Method for isolating adeno-associated virus capsids, compositions obtained by said method and uses thereof

Abstract

The present disclosure provides a method for separating adeno-associated virus capsids in which genetic material is fully packaged from adeno-associated virus capsids in which genetic material is not fully packaged, comprising the steps of: a) applying a liquid sample containing adeno-associated virus capsids to a chromatographic material, wherein the liquid sample has a purity of at least 90% and a purity of at least 10 12 adeno-associated virus capsids at a concentration of adeno-associated virus capsids / ml, at least 10% of the adeno-associated virus capsids being fully packaged adeno-associated virus capsids with genetic material; the chromatographic material comprising a strong or partially strong anion exchange chromatographic material comprising a support and a ligand for binding to the adeno-associated virus capsids, the chromatographic material comprising a surface extender linking the ligand to the support, the surface extender being a polymer, the polymer being selected from the group consisting of (i) a polysaccharide having a naturally occurring backbone, such as starch, cellulose, dextran, or agarose, and and (ii) a polymer with a synthetic backbone, such as polyvinyl alcohol, polyacrylamide, polymethacrylamide, or polyvinyl ether; and (b) eluting the adeno-associated virus capsids in which the genetic material is fully packaged from the chromatographic material, wherein the adeno-associated virus capsids eluted in step (b) are eluted in an eluate fraction, the combined eluate fraction comprising at least 50% of the adeno-associated virus capsids of the liquid sample added in step (a), of which at least 60% of the adeno-associated virus capsids are fully packaged in the genetic material. Further disclosed are compositions, including pharmaceutical compositions, obtainable by the separation method, and uses of such compositions, and the use of an anionic chromatographic material for the separation of adeno-associated virus capsids.

Description

[Technical field]

[0001] The present disclosure relates to the field of adeno-associated capsid separation and is directed to methods for separating adeno-associated viral capsids in which genetic material is fully packaged from adeno-associated viral capsids in which genetic material is not fully packaged, and the use of anion exchange chromatography materials for such separation. Further disclosed are compositions, including pharmaceutical compositions, obtainable by said methods and uses of such compositions. [Background technology]

[0002] Adeno-associated virus (AAV) is a non-enveloped virus that has a linear single-stranded DNA (ssDNA) genome and 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. AAV vectors are one of the most attractive gene transfer tools for developing novel gene therapies for muscle diseases and other disorders. Most of the early AAV gene transfer studies used AAV serotype 2 (AAV2). To further improve the efficiency and specificity of AAV-mediated gene transfer, numerous AAV serotypes and variants have been developed by viral genome engineering and / or capsid modification. The use of serotypes such as AAV8 and AAV9 has increased in recent years. The target organ determines the choice of serotype. In order to use AAV particles as vectors in therapy, it is necessary to purify the viral particles from cellular impurities such as DNA after transfection. Ultracentrifugation is efficient but not scalable. Typically, several filtration steps and several chromatography steps are used to separate AAV particles from cell cultures (see, for example, Non-Patent Document 1).

[0003] The therapeutic efficacy of AAV vectors depends on a high percentage of viral particles in which the genetic material of interest is fully packaged. Upstream expression systems produce a mixture of fully packaged AAV particles (containing the genetic material of interest), empty AAV particles, and AAV particles in which the genetic material of interest is partially packaged, along with impurities. Therefore, it is necessary to enrich fully packaged AAV particles in the purification process. However, there are several challenges associated with achieving efficient and scalable separation of fully packaged and empty adeno-associated virus capsids, including: - The large diversity of capsids (serotypes and variants) and cell culture differences in yield of intact capsids, which means that extensive optimization is required for the purification of each serotype or variant of adeno-associated virus. - Small differences between fully packaged and empty capsids related to some parameters relevant for purification, e.g., isoelectric point. - In addition to fully packaged and empty capsids, partially packaged capsid variants are also produced in infected host cells, and there are indications that such partially packaged, and therefore less therapeutically effective, capsids may in part be coeluted with fully packaged capsids.

[0004] Therefore, novel purification strategies are needed to speed up the purification process, reduce costs, and provide large-scale methods for downstream processing of various adeno-associated virus serotypes. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 6,428,707 [Patent Document 2] U.S. Patent No. 6,602,990 [Patent Document 3] US Patent Application Publication No. 2014 / 0296464 [Patent Document 4] U.S. Patent Application Publication No. 2016 / 0288089 [Patent Document 5] International Publication No. 2018 / 011600 [Patent Document 6] International Publication No. 2018 / 037244 [Patent Document 7] International Publication No. 2013 / 068741 [Patent Document 8] International Publication No. 2015 / 052465 [Patent Document 9] U.S. Patent No. 7867784

Non-licensed documents

[0006]

Non-patent document 1

Non-patent document 2

Non-patent document 3

Non-patent document 4

[0007] The objective of the present disclosure is to provide a scalable solution that results in improved separation of fully packaged and non-fully packaged adeno-associated virus capsids. This is accomplished by obtaining improved resolution of fully packaged and non-fully packaged capsids when performing the separation method as disclosed herein, which results in achieving a composition with an improved ratio of fully packaged capsids to non-fully packaged capsids. The focus of the present disclosure is the polishing step of the separation method, also referred to as secondary or final purification. [Means for solving the problem]

[0008] More particularly, a first aspect of the disclosure provides a method for separating adeno-associated virus capsids in which genetic material is fully packaged from adeno-associated virus capsids in which genetic material is not fully packaged, the method comprising: a) adding a liquid sample containing adeno-associated virus capsids to a chromatographic material, the liquid sample having a purity of at least 90% and a purity of at least 10 12 adeno-associated virus capsids at a concentration of adeno-associated virus capsids / ml, at least 10% of the adeno-associated virus capsids being fully packaged adeno-associated virus capsids in which the genetic material is; the chromatographic material comprises a strong or partial strong anion exchange chromatographic material comprising a support and a ligand for binding to the adeno-associated virus capsid; The chromatographic material comprises a surface extender that links the ligand to the support, the surface extender being a polymer, the polymer comprising: (i) polymers with naturally occurring backbones, such as polysaccharides, such as starch, cellulose, dextran, or agarose, and (ii) Polymers with synthetic backbones, such as polyvinyl alcohol, polyacrylamide, polymethacrylamide, or polyvinyl ether. A process selected from the following: b) eluting the adeno-associated virus capsids in which the genetic material is fully packaged from the chromatographic material. wherein the adeno-associated virus capsids eluted in step (b) are eluted in an eluate fraction, the combined eluate fraction comprising at least 50% of the adeno-associated virus capsids of the liquid sample added in step (a), at least 60% of the adeno-associated virus capsids being fully packaged with genetic material.

