2, such as from about 1 to about 20 mM, such as from about 2 to about 10 mM, or about 0.5, 1.0, 1.5, 2.0, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 mM, of MgCl2.
Where said compound which improves separation is a detergent, it may for example be selected from poloxamer, such as poloxamer 188 or Pluronic™ F68, and polysorbate, such as Tween 20 or Tween 80.
As described in Example 1 below, a non-limiting example of a suitable buffer system to be applied in the above-disclosed methods include a buffer A and a buffer B, both containing 20 mM Bis-Tris Propane (BTP) pH 9.0 and 2 mM MgCl2, and buffer B additionally comprising 250 mM sodium acetate (NaOAc) as elution salt. Buffer A is applied in step (a), (e), and (l), respectively. A step gradient of buffer B is applied in step (b) of the pre-screening method. The first and second value of conductivity or conductivity-related parameter are achieved by applying a mixture of buffer A and buffer B of suitable proportions in steps (f) and (g) of the method of
As described in Example 2 below, another non-limiting example of a suitable buffer system to be applied in the above-disclosed methods include a buffer A and a buffer B, both containing 20 mM Bis-Tris Propane (BTP) pH 7.0, 1% sucrose and 0.1% Pluronic, and buffer B additionally comprising 20 mM MgCl2.
As described in Example 3 below, another non-limiting example of a suitable buffer system to be applied in the above-disclosed methods include a buffer A and a buffer B, both containing 20 mM Bis-Tris Propane (BTP) pH 7.0 or 9.5 respectively, 18 mM MgCl2, 1% sucrose and 0.1% Pluronic, and buffer B additionally comprising 400 mM NaCl.
The chromatography material applied in the herein disclosed methods comprises a support to which the ligand is coupled. The support may be made from an organic or inorganic material and may be porous or non-porous. In one embodiment, the support is prepared from a native polymer, such as cross-linked carbohydrate material, e.g. agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, pectin, starch, etc. The native polymer supports are easily prepared and optionally cross-linked according to standard methods, such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964). In an especially advantageous embodiment, the support is a kind of relatively rigid but porous agarose, which is prepared by a method that enhances its flow properties, see e.g. U.S. Pat. No. 6,602,990 (Berg). In an alternative embodiment, the support is prepared from a synthetic polymer or copolymer, such as cross-linked synthetic polymers, e.g. styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters, vinyl amides etc. Such synthetic polymers are easily prepared and optionally cross-linked according to standard methods, see e.g. “Styrene based polymer supports developed by suspension polymerization” (R Arshady: Chimica e L'Industria 70(9), 70-75 (1988)). Native or synthetic polymer supports are also available from commercial sources, such as Cytiva, Sweden, for example in the form of porous particles. In yet an alternative embodiment, the support is prepared from an inorganic polymer, such as silica. Inorganic porous and non-porous supports are well known in this field and easily prepared according to standard methods.
The support of the chromatography material may be in the form of particles, such as substantially spherical, elongated or irregularly formed particles.
Where the chromatography material is in the form of particles, the particles may be particles having a homogeneous porosity and being at least partly permeable to adeno-associated virus capsids.
Herein, the term “homogeneous porosity” is intended to mean that a particle having a homogeneous porosity has a homogeneous porosity throughout its entire structure or volume, such that each particle is at least partly permeable to adeno-associated virus capsids throughout its entire structure or volume. In other words, a particle having a homogeneous porosity has a porosity which permits adeno-associated virus capsids to diffuse, completely or at least partly, through its pores, throughout the entire structure or volume of the particle.
Adeno-associated viruses are approx. 20-25 nm in diameter. Since a capsid is the shell of a virus particle, and since adeno-associated viruses do not have a lipoprotein bilayer envelope surrounding the capsid, the size of an adeno-associated virus capsid is approx. 20-25 nm in diameter.
Accordingly, where the chromatography material is in the form of particles having a homogeneous porosity and being at least partly permeable to adeno-associated virus capsids, each particle may suitably comprise pores of a diameter which is >25 nm, i.e., larger than the diameter of the adeno-associated virus capsids to be separated, thereby enabling diffusion of capsids within the entire particle. It is to be understood that for the specific purposes of the present disclosure, i.e., to separate adeno-associated virus capsids, a diameter >25 nm may be of any size >25 nm, including but not limited to 30, 50, 75, 100, 150, or 200 nm.