[0009] The method disclosed above comprises the steps of: separating the eluate fraction containing the adeno-associated virus capsids in which the genetic material is fully packaged, which is eluted in step (b), c1) concentrating the adeno-associated virus capsid to a pharma- ceutical suitable dose; c2) replacing the buffer applied in step (b) of the above mentioned separation method with a pharma- ceutically acceptable buffer, and / or c3) Sterilizing the eluate fraction containing the adeno-associated virus capsid to obtain a pharmaceutical composition comprising the adeno-associated virus capsid.

[0010] The present disclosure further provides a method for preventing or treating an organ or tissue associated disease or disorder in a subject, comprising administering to the subject a pharmaceutical composition comprising adeno-associated virus capsids obtained by carrying out the above-mentioned separation method, wherein in the pharmaceutical composition, the ratio of adeno-associated virus capsids in which the genetic material is fully packaged to adeno-associated virus capsids in which the genetic material is not fully packaged is at least 3:2.

[0011] In addition, the present disclosure is directed to a composition comprising adeno-associated virus capsids obtained by carrying out a method for separating fully packaged from non-fully packaged adeno-associated virus capsids, as described in detail above, wherein the ratio of fully packaged adeno-associated virus capsids in which the genetic material is present to non-fully packaged adeno-associated virus capsids in which the genetic material is present is at least 3:2.

[0012] The present disclosure also provides a pharmaceutical composition comprising adeno-associated virus capsids obtained by carrying out the above disclosed separation method comprising one or more of steps c1-c3 (as described in detail above), wherein in the pharmaceutical composition the ratio of adeno-associated virus capsids in which the genetic material is fully packaged to adeno-associated virus capsids in which the genetic material is not fully packaged is at least 3:2.

[0013] There is further provided a pharmaceutical composition as described above for use in therapy, optionally for use in gene therapy.

[0014] The disclosure, as further described in detail below, includes a support, a ligand, and a surface extender linking the ligand to the support for separating adeno-associated viral capsids in which genetic material is fully packaged from adeno-associated viral capsids in which genetic material is not fully packaged, the surface extender having formula IV:

[0015] [ka]

[0016] Also provided is the use of an anion exchange chromatography material defined by:

[0017] In particular, the present disclosure is directed to the isolation and use of adeno-associated virus capsids of adeno-associated virus serotypes 1, 2, 4, 5, 6, 7, 8, 9, and 10 (AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10) or variants thereof.

[0018] Preferred aspects of the present disclosure are set out below in the detailed description and dependent claims. [Brief description of the drawings]

[0019] [Figure 1] 1 is a flow chart of a method for isolating adeno-associated virus capsids according to the present disclosure. [Diagram 2] 2 is a flow chart of the method for isolating adeno-associated virus capsids according to FIG. 1, further comprising an additional step (a1) prior to step (a). [Diagram 3] 2 is a flow chart of a method for isolating adeno-associated virus capsids according to FIG. 1, optionally including step (a1) before step (a) and further including one or more additional steps (c1), (c2) and / or (c3) after step (b). [Figure 4] FIG. 1 is a graph showing elution curves for fully packaged and empty capsids following the separation method as described in Example 1 below. [Diagram 5] FIG. 1 is a graph showing elution curves for fully packaged and empty capsids following the separation method as described in Example 1 below. [Figure 6] 1 is a graph showing elution curves for fully packaged and empty capsids following the separation method as described in Example 4 below. [Figure 7] 1 is a graph showing elution curves for fully packaged and empty capsids following the separation method as described in Example 4 below. [Figure 8] 1 is a graph showing elution curves for fully packaged and empty capsids following the separation method as described in Example 5 below. [Figure 9] FIG. 1 is a graph showing elution curves for fully packaged and empty capsids following the separation method as described in Example 6 below. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present disclosure provides a method for separating adeno-associated virus capsids in which genetic material has been fully packaged from adeno-associated virus capsids in which genetic material has not been fully packaged, as shown in FIG. a) adding a liquid sample containing adeno-associated virus capsids to a chromatographic material, the liquid sample having a purity of at least 90% and a purity of at least 10 12 adeno-associated virus capsids at a concentration of adeno-associated virus capsids / ml, at least 10% of the adeno-associated virus capsids being fully packaged adeno-associated virus capsids in which the genetic material is; the chromatographic material comprises a strong or partial strong anion exchange chromatographic material comprising a support and a ligand for binding to the adeno-associated virus capsid; The chromatographic material comprises a surface extender that links the ligand to the support, the surface extender being a polymer, the polymer comprising: (i) polymers with naturally occurring backbones, such as polysaccharides, such as starch, cellulose, dextran, or agarose, and (ii) Polymers with synthetic backbones, such as polyvinyl alcohol, polyacrylamide, polymethacrylamide, or polyvinyl ether. A process selected from the following: b) eluting the adeno-associated virus capsids in which the genetic material is fully packaged from the chromatographic material. wherein the adeno-associated virus capsids eluted in step (b) are eluted in an eluate fraction, which combined eluate fraction comprises at least 50% of the adeno-associated virus capsids of the liquid sample added in step (a), at least 60% of the adeno-associated virus capsids having fully packaged genetic material, thereby solving or at least alleviating problems associated with existing methods for separating fully packaged from non-fully packaged adeno-associated virus capsids.

[0021] Notable advantages of the presently disclosed methods include that they are suitable for large-scale separation of fully packaged capsids and not fully packaged capsids, and provide an improved ratio of fully packaged capsids to not fully packaged capsids compared to prior art methods. By practicing the presently disclosed methods, it is possible to obtain compositions having a ratio of fully packaged capsids to not fully packaged capsids of at least 3:2. More specifically, the presently disclosed methods provide an improved ratio of fully packaged capsids to empty capsids, and an improved ratio of fully packaged capsids to partially packaged capsids.

[0022] "Virus particle" is used herein to refer to a complete infectious virus particle. It comprises a core containing the viral genome (i.e., the viral genome) in either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) form, which is surrounded by a morphologically defined shell. The shell is called the 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, with the aim of generating viral vectors for various applications, such as therapeutics, the genome of the viral particle is modified to contain a gene insert containing the genetic material of interest. The modified viral particle is allowed to infect host cells in cell culture, the viral particles grow in said host cells, and the viral particles are then purified from the cell culture by any means of separation and purification. In the present specification, the viral particles isolated from the cell culture by the methods disclosed herein may alternatively be referred to as "target molecules" or "targets". "Viral particle" is intended to mean a type of viral particle, and it should be understood that the singular form of the term can encompass a large number of individual viral particles. As used herein, the term "viral particle" may be used interchangeably with the terms "vector" and "capsid," respectively, as further defined below.

[0023] The term "vector" is used herein to refer to a viral particle, usually a recombinant viral particle, intended to be used to achieve gene transfer to modify a particular cell type or tissue. Viral particles can be engineered to provide vectors that express, for example, therapeutic genes. Several types of viruses are currently being investigated for use in delivering genetic material (e.g., genes) to cells to provide transient or permanent transgene expression. These include adenoviruses, retroviruses (gamma-retroviruses and lentiviruses), poxviruses, adeno-associated viruses (AAV), baculoviruses, and herpes simplex viruses. In the present specification, the term "vector" can be used interchangeably with the terms "viral particle" and "capsid," respectively.