Further, it is to be understood that a particle having a homogeneous porosity throughout its entire structure or volume nevertheless may comprise pores of different sizes, both pores that are large enough to easily allow capsids to diffuse within the particle and pores that are small enough not to allow diffusion of capsids. This diversity of pore size can be measured by the diffusion coefficient of a molecule of a well-defined molecular weight and hydrodynamic size. As a non-limiting example, dextran, which has a molecular weight of 140-225 kDa or a hydrodynamic diameter of 20-25 nm (i.e., a diameter of the same size as adeno-associated virus capsids), can be used to evaluate the degree of diffusion of adeno-associated virus capsids within the pores of the particles.
The chromatography materials Capto Q and Capto DEAE, advantageously used in Examples 1-3 herein, comprise a support in the form of substantially spherical particles or beads, which have a diameter of approx. 90 μm. This type of particle is a non-limiting example of a particle having a homogeneous porosity (i.e., throughout its entire structure or volume) and being at least partly permeable to adeno-associated virus capsids (i.e., throughout its entire structure or volume).
Suitable particle sizes of a chromatography material for use in the presently disclosed methods may be in a diameter range of 5-500 μm, such as 10-100 μm, e.g., 30-90 μm. In the case of essentially spherical particles, the average particle size may be in the range of 5-1000 μm, such as 10-500. In a specific embodiment, the average particle size is in the range of 10-200 μm. The skilled person in this field can easily choose the suitable particle size and porosity depending on the process to be used. For example, for a large-scale process, for economic reasons, a more porous but rigid support may be preferred to allow processing of large volumes, especially for the capture step. In chromatography, process parameters such as the size and the shape of the column will affect the choice. In an expanded bed process, the matrix commonly contains high density fillers, preferably stainless-steel fillers. For other processes other criteria may affect the nature of the matrix.
The chromatography material may be dried, such as dried particles which upon use are soaked in liquid to retain their original form. For example, such a dried chromatography material may comprise dried agarose particles.
The chromatography material may be in the form of magnetic particles, i.e., magnetic adsorbent beads. The term “magnetic particle” is defined herein as a particle which is able to be attracted by a magnetic field. At the same time, magnetic particles for use in the presently disclosed method shall not aggregate in the absence of a magnetic field. In other words, the magnetic particles shall behave like superparamagnetic particles. The particle may have any symmetric shape, such as a sphere or a cube, or any asymmetric shape. Spherical magnetic particles are often called magnetic beads. It is to be understood that the terms “magnetic particle”, “magnetic bead”, “Mag particle”, “Mag bead”, “magparticle” and “magbead” may be used interchangeably herein, without limiting the scope to magnetic particles having a spherical shape. Separation of biomolecules by use of magnetic adsorbent beads is known in the art. Magnetic particles suitable for use in the presently disclosed method have been described in WO2018122089, which is hereby incorporated by reference in its entirety. A non-limiting example of magnetic particles which may be used in the presently disclosed methods are Mag Sepharose™ PrismA (Cytiva, Sweden).
The support of the chromatography material may alternatively take any other shape conventionally used in separation, such as monoliths, filters or membranes, capillaries, chips, nanofibers, surfaces, etc.
Where the support of the chromatography material comprises a monolith, a suitable pore diameter in the monolith for the purpose of separating adeno-associated virus capsids ranges from a minimum pore diameter of >25 nm, i.e., larger than the diameter of the capsids to be separated, and up to a maximum pore diameter of about 5 m, such as about 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 m.
Where the support of the chromatography material comprises nanofibers, such nanofibers may for example comprise electrospun polymer nanofibers. When in use, such nanofibers form a stationary phase comprising a plurality of pores through which a mobile phase can permeate.
The support of the chromatography material may comprise a membranous structure, such as a single membrane, a pile of membranes or a filter. The membrane may be an adsorptive membrane. Where the support of the chromatography material comprises a membranous structure, a suitable pore diameter in the membranous structure for the purpose of separating adeno-associated virus capsids ranges from a minimum pore diameter of >25 nm, i.e., larger than the diameter of the capsids to be separated, and up to a maximum pore diameter of about 5 m, such as about 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 m. Where the chromatography material comprises a membranous structure, such membranous structure may for example comprise a nonwoven web of polymer nanofibers.
Non-limiting examples of suitable polymers may be selected from polysulfones, polyamides, nylon, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, and polyethylene oxide, and mixtures thereof.
Alternatively, the polymer may be a cellulosic polymer, such as selected from a group consisting of cellulose and a partial derivative of cellulose, particularly cellulose ester, cross-linked cellulose, grafted cellulose, or ligand-coupled cellulose. Cellulose fiber chromatography (known as Fibro chromatography; Cytiva, Sweden) is an ultrafast chromatography purification for short process times and high productivity, which utilizes the high flow rates and high capacities of cellulose fiber. Where the support of the chromatography material comprises cellulose fibers such as Fibro, a suitable pore diameter in the cellulose fiber for the purpose of separating adeno-associated virus capsids ranges from a minimum pore diameter of >25 nm, i.e., larger than the diameter of the capsids to be separated, and up to a maximum pore diameter of about 5 μm, such as about 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 μm.
The term “membrane chromatography” has its conventional meaning in the field of bioprocessing. In membrane chromatography there is binding of components of a fluid, for example individual molecules, associates or particles, to the surface of a solid phase in contact with the fluid. The active surface of the solid phase is accessible for molecules by convective transport. The advantage of membrane adsorbers over packed chromatography columns is their suitability for being run with much higher flow rates. This is also called convection-based chromatography. A convection-based chromatography matrix includes any matrix in which application of a hydraulic pressure difference between the inflow and outflow of the matrix forces perfusion of the matrix, achieving substantially convective transport of substance(s) into the matrix or out of the matrix, which is effected very rapidly at a high flow rate. Convection-based chromatography and membrane adsorbers are described in for example US20140296464A1, US20160288089A1, WO2018011600A1, WO2018037244A1, WO2013068741A1, WO2015052465A1, U.S. Pat. No. 7,867,784B2, hereby incorporated by reference in their entirety.
In the herein disclosed pre-screening method, methods for separating fully packaged capsids from not fully packaged adeno-associated virus capsids, and uses of chromatography material for separating full from empty capsids, the chromatography material referred to may advantageously be a polishing chromatography material, meaning that the chromatography material is applied in a polishing step.
The term “polishing step” refers in the context of liquid chromatography to a final purification step, wherein trace impurities are removed to leave an active, safe product. Impurities removed during the polishing step are often conformers of the target molecule, i.e., forms of the target molecule having particular molecular conformations, or suspected leakage products. A polishing step may alternatively be called “secondary purification step”.
Further, the liquid sample added in step (a), step (e), and step (I) respectively, of the herein disclosed methods, may advantageously be a pre-purified liquid sample.
The present disclosure further provides a method for separating fully packaged adeno-associated virus capsids from not fully packaged adeno-associated virus capsids, comprising performing the steps (a)-(g) or alternatively steps (I)-(III) as described in detail above, the method further comprising a step (al) which comprises pre-purifying adeno-associated virus capsids by separating adeno-associated virus capsids from an adeno-associated virus capsid-containing cell culture harvest, thereby obtaining a pre-purified liquid sample comprising adeno-associated virus capsids, before adding said pre-purified liquid sample comprising adeno-associated virus capsids to the chromatography material according to step (a), step (e), or step (I), respectively, of the methods described above.
Such a pre-purifying step (al) may alternatively be called a “capture step” and refers in the context of liquid chromatography to the initial step(s) of a separation procedure. Most commonly, a capture step includes clarification (e.g. by filtration, centrifugation, or precipitation), and normally also concentration and/or stabilisation of the sample, and a significant purification from soluble impurities, for example by applying chromatography after the clarification, concentration, and stabilisation of sample. After the capture step, an intermediate purification may follow, which further reduces remaining amounts of impurities such as host cell proteins, DNA, viruses, endotoxins, nutrients, components of a cell culture medium, such as antifoam agents and antibiotics, and product-related impurities, such as aggregates, misfolded species, and aggregates.
Such a pre-purifying step may comprise subjecting the adeno-associated virus capsid-containing cell culture harvest to one or more of the following non-limiting examples of purification methods:
Non-limiting examples of chromatography materials suitable to apply in a pre-purifying step include affinity chromatography material, ion exchange chromatography material, and size-exclusion chromatography material, respectively. The chromatography material may be functionalized with a positively charged group, such as a quaternary amino, quaternary ammonium, or amine group, or a negatively charged group, such as a sulfonate or carboxylate group. The chromatography material may be functionalized with an ion exchanger group, an affinity peptide/protein-based ligand, a hydrophobic interaction ligand, an IMAC ligand, or a DNA based ligand such as Oligo dT.