[0024] The term "capsid" refers to the shell of a viral particle. The capsid surrounds the core of the viral particle and should usually contain the viral genome. Modified (recombinant) capsids, as produced in the upstream process of production, are expected to contain the complete viral genome, which contains the genetic material of interest for one or more applications, e.g., for various therapeutic applications. However, due to low packaging efficiency, the constructed capsids do not necessarily contain genetic material or only encapsidate truncated gene fragments, resulting in so-called empty and partially filled capsids, respectively. These capsids have no therapeutic function, but still compete to bind to receptors during cell-mediated processes. This can reduce the overall therapeutic efficacy and cause undesirable immune responses. As a result, tracking these capsids throughout the production process is important to ensure consistent product quality and appropriate administration responses (Non-Patent Document 2). In up to 20-30% of the population of artificially produced virus particles in cell culture, the capsid is only partially filled with genetic material. Moreover, in up to 98% of artificially produced virus particles, the capsid does not contain any part of the viral genome at all, i.e. is empty. However, typically between 80%-90% of artificially produced virus particles have empty capsids, and the best case currently achieves only 50% empty capsids.

[0025] As used herein, the term "capsid" may be used interchangeably with the terms "vector" and "viral particle," respectively. In the context of this disclosure, a capsid may or may not contain genetic material.

[0026] The term "genetic material of interest" is intended to mean genetic material that is deemed suitable and valuable in the field of bioprocessing to be produced by viral replication and purified so that it can be used in various applications, including but not limited to therapeutic applications. As a non-limiting example, genetic material of interest can include therapeutically relevant genetic material, such as therapeutically relevant nucleotide sequences.

[0027] The term "capsid in which the genetic material is fully packaged" refers to a capsid that is correctly produced (by a host cell), or in other words, The capsid containing the complete viral genome, or in other words, The capsid contains 100% of the viral genome, or in other words, Capsid containing a functional viral genome is used herein to represent

[0028] The viral genome comprises a genetic insert which contains the genetic material of interest as defined elsewhere herein.

[0029] A capsid that contains a complete viral genome may alternatively be referred to herein as a "complete capsid" or a "completely packaged capsid." The terms "complete capsid," "completely packaged capsid," and "capsid in which the genetic material is fully packaged" may be used interchangeably throughout this document.

[0030] The term "capsid in which the genetic material is not fully packaged" refers to a capsid that has not been properly produced (by the host cell), or in other words, Capsids that do not contain the complete viral genome, or in other words, Capsids that contain less than 100% of the viral genome is used herein to represent

[0031] Capsids in which the genetic material is not fully packaged are either partially filled with genetic material or are not filled with any genetic material at all.

[0032] The term "capsid in which genetic material is not completely packaged" encompasses the terms "partially filled capsid" and "empty capsid" as defined below.

[0033] A "partially filled capsid" refers to a capsid that contains a portion of the viral genome, such as a missing portion of the viral genome, or in other words, A capsid containing a partial viral genome, or in other words, Capsids containing incomplete viral genomes, or in other words, Capsids containing defective viral genomes, or in other words, A capsid that comprises more than 0% and less than 100% of the complete viral genome, for example, about 1% to about 99%, for example, about 5% to about 95%, for example, about 10% to about 90%, or for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% of the complete viral genome. As defined herein, partially filled capsids are incorrectly generated capsids, and therefore it is desirable to separate and remove as many partially filled capsids as possible from a population of capsids before using the population of capsids for its intended use, e.g., therapeutic use. As used herein, partially filled capsids may alternatively be referred to as "intermediate capsids."

[0034] An "empty capsid" is a capsid that does not contain any part of the viral genome, i.e., contains 0% of the viral genome, or in other words, Empty capsids are defined herein as capsids that are not filled with any genetic material. Thus, empty capsids do not contain any genetic material of interest. Therefore, it is desirable (e.g., may be required due to clinical regulations) to separate and remove as many empty capsids as possible from a population of capsids before using the population of capsids for its intended use, e.g., therapeutic use.

[0035] Before using a population of viral particles for its intended use, e.g., therapeutic use, it may be desirable (and may even be required, e.g., due to clinical regulations) to enrich for complete capsids, i.e., to increase the percentage of complete capsids at the expense of the percentage of partially filled and empty capsids.

[0036] The percentage of full and empty capsids in a population of capsids can be estimated or analyzed in several ways known in the art, some of which are briefly described below.

[0037] 1:A260:280 in the chromatogram will give an estimate of the percentage of complete capsids present in the peak (a ratio of 1-1.5 indicates enrichment in complete capsids, while a ratio of 0.5-0.7 contains mostly empty capsids).

[0038] 2. qPCR:ELISA ratio. qPCR quantifies viral genomes and ELISA quantifies total viral particles. The ratio of the two assays with variation will be less precise and uncertain. Orthogonal analysis is required for confirmation (see 3, 4 or 5 below).

[0039] 3. Analytical anion exchange to separate full and empty capsids (A260:280 ratio and peak area to calculate percentages). Precision dependent on peak definition.

[0040] 4. Analytical Ultracentrifugation (AUC): Detects and quantifies particles of different densities (corresponding to full, partially filled, and empty capsids). This is currently known in the art as the "gold standard". However, ultracentrifugation is not scalable and therefore not suitable for the analysis of large batches of capsids.

[0041] 5. Transmission Electron Microscopy (TEM). Image analysis to count particles (full, partially filled and empty capsids). Can introduce artifacts from sample preparation.

[0042] Some methods for estimating or analyzing the percentage of full and empty capsids in a population of capsids are described in more detail in Xiaotong Fu et al., Analytical Strategies for Quantification of Adeno-Associated Virus Empty Capsids to Support Process Development, Human gene therapy methods, 2019, 30(4):144-152, which is hereby incorporated by reference.

[0043] The term "liquid sample" as used herein should be understood to encompass any type of sample obtained from a cell culture, or a fluid derived from a cell culture, which has been at least partially purified by any means of separation and purification.

[0044] The term "separation matrix" is used herein to describe a material that includes a support to which one or more ligands that contain functional groups are coupled. The functional groups of the ligands bind to compounds, also referred to herein as analytes, that are to be separated from the liquid sample and / or from other compounds present in the liquid sample. The separation matrix may further include compounds that couple the ligands to the support. The terms "linker", "extender", and "surface extender" may be used to describe such compounds, as further described below. The term "resin" is sometimes used for separation matrices in the art. The terms "chromatographic material" and "chromatographic matrix" are used herein to describe types of separation matrices.

[0045] The term "surface" as used herein means all external surfaces, which in the case of porous supports includes the outer surfaces and the pore surfaces.