Herein, the term “cell culture” refers to a culture of cells or a group of cells being cultivated, wherein the cells may be any type of cells, such as bacterial cells, viral cells, fungal cells, insect cells, or mammalian cells. A cell culture may be unclarified, i.e., comprising cells, or may be cell-depleted, i.e., a culture comprising no or few cells but comprising biomolecules released from the cells before removing the cells. Further, an unclarified cell culture may comprise intact cells, disrupted cells, a cell homogenate, and/or a cell lysate.
The term “cell culture harvest” is used herein to denote a cell culture which has been harvested and removed from the vessel or equipment, in which the cells have been cultivated.
The term “separation device” has its conventional meaning in the field of bioprocessing and is to be understood as encompassing any type of separation device which is capable of and suitable for separating and purifying compounds from a fluid containing by-products from the production of the compounds. A separation device may comprise a separation matrix, as further defined elsewhere herein.
Non-limiting examples of separation devices suitable for use in the polishing step according to the presently disclosed method include chromatography columns and membrane devices, as further described elsewhere herein. Such separation devices may suitably comprise chromatography material in the form of a strong anion exchange chromatography material comprising a ligand as defined by Formula I, II or III, as described in detail elsewhere herein.
Non-limiting examples of separation devices suitable for use in a capture step, or pre-purification step, as described herein, are filtration apparatuses, chromatography columns and membrane devices. Chromatography columns suitable for use in the capture step may for example be packed with affinity chromatography material, ion exchange chromatography material, mixed mode chromatography material or hydrophobic interaction chromatography material.
The herein disclosed method for separating fully packaged adeno-associated virus capsids from not fully packaged adeno-associated virus capsids may further comprise subjecting the eluate fractions comprising adeno-associated virus capsids fully packaged with genetic material, eluted in step (g) or step (III), respectively, of the methods as described above, to one or more of the following steps:
A person skilled in the art understands that the pharmaceutically relevant dose will depend on various factors such as, but not limited to, the disease or disorder to be treated as well as the weight and condition of the subject to be treated with a pharmaceutical composition.
Pharmaceutically acceptable buffers are well known in the art and can easily be chosen by the skilled person.
For the resulting composition to fulfil all regulatory requirements for pharmaceutical compositions, normally all of the above-listed three steps h1-h3 have to be performed.
In the above-disclosed methods, the adeno-associated virus capsids may advantageously be capsids of adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 9 (AAV9), or adeno-associated virus serotype 10 (AAV10), or a variant thereof.
The term “variant” in relation to an adeno-associated virus (AAV) serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, as listed above, is intended to mean a modified or engineered AAV, in which the capsid structure has been modified to improve clinical performance, for example towards a specific target organ. As a non-limiting example, an AAV8 variant comprises capsid parts of AAV8 and may additionally comprise capsid parts of other AAV serotypes than AAV8, such as AAV5. However, an AAV8 variant as referred to herein must retain a significant structural similarity to a non-modified AAV8 capsid, such as retaining at least 50%, such as 60%, 70%, 80%, or 90%, of the external surface structure of a non-modified AAV8 capsid. This applies equally to a variant of AAV serotype 1, 2, 3, 4, 5, 6, 7, 9, or 10, as compared to a non-modified AAV serotype 1, 2, 3, 4, 5, 6, 7, 9, or 10, respectively. Further, as a non-limiting example, in the context of purification or separation of a variant of AAV8, a “variant” is herein defined as an adeno-associated virus which has a functionally equivalent binding capacity to the ligand of a specified chromatography material, compared to the binding capacity of the original AAV8 to said specified chromatography material. This applies equally to a variant of AAV serotype 1, 2, 3, 4, 5, 6, 7, 9, or 10, as compared to the original AAV serotype 1, 2, 3, 4, 5, 6, 7, 9, or 10, respectively. The specified chromatography material may, for example, be a strong anion exchange chromatography material as disclosed in more detail elsewhere herein. A variant of an adeno-associated virus may for example be obtained by spontaneous mutation, or by engineered modification (i.e., obtained by human interaction), of one or more nucleotides of the genome of the adeno-associated virus.
According to a currently preferred embodiment, in the separation method as illustrated in
wherein the elution buffer of steps (b), (f), optionally (f′), and (g) comprises sodium acetate. Said method may be applied for separation of AAV capsids of any serotype or variant as described above. In particular, the capsids to be separated may be capsids of the AAV9 serotype or a variant thereof.