[0046] As used herein, the term "strong anion exchange chromatographic material" is intended to mean a chromatographic material that includes a ligand that includes a quaternized amine group. A quaternary amine group is a strong anion exchange group that is always positively charged regardless of the pH to which it is exposed. For DEAE-based types of chromatographic materials, the degree of quaternization of the amine groups can vary among the amine groups contained in the chromatographic material. A degree of quaternization of about 12% to about 100% of the amine groups in the entire chromatographic material is believed to result in a chromatographic material that generally behaves like a strong or at least partially strong anion exchange chromatographic material, since at least 12% of all these amine groups are always charged. In contrast to quaternized amine groups, almost all other ion exchange groups are weak, i.e., their charge varies from completely charged to uncharged within the reasonable pH range of use (e.g., pH 2-11), and have a neutral charge (equal amount of + and - charges) at pI.

[0047] Capto Q (Cytiva, Sweden) is a non-limiting example of a strong anion exchange chromatographic material with about 100% quaternized amine groups. Capto DEAE (Cytiva, Sweden) is a non-limiting example of a strong or partially strong anion exchange chromatographic material with a degree of quaternization of about 15% of the amine groups.

[0048] The separation matrix can be contained in any type of separation device as further defined elsewhere herein. As a non-limiting example, the chromatographic material can be packed into a chromatographic column and then the liquid sample can be added to the chromatographic material contained in the chromatographic column.

[0049] In this context, a "ligand" is a molecule that has a known or unknown affinity for a given analyte and includes any functional group or capture agent immobilized on its surface, whereas an "analyte" includes any specific binding partner for a ligand. The term "ligand" may be used interchangeably herein with the terms "specific binding molecule," "specific binding partner," "capture molecule," and "capture agent." Molecules in a liquid sample that interact with a ligand are referred to herein as "analytes." Analytes of interest according to the present disclosure are adeno-associated virus capsids, more specifically, adeno-associated virus capsids with or without fully packaged genetic material. Thus, the terms "analyte," "adeno-associated virus capsid," and "capsid" may be used interchangeably herein.

[0050] In the methods disclosed herein for separating fully packaged capsids from not fully packaged capsids, the chromatographic material used comprises a linker that connects the ligand to the support, i.e., the coupling of the ligand to the support is achieved by introducing a linker between the support and the ligand. The coupling can be carried out according to any conventional covalent coupling method, such as by using epichlorohydrin; epibromohydrin; allyl-glycidyl ether; bis-epoxides such as butanediol diglycidyl ether; halogen-substituted aliphatic substances such as di-chloro-propanol; and divinyl sulfone. Other non-limiting examples of suitable linkers are polyethylene glycols (PEGs) having 2-6 carbon atoms, carbohydrates having 3-6 carbon atoms, and polyhydric alcohols having 3-6 carbon atoms. All of these methods are well known in the art and are easily carried out by the skilled artisan.

[0051] The ligand is preferably coupled to the support via a longer linker molecule, also known as a "surface extender" or simply an "extender". Extenders are well known in the art and are commonly used to sterically increase the distance between the ligand and the support. Extenders may be represented as tentacles or flexible arms. For a more detailed description of possible chemical structures, see, for example, US Pat. No. 5,399,363, which is incorporated herein by reference. Briefly, the extender may be in the form of a polymer, such as a homopolymer or a copolymer. The hydrophilic polymeric extender may be of synthetic origin, i.e. with a synthetic backbone, or of biological origin, i.e. a biopolymer with a naturally occurring backbone. Typical synthetic polymers are polyvinyl alcohol, polyacrylamide and polymethacrylamide, polyvinyl ethers, etc. Typical biopolymers are polysaccharides, such as starch, cellulose, dextran, agarose, etc. The results described in Example 1 herein surprisingly show that chromatographic materials containing surface extenders provide improved separation of intact and empty AAV capsids compared to the same chromatographic materials without surface extenders.

[0052] The term "eluting agent" is used in its conventional sense in the art, ie a buffer of suitable pH and / or ionic strength to release one or more compounds from the separation matrix.

[0053] The term "eluate" is used in its conventional sense in the art, ie, the portion of a liquid sample that is eluted from a chromatography column after the liquid sample has been loaded onto the chromatography column.

[0054] As mentioned above, in the method for separating fully packaged capsids from not fully packaged capsids, the liquid sample applied to the chromatographic material in step (a) has a purity of at least 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and a purity of at least 10 12 , e.g., 10 13 , 10 14 , or 10 15 adeno-associated virus capsids at a concentration of adeno-associated virus capsids / ml, of which at least 10%, e.g., 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% are adeno-associated virus capsids in which the genetic material is fully packaged. With respect to the purity of adeno-associated virus capsids in a liquid sample, a purity of at least 90%, e.g., up to 99%, is intended to mean that at least 90%, e.g., up to 99%, of the biological material in the liquid sample is represented by adeno-associated virus capsids (including full, empty, and partially filled capsids), while the remainder, up to 10%, e.g., 1%, is represented by host cell proteins and DNA.

[0055] The objective of step (b) of the method disclosed above is to obtain fully packaged capsids of the highest possible purity. Those skilled in the art will readily understand that this can be achieved by applying a variety of different separation conditions. A non-limiting example of a separation condition for obtaining fully packaged capsids of the highest possible purity is one that allows the binding of incompletely packaged capsids to a chromatographic material, but does not allow the binding of incompletely packaged capsids to a chromatographic material. (i) allowing fully packaged capsids to substantially flow through the chromatographic material (i.e., fully packaged capsids that do not substantially bind to the chromatographic material); or (ii) separation conditions that allow fully packaged capsids to bind to the chromatographic material and subsequently elute them from the chromatographic material. It should be understood that in the bind-elute process described in item (ii), fully packaged capsids may be eluted from the chromatographic material before or after non-fully packaged capsids, depending on which separation conditions are applied.

[0056] As mentioned above, there are slight differences between fully packaged and not fully packaged capsids in relation to some parameters relevant for purification, such as their isoelectric points. This often leads to (at least partial) co-elution of fully packaged and not fully packaged capsids. Thus, in reality, the adeno-associated virus capsids eluted in step (b) of the method disclosed above will not be completely separated into full, empty, and partially filled capsids. However, there will be an eluate fraction that contains a substantially higher percentage of full capsids than in the liquid sample applied to the chromatographic material in step (a). More specifically, as disclosed above, the adeno-associated virus capsids eluted in step (b), i.e., adeno-associated virus capsids with fully packaged genetic material, are eluted in an eluate fraction, which combined eluate fraction comprises at least 50%, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, of the adeno-associated virus capsids of the liquid sample added in step (a), of which at least 60%, e.g., 65%, 70%, 75%, 80%, 85%, or 90% of the adeno-associated virus capsids are fully packaged with genetic material. Non-limiting examples of intact capsid recovery and purification achieved by the methods disclosed herein include at least 50% recovery of capsids from the liquid sample added in step (a), of which at least 60% are intact capsids, e.g., at least 70% recovery of capsids from the liquid sample added in step (a), of which at least 80% are intact capsids. In Example 1, further described below, results show at least 60% recovery of capsids from the harvest, of which at least 60% are intact capsids. Currently, there are no publicly available large-scale methods that provide recovery and purification as high as the methods disclosed herein.

[0057] As mentioned above, the chromatographic material applied in the methods disclosed herein includes a strong or partial strong anion exchange chromatographic material comprising a support and a ligand for binding to the adeno-associated virus capsid.

[0058] The ligand for the strong anion exchange chromatography material has the following formula I:

[0059] [ka]

[0060] (In the formula, R1 is selected from C1-C3 alkyl, and R2 and R3 are independently selected from C1-C3 alkyl, CH2OH, and CH2CHOHCH3. It can be defined by:

[0061] As a non-limiting example, each of R1, R2, and R3 is CH3.

[0062] There are currently available chromatographic materials comprising ligands defined by formula I, where each of R1, R2, and R3 is CH3, such as the chromatographic material available under the name Capto Q provided by Cytiva, Sweden (www.cytivalifesciences.com). Capto Q further comprises dextran as a surface extender and is a chromatographic medium for high-resolution polishing steps in industrial purification processes, such as for the purification of monoclonal antibodies. However, so far it has not been used for the separation of fully packaged and non-fully packaged adeno-associated capsids.

[0063] According to another non-limiting example, R1 and R2 are ethyl and R3 is methyl.

[0064] By yet another non-limiting example, R1 and R2 are methyl and R3 is CH2CHOHCH3.

[0065] The density of the ligand defined by formula I can 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 strong anion exchange chromatography material.

[0066] Alternatively, the ligand of the strong or partially strong anion exchange chromatography material may be of the following formula II:

[0067] [ka]

[0068] (In the formula, m is an integer from 1 to 3; R1 and R2 are independently selected from C1-C3 alkyl, R3 and R4 are independently selected from C1-C3 alkyl and CH2CHOHCH3, and R5 is selected from hydrogen, C1-C3 alkyl and CH2CHOHCH3. where m is 1, the ligand of the strong or partially strong anion exchange chromatography material can be defined by the following formula III:

[0069] [ka]

[0070] (In the formula, n is an integer of 0 to 3, provided that when n is 0, R3 and R4 are independently selected from C1-C3 alkyl, and R5 is hydrogen or CH2CHOHCH3. is defined as follows:

[0071] As a non-limiting example, the ligand may be defined by Formula III and have the following structures (i)-(iv): (i) the structure in which n is 0, R3 and R4 are ethyl, and R5 is hydrogen or CH2CHOHCH3; (ii) the structure in which n is 1, R1, R2, R3, and R4 are ethyl, and R5 is hydrogen or CH2CHOHCH3; (iii) the structure, in which n is 2, each of R1 and R2 is ethyl, R3 and R4 are ethyl, and R5 is hydrogen or CH2CHOHCH3; (iv) the structure in which n is 3, each of R1 and R2 is ethyl, R3 and R4 are ethyl, and R5 is hydrogen or CH2CHOHCH3. Includes a combination of two or more of the following:

[0072] One currently available chromatographic material comprising a ligand defined by formula III and comprising a combination of structures (i)-(iv) referred to above is a chromatographic resin called Capto DEAE (Cytiva, Sweden). Capto DEAE further comprises dextran as a surface extender and is a chromatographic medium for high-resolution polishing steps in industrial purification processes, e.g., for the purification of monoclonal antibodies. However, to date it has not been used for the separation of fully packaged and non-fully packaged adeno-associated capsids.

[0073] According to another non-limiting example, the ligand is defined by formula III, m is 1, n is 1, 2, or 3, each of R1, R2, R3, and R4 is methyl, and R5 is hydrogen.

[0074] According to yet another non-limiting example, the ligand is defined by formula III, m is 1, n is 1, 2, or 3, each of R1, R2, R3, and R4 is methyl, and R5 is CH2CHOHCH3.

[0075] According to another non-limiting example, the ligand is defined by formula III, m is 1, and the ligand has the following structures (i)-(iv): (i) the structure in which n is 0, R3 and R4 are methyl, and R5 is hydrogen or CH2CHOHCH3; (ii) the structure in which n is 1, R1, R2, R3, and R4 are methyl, and R5 is hydrogen or CH2CHOHCH3; (iii) the structure, in which n is 2, each of R1 and R2 is methyl, R3 and R4 are methyl, and R5 is hydrogen or CH2CHOHCH3; (iv) the structure in which n is 3, R1 and R2 are methyl, R3 and R4 are methyl, and R5 is hydrogen or CH2CHOHCH3. Includes a combination of two or more of the following:

[0076] The density of the ligand defined by Formula II or Formula III can 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 strong or partially strong anion exchange chromatography material.

[0077] As described above, the chromatographic material comprises a surface extender that links the ligand to the support, the surface extender being a polymer, the polymer being (i) polymers with naturally occurring backbones, such as polysaccharides, such as starch, cellulose, dextran, or agarose, and (ii) Polymers with synthetic backbones, such as polyvinyl alcohol, polyacrylamide, polymethacrylamide, or polyvinyl ether. is selected from.

[0078] As a non-limiting example, the surface extender is dextran. The dextran can have a molecular weight of about 10 to about 2000 kDa, e.g., about 10, 40, 70, 250, 750, or 2000 kDa, e.g., 40 kDa. The density of the dextran can be about 5 to about 30 mg of dextran per ml of chromatography material. It should be understood that the amount of dextran immobilized on the chromatography material can vary depending, for example, on the molecular weight of the dextran immobilized. Typically, a decreasing amount is required to increase the molecular weight of the dextran.

[0079] Steps (a) and (b) of the method disclosed above may comprise applying a buffer having a pH of about 6.0 to about 10.5, such as about 7.0 to about 10.0, such as about 7.5 to about 9.5, or about 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, or 10.5. By way of non-limiting example, as described in Example 1 below, a pH of about 9.5 may be applied to a chromatographic material comprising a ligand defined by Formula I. Additionally, a pH of about 7.5 may be applied to a chromatographic material comprising a ligand defined by Formula II or Formula III.

[0080] The buffer is suitably selected from buffers commonly recommended for anion exchange chromatography and may include, for example, tris(hydroxymethyl)amino-methane (i.e., Tris), 1,3-bis(tris(hydroxymethyl)methylamino)propane (i.e., Bis-Trispropane), triethanolamine, N-methyldiethanolamine, diethanolamine, 1,3-diaminopropane, or ethanolamine. A person skilled in the art can select a suitable concentration for any one of the buffers listed above.