According to another currently preferred embodiment, in the separation method as illustrated in
wherein the elution buffer of steps (II), optionally (II′), and (III) comprises sodium acetate. Said method may be applied for separation of AAV capsids of any serotype or variant as described above. In particular, the capsids to be separated may be capsids of the AAV9 serotype or a variant thereof.
The present disclosure further provides use of an anion exchange chromatography material comprising a support, a ligand, and a surface extender connecting the ligand to the support, and being defined by Formula IV:
In the herein disclosed use, the adeno-associated virus capsids fully packaged with genetic material may be separated from adeno-associated virus capsids not fully packaged with genetic material by performing steps (a)-(d) as described above, and further by performing the steps:
Preferably, the elution buffer of step (b) of the above-described use, and where applicable also the elution buffer of steps (f) and (g) of the above-described use, comprises sodium acetate. Said use may be applied to separation of AAV capsids on any serotype or variant as described above. In particular, the capsids to be separated may be capsids of the AAV9 serotype or a variant thereof.
Devices or compositions “comprising” one or more recited components may also include other components not specifically recited. The term “comprising” includes as a subset “consisting essentially of” which means that the device or composition has the components listed without other features or components being present. Likewise, methods “comprising” one or more recited steps may also include other steps not specifically recited.
The singular “a” and “an” shall be construed as including also the plural.
The following currently available anion exchange chromatography materials gave improved results, as described further below:
Further, the currently available Capto Q ImpRes (Cytiva, Sweden) was also tested as described below. The support material of the ImpRes resin consists of substantially spherical particles or beads, which have a diameter of 40 μm.
Each resin was packed in a Tricorn 5 column (2 mL) according to the packing instructions. The runs were performed using an Akta Pure P25 system (P25-20031) with a flowrate of 1 CV/min (i.e., 2 mL/min), with the mixer of the system disconnected in order to minimize the dead volume and to get sharp conductivity steps. The sample was applied to the previously equilibrated column using a capillary loop. Typically, samples applied to each resin comprised affinity purified, or affinity and size exclusion purified, AAV2, AAV5, AAV8 or AAV9, respectively, at a concentration of approx. 5×1012 AAV capsids, containing a mixture of full and empty capsids (>5% full capsids, as follows: AAV2 7-10%, AAV5 47%, AAV8 11-35%, AAV9 40%). The material needs to have low conductivity (1-3 mS/cm) to ensure binding of AAV to the anion exchange ligand.
The 280 and 260 nm UV absorbance were monitored during the runs and the 260/280 ratios were used as a diagnostic tool to navigate in the chromatogram and distinguish between full and empty capsid populations. The chromatograms were analyzed using the Evaluation package of Unicorn. A 260/280 ratio above 1.2 is considered to indicate 100% full capsids, and a 260/280 ratio of approx. or below 0.6-0.7 is considered to indicate 100% empty capsids. Blank runs with the buffers without AAV were performed to subtract any background signal if needed, to ensure removal of potential UV signals from the buffers.
Currently available cation exchange resin, Capto S, and prototype cation exchange resin, Capto CM Dx ImpRes, were evaluated with acetate buffer at pH 4.5 and 5, with and without additives (0.1% poloxamer 188, 1% sucrose, with different elution salts (NaCl, NaOAc, NH4Cl or NH4SO4 up to 500 mM) and additive salts (MgCl2 and MgSO4 up to 20 mM), by applying an isocratic elution or continuous gradient elution, respectively. None of the above-mentioned conditions or resins resulted in a good baseline separation of full and empty capsids (data not shown).
Each of the currently available anion exchange resins with dextran extenders, Capto DEAE (partially strong anion exchange) and Capto Q (strong anion exchange), were evaluated using a buffer system including a buffer A and a buffer B, both containing 20 mM Bis-Tris Propane (BTP) pH 9.0 and 2 mM MgCl2, and buffer B additionally comprising 250 mM sodium acetate (NaOAc) as elution salt.
In
The step gradient applied in the pre-screening method comprised short steps (3 CV) of increasing concentration of NaOAc, more particularly a 12.5 mM increase per step, i.e., an increase in concentration of 5% per step of the 250 mM NaOAc of buffer B, which corresponds to an increase in conductivity of approx. 1.2-1.5 mS/cm per step.