[0081] In the method disclosed above, step (b) may comprise applying a buffer, optionally one of the buffers mentioned above, which comprises a compound that improves the separation of capsids in which the genetic material is fully packaged and capsids in which the genetic material is not fully packaged. This compound may or may not be present in the buffer applied in step (a). Without being bound by theory, such a compound may improve the separation, for example, by affecting the capsid-ligand interaction or the capsid-capsid interaction. The compound that improves the separation may be selected, for example, from carbohydrates, divalent metal ions, and detergents.

[0082] When the compound that improves separation is a carbohydrate, it may be selected, for example, from sucrose, sorbitol, and polysaccharides.

[0083] When the compound that improves separation is a divalent metal ion, it can be, for example, Mg 2+ , Fe 2+ , and Mn 2+ The metal ion may be selected from the group consisting of: 1) a cation, 2) a phosphate buffer, 3) a phosphate buffer, 4) a phosphate buffer, 5) a phosphate buffer, 6) a phosphate buffer, 7) a phosphate buffer, 8) a phosphate buffer, 9) a phosphate buffer, 10) a phosphate buffer, 11) a phosphate buffer, 12) a phosphate buffer, 13) a phosphate buffer, 14) a phosphate buffer, 15) a phosphate buffer, 16) a phosphate buffer, 17) a phosphate buffer, 18) a phosphate buffer, 19) a phosphate buffer, 20) a phosphate buffer, 25) a phosphate buffer, 26) a phosphate buffer, 27) a phosphate buffer, 28) a phosphate buffer, 29) a phosphate buffer, 30) a phosphate buffer, 31) a phosphate buffer, 32) a phosphate buffer, 33) a phosphate buffer, 34) a phosphate buffer, 35) a phosphate buffer, 36) a phosphate buffer, 37) a phosphate buffer, 38) a phosphate buffer, 39) a phosphate buffer, 40) a phosphate buffer, 41) a phosphate buffer, 42) a phosphate buffer, 43) a phosphate buffer, 44) a phosphate buffer, 45) a phosphate buffer, 46) a phosphate buffer, 47) a phosphate buffer, 48) a phosphate buffer, 49) a phosphate buffer, 50) a phosphate buffer, 51) a phosphate buffer, 52) a phosphate buffer, 53) a phosphate buffer, 54) a phosphate buffer, 55) a phosphate buffer, 56) a phosphate buffer, 57) a phosphate buffer, 58) a phosphate buffer, 59) a phosphate buffer, 58) a phosphate buffer, 59)

[0084] When the compound improving separation is a surfactant, it may be selected, for example, from poloxamers, such as Poloxamer 188 or Pluronic™ F68, and polysorbates, such as Tween 20 or Tween 80.

[0085] In the above method, step (b) may include a step of applying a buffer, optionally one of the buffers mentioned above, which contains a compound that can help elute the capsids bound to the chromatographic material. This compound is not present in the buffer applied in step (a). A non-limiting example of such a compound is a salt, such as a salt of a monovalent metal ion. More specifically, the salt may be a kosmotropic salt. A salt in an aqueous medium is defined as kosmotropic (ordering) if it contributes to the stability and structure of water-water interactions. In contrast, chaotropic (disordering) salts have the opposite effect of disrupting the water structure, increasing the solubility of non-polar solvent particles, and destabilizing solute aggregates. Cosmotropes make water molecules interact well, which in fact stabilizes intramolecular interactions in macromolecules such as proteins (Moelbert S et al.). For example, a scale can be established 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 out proteins (Hyde A et al.).

[0086] More specifically, the cosmotropic salt is: (i) CO3 2- , SO4 2- , S2O3 2- , H2PO4 - , HPO4 2- , acetic acid - , Citric Acid - , and Cl - and (ii) an anion selected from the group consisting of NH + , K + , Na + , and Li +In a currently preferred embodiment, the salt is sodium acetate (NaOAc). Non-limiting examples of suitable concentrations of NaOAc include 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 should be understood that other salts consisting of combinations of anions as listed in (i) and cations as listed in (ii) may alternatively be used to elute capsids. Non-limiting examples are NaCl, LiCl, KCl, or other equivalent metal salts suitable for use in salt elution, as known in the art. Non-limiting examples of suitable concentrations of NaCl include about 5 mM to about 2 M NaCl, 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 NaCl. Additionally, step (b) may include applying a gradient of such a compound to improve elution of the adeno-associated virus capsids in which the genetic material is fully packaged from the chromatographic material. Such a gradient may be a linear gradient or a step gradient, or a combination thereof.

[0087] Non-limiting examples of suitable buffers applied in step (b) may include 20 mM Bis-Tris Propane (BTP), pH 9.5 (for ligands defined by Formula I) or pH 7.5 (for ligands defined by Formula II or III), 2 mM MgCl2, 1% sucrose, and 0.1% poloxamer 188.

[0088] The chromatographic materials applied in the methods disclosed herein include supports to which ligands are coupled. The supports may be made of organic or inorganic materials and may be porous or non-porous. In one embodiment, the supports are prepared from natural polymers such as cross-linked carbohydrate materials, e.g., agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, pectin, starch, etc. Natural polymer supports are easily prepared and optionally cross-linked according to standard methods such as inverse suspension gelation (Non-Patent Document 3). In a particularly advantageous embodiment, the support is a type of relatively rigid but porous agarose, which is prepared by methods that enhance its flow properties, see for example US Patent No. 5,399,993 (Berg). In an alternative embodiment, the support is prepared from synthetic polymers or copolymers, such as cross-linked synthetic polymers, e.g., styrene or styrene derivatives, divinylbenzene, acrylamide, acrylate esters, methacrylate esters, vinyl esters, vinyl amides, etc. Such synthetic polymers are easily prepared and optionally crosslinked according to standard methods, see for example, J. Am. Chem. Soc. 1999, 143: 1311-1323 (2002). Natural 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 the art and are easily prepared according to standard methods.

[0089] The support for the chromatographic material may be in the form of particles, such as particles that are substantially spherical, elongated or irregular in shape.

[0090] When the chromatographic material is in the form of particles, the particles can be particles that have homogenous porosity and are at least partially permeable to the adeno-associated virus capsid.

[0091] As used herein, the term "homogeneous porosity" is intended to mean that a particle with homogeneous porosity has homogeneous porosity throughout its entire structure or volume, such that each particle is at least partially permeable to the adeno-associated virus capsid throughout its entire structure or volume. In other words, a particle with homogeneous porosity has a porosity that allows the adeno-associated virus capsid to diffuse completely or at least partially through its pores throughout the entire structure or volume of the particle.

[0092] The adeno-associated virus has a diameter of approximately 20 to 25 nm. The capsid is the shell of the virus particle, and the adeno-associated virus does not have a lipoprotein bilayer envelope surrounding the capsid, so the size of the adeno-associated virus capsid is approximately 20 to 25 nm in diameter.

[0093] Thus, when the chromatographic material is in the form of particles having homogeneous porosity and at least partially permeable to adeno-associated virus capsids, each particle can suitably contain pores with a diameter of >25 nm, i.e., larger than the diameter of the adeno-associated virus capsid to be separated, thereby allowing diffusion of the capsid throughout the particle. For the specific purposes of the present disclosure, i.e., to separate adeno-associated virus capsids, it should be understood that a diameter >25 nm can be any size >25 nm, including, but not limited to, 30, 50, 75, 100, 150, or 200 nm.

[0094] Furthermore, it should be understood that a particle with homogenous porosity throughout its entire structure or volume may nevertheless contain pores of different sizes, both large enough to readily allow the capsid to diffuse into the particle and small enough not to allow diffusion of the capsid. This diversity of pore sizes can be measured by the diffusion coefficients of molecules of well-defined molecular weight and hydrodynamic size. As a non-limiting example, dextran with a molecular weight of 140-225 kDa or a hydrodynamic diameter of 20-25 nm (i.e., a diameter of the same size as the adeno-associated virus capsid) can be used to assess the degree of diffusion of the adeno-associated virus capsid within the pores of the particle.

[0095] The chromatography materials Capto Q and Capto DEAE advantageously used in Example 1 herein comprise supports in the form of substantially spherical particles or beads with a diameter of approximately 90 μm. This type of particle is a non-limiting example of a particle that has homogeneous porosity (i.e., throughout its entire structure or volume) and is at least partially permeable to the adeno-associated virus capsid (i.e., throughout its entire structure or volume).

[0096] Suitable particle sizes of chromatographic materials for use in the methods disclosed herein may be in the diameter range of 5-500 μm, e.g. 10-100 μm, e.g. 30-90 μm. For essentially spherical particles, the average particle size may be in the range of 5-1000 μm, e.g. 10-500. In certain embodiments, the average particle size is in the range of 10-200 μm. Those skilled in the art can easily select the appropriate particle size and porosity depending on the process used. For example, for large-scale processes, more porous but stiff supports may be preferred for economic reasons, especially to allow the processing of large volumes for the capture step. In chromatography, process parameters such as the size and shape of the column will influence the choice. In expanded bed processes, the matrix generally contains a dense packing, preferably a stainless steel packing. For other processes, other criteria may influence the nature of the matrix.

[0097] The chromatographic material may be dry, e.g., dry particles that are immersed in a liquid during use to retain their original morphology, e.g., such dry chromatographic material may include dry agarose particles.

[0098] The support of the chromatographic material may alternatively take any other form conventionally used in separations, such as a monolith, a filter or membrane, a capillary, a chip, a nanofiber, a surface, etc.

[0099] Where the chromatographic material support comprises a monolith, pore diameters within the monolith suitable for purposes of separating adeno-associated virus capsids range from a minimum pore diameter of >25 nm, i.e., a diameter greater than the diameter of the capsids to be separated, to a maximum pore diameter of about 5 μm, e.g., about 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 μm.

[0100] When the support of the chromatographic material comprises nanofibers, such nanofibers may comprise, for example, electrospun polymer nanofibers. In use, such nanofibers form a stationary phase comprising a plurality of pores through which the mobile phase can permeate.

[0101] The support of the chromatographic material may comprise a membrane structure, such as a single membrane, multiple membranes or a filter. The membrane may be an adsorptive membrane. When the support of the chromatographic material comprises a membrane structure, the pore diameter within the membrane structure suitable for separating adeno-associated virus capsids ranges from a minimum pore diameter of >25 nm, i.e., a diameter larger than the diameter of the capsid to be separated, to a maximum pore diameter of about 5 μm, e.g., about 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 μm. When the chromatographic material comprises a membrane structure, such a membrane structure may comprise, for example, a non-woven web of polymeric nanofibers.

[0102] Non-limiting examples of suitable polymers may be selected from polysulfone, polyamide, nylon, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, and polyethylene oxide, and mixtures thereof.

[0103] Alternatively, the polymer may be a cellulosic polymer, for example selected from the group consisting of cellulose and partial derivatives of cellulose, in particular cellulose esters, crosslinked cellulose, grafted cellulose, or ligand-coupled cellulose. Cellulose fiber chromatography (known as Fibro chromatography; Cytiva, Sweden) is an ultra-fast chromatographic purification method for short process times and high productivity, taking advantage of the high flow rate and high capacity of cellulose fibers. When the support of the chromatographic material comprises cellulose fibers such as Fibro, the pore diameters in the cellulose fibers suitable for the purpose of separating adeno-associated virus capsids range from a minimum pore diameter of >25 nm, i.e., a diameter larger than the diameter of the capsid to be separated, to a maximum pore diameter of about 5 μm, for example, about 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 μm.

[0104] The term "membrane chromatography" has its conventional meaning in the field of bioprocessing. In membrane chromatography, there is binding of components of a fluid, e.g., 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 to molecules by convective transport. The advantage of membrane adsorbers over packed chromatography columns is their suitability to be operated at much higher flow rates. This is also called convection-based chromatography. Convection-based chromatography matrices include any matrix in which application of a hydraulic pressure difference between the inflow and outflow of the matrix achieves substantial convective transport of substances into or out of the matrix, such that perfusion of the matrix is ​​strong and is accomplished very rapidly at high flow rates. Convection-based chromatography and membrane adsorbers are described, for example, in U.S. Pat. No. 5,399,623; U.S. Pat. No. 5,493,361; U.S. Pat. No. 5,493,623; U.S. Pat. No. 5,493,733; U.S. Pat. No. 5,493,735 ... and U.S. Pat. No. 5,523,363, which are incorporated herein by reference in their entirety.

[0105] In the method disclosed herein for separating adeno-associated virus capsids in which the genetic material is fully packaged from adeno-associated virus capsids in which the genetic material is not fully packaged, the chromatographic material used, the chromatographic material referred to in steps (a) and (b) of the method, may advantageously be a polishing chromatographic material, meaning that the chromatographic material is applied in a polishing step.

[0106] The term "polishing step", in the context of liquid chromatography, refers to a final purification step in which trace impurities are removed to leave an active and safe product. The impurities removed during the polishing step are often conformers of the target molecule, i.e., forms of the target molecule with a particular molecular conformation, or spurious breakthrough products. The polishing step may alternatively be referred to as a "secondary purification step".

[0107] Furthermore, the liquid sample added in step (a) of the method disclosed herein for separating adeno-associated virus capsids in which genetic material is fully packaged from adeno-associated virus capsids in which genetic material is not fully packaged may advantageously be a pre-purified liquid sample.

[0108] The present disclosure further provides a method for separating fully packaged adeno-associated virus capsids from non-fully packaged adeno-associated virus capsids, comprising carrying out the method as shown in FIG. 1 and described in detail above, further comprising step (a1) comprising pre-purifying the adeno-associated virus capsids by separating them from an adeno-associated virus capsid-containing cell culture harvest as illustrated in FIG. 2, thereby obtaining a pre-purified liquid sample comprising adeno-associated virus capsids, and then adding said pre-purified liquid sample comprising adeno-associated virus capsids to a chromatographic material according to step (a) of the method as shown in FIG. 1.