Based on the chromatogram of
Also based on the chromatogram of
Here, it is noted that the second value of conductivity could instead have been chosen so as to correspond to the concentration of NaOAc applied when eluting the last peak containing empty and/or full capsids or any higher value. Thus, the second value of conductivity could have been approx. 3.6-4.5 mS/cm, corresponding to 15% of buffer B, i.e., 37.5 mM NaOAc as applied when eluting peak no. 3, or any higher value, i.e., ≥3.6 mS/cm, corresponding to ≥37.5 mM NaOAc.
Based on the chromatogram of
Also based on the chromatogram of
Here, it is noted that the second value of conductivity could instead have been chosen so as to correspond to the concentration of NaOAc applied when eluting the last peak containing empty and/or full capsids or any higher value. Thus, the second value of conductivity could have been approx. 9.6-12.0 mS/cm, corresponding to 40% of buffer B, i.e., 100 mM NaOAc as applied when eluting peak no. 3, or any higher value, i.e., >9.6 mS/cm, corresponding to ≥100 mM NaOAc.
Based on the chromatogram of
The first and second value of conductivity as determined in the above-described pre-screening method were applied in a subsequent two-step elution method for separating full capsids from empty capsids.
When using the Capto Q resin, all tested serotypes (AAV2, AAV5, AAV8, and AAV9) resulted in a good separation of full and empty capsids.
In the AAV9 sample tested, the peaks containing empty and/or full capsids were also analyzed with qPCR, showing very small amounts (0.3%) of full capsids in the empty peak and an overall 91% viral genome recovery in the full peak.
AAV9 capsids were separated on the Capto DEAE resin by applying the conditions established in the pre-screening method described above, i.e., a first value of conductivity corresponding to 0% of buffer B and a second value of conductivity corresponding to 4% of buffer B. The resulting separation of full and empty capsids was as shown in
Capto Q ImpRes (without Dextran Extenders)
Capto Q ImpRes resin was evaluated for separation of AAV9 and AAV5, respectively, under conditions identical to those described above, except that a flowrate of 1 ml/min was applied due to high delta column pressures. The resin did not work for AAV5 but worked adequately for pre-screening and 2-step elution for separation of AAV9 full capsids from AAV9 empty capsids (results not shown). However, Capto Q ImpRes (without extenders) does not bind AAV9 empty capsids (which thereby elute in the flow-through) and only binds AAV9 full capsids weakly, and thus provides a less robust separation method than Capto Q (with extenders).
The Capto Q resin was evaluated for separation of AAV9 and AAV5, respectively, by applying a pre-screening method followed by a 2-step elution as described in Example 1, with the difference that buffer A and buffer B of the buffer system both included 20 mM Bis-Tris Propane (BTP) pH 7.0 (AAV5) or pH 9.5 (AAV9), 1% sucrose and 0.1% Pluronic, and buffer B additionally comprising 20 mM MgCl2.
Based on the chromatogram of
Further, based on the chromatogram of
Alternatively, the second value of conductivity could have been chosen to be higher than the concentration of MgCl2 applied when eluting the last peak containing empty and/or full capsids, i.e., peak no. 5.
The first and second value of conductivity as determined in the above-described pre-screening method were applied in a subsequent two-step elution method for separating full capsids from empty capsids.
An alternative buffer system was also tested, including buffer A and buffer B having a higher pH, both containing 20 mM Bis-Tris Propane (BTP) pH 9.5, 1% sucrose and 0.1% Pluronic, and buffer B additionally comprising 30 mM MgCl2, i.e., a higher concentration of MgCl2. Here, in the pre-screening method, 3.5% steps of increasing MgCl2 were applied. The first and second conductivity values determined corresponded to 68% and 90% of buffer B, respectively. Said conductivity values were then applied in the two-step elution method.
The above-described conditions resulted in successful baseline separation of AAV5 (results for the alternative buffer system not shown). AAV9 binds less strongly to anion exchange, however conditions involving higher pH (such as 9.5), and application of small conductivity increase elution steps resulted in acceptable performance (results not shown).
Experimental designs for separation of fully packaged AAV9 capsids from empty and partially packaged AAV9 capsids are performed with equipment and samples as in Example 1 and Example 2 above, and further by use of anion exchange chromatography material as in Example 1 and Example 2, with the following variations:
Experimental designs for separation of full capsids from empty capsids of adeno-associated virus serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV10, and variants thereof, are performed according to the variable conditions of Examples 1-4 above.
It is to be understood that the present disclosure is not restricted to the above-described exemplifying embodiments thereof and that several conceivable modifications of the present disclosure are possible within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2206123.8 | Apr 2022 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/060871 | 4/26/2023 | WO |