[0109] Such a pre-purification step (a1) may alternatively be called a "capture step" and, in the context of liquid chromatography, refers to an initial step of the separation procedure. Most commonly, the capture step involves clarification (e.g. by filtration, centrifugation, or precipitation) and usually also concentration and / or stabilization of the sample, and after sample clarification, concentration, and stabilization, significant purification from soluble impurities, for example by applying chromatography. The capture step may be followed by intermediate purification, which further reduces the residual amounts of impurities such as host cell proteins, DNA, viruses, endotoxins, nutrients, components of cell culture media such as antifoaming agents and antibiotics, and product-related impurities such as aggregates, misfolded species, and aggregates.

[0110] Such a pre-purification step may include subjecting the adeno-associated virus capsid-containing cell culture harvest to one or more of the following non-limiting examples of purification methods: (i) affinity chromatography; (ii) ion exchange chromatography; (iii) precipitation or tangential flow filtration (TFF) followed by size exclusion chromatography, for example by using Capto Core 400 chromatographic material (Cytiva, Sweden), which combines capsid flow-through and binding of impurities to the chromatographic material; (iv) TFF followed by ion exchange chromatography; and (v) TFF followed by ion exchange chromatography and Capto Core.

[0111] Non-limiting examples of chromatographic materials suitable for application in the pre-purification step of the method illustrated in Figure 2 and described above include affinity chromatographic materials, ion exchange chromatographic materials, and size exclusion chromatographic materials, respectively. The chromatographic materials may be functionalized with positively charged groups such as quaternary amino, quaternary ammonium, or amine groups, or negatively charged groups such as sulfonate or carboxylate groups. The chromatographic materials may be functionalized with ion exchange groups, affinity peptide / protein-based ligands, hydrophobic interaction ligands, IMAC ligands, or DNA-based ligands such as Oligo dT.

[0112] As used herein, the term "cell culture" refers to a culture of cells or a group of cultured cells, where the cells can be any type of cell, such as bacterial cells, viral cells, fungal cells, insect cells, or mammalian cells. The cell culture may be unclarified, i.e., contain cells, or may be cell-depleted, i.e., cultures that contain no or few cells, but contain biomolecules released from the cells prior to cell removal. Additionally, unclarified cell cultures may contain intact cells, disrupted cells, cell homogenates, and / or cell lysates.

[0113] The term "cell culture harvest" is used herein to refer to a cell culture that has been harvested and removed from the vessel or device in which the cells were cultured.

[0114] The term "separation device" has its conventional meaning in the art of bioprocessing and should be understood to encompass any type of separation device capable of and suitable for separating and purifying a compound from a fluid containing a by-product from the production of the compound. A separation device may include a separation matrix as further defined elsewhere herein.

[0115] Non-limiting examples of separation devices suitable for use in the polishing step according to the methods disclosed herein include chromatographic columns and membrane devices as further described elsewhere herein. Such separation devices may suitably comprise a chromatographic material in the form of a strong or partially strong anion exchange chromatographic material comprising a ligand as defined by formula I, II or III, as described in detail elsewhere herein.

[0116] Non-limiting examples of separation devices suitable for use in the capture step or pre-purification step as described herein are filtration devices, chromatography columns and membrane devices. Chromatography columns suitable for use in the capture step can be packed with, for example, affinity chromatography material, ion exchange chromatography material, mixed mode chromatography material or hydrophobic interaction chromatography material.

[0117] As illustrated in FIG. 3, the method disclosed herein for separating fully packaged adeno-associated virus capsids from non-fully packaged adeno-associated virus capsids comprises: separating the eluate fraction containing the adeno-associated virus capsids in which the genetic material is fully packaged, which is eluted in step (b) of the method as described above, from the c1) concentrating the adeno-associated virus capsid to a pharma- ceutical suitable dose; c2) replacing the buffer applied in step (b) of the process with a pharma- ceutically acceptable buffer, and / or c3) Sterilizing the eluate fraction containing the adeno-associated virus capsid to obtain a pharmaceutical composition comprising the adeno-associated virus capsid.

[0118] Those skilled in the art will appreciate that the pharma- ceutically appropriate dose will depend on a variety of factors, including, but not limited to, the disease or disorder being treated and the weight and condition of the subject being treated with the pharmaceutical composition.

[0119] Pharmaceutically acceptable buffers are well known in the art and can be readily selected by one of ordinary skill in the art.

[0120] In order for the resulting composition to meet all regulatory requirements for pharmaceutical compositions, usually all three steps c1 to c3 listed above must be carried out.

[0121] In the methods disclosed above, the adeno-associated virus capsid may advantageously be a capsid of adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 9 (AAV9), or adeno-associated virus serotype 10 (AAV10), or a variant thereof.

[0122] The term "variant" in relation to adeno-associated virus (AAV) serotypes 1, 2, 4, 5, 6, 7, 8, or 10 as listed above is intended to mean an AAV in which the capsid structure has been modified, altered, or engineered to improve clinical performance, e.g., to target a specific target organ. As a non-limiting example, an AAV8 variant includes the capsid portion of AAV8 and may further include the capsid portion of other AAV serotypes other than AAV8, such as AAV5. However, an AAV8 variant as referred to herein must retain significant structural similarity to the unmodified AAV8 capsid, e.g., retaining at least 50%, e.g., 60%, 70%, 80%, or 90% of the outer surface structure of the unmodified AAV8 capsid. This applies equally to variants of AAV serotypes 1, 2, 4, 5, 6, 7, or 10, compared to unmodified AAV serotypes 1, 2, 4, 5, 6, 7, or 10, respectively. Further, by way of non-limiting example, in the context of purification or separation of variants of AAV8, a "variant" is defined herein as an adeno-associated virus that has a functionally equivalent binding ability to a ligand of a designated chromatographic material, compared to the binding ability of the original AAV8 to said designated chromatographic material. This applies equally to variants of AAV serotypes 1, 2, 4, 5, 6, 7, or 10, compared to the original AAV serotypes 1, 2, 4, 5, 6, 7, or 10, respectively. The designated chromatographic material may be, for example, a strong or partially strong anion exchange chromatographic material, as disclosed in more detail elsewhere herein. Variants of the adeno-associated virus can be obtained, for example, by natural mutation or by engineered modification (ie, obtained by human interaction) of one or more nucleotides of the genome of the adeno-associated virus.

[0123] According to a currently preferred embodiment, in the separation methods as illustrated in Figures 1, 2, and 3, respectively, the chromatographic material comprises a compound represented by formula IV:

[0124] [ka]

[0125] and the elution buffer of step (b) comprises sodium acetate. The method can be applied to the separation of AAV capsids of any serotype or variant as described above. In particular, the capsids to be separated can be capsids of the AAV9 serotype or variants thereof.

[0126] The present disclosure includes a support, a ligand, and a surface extender linking the ligand to the support for separating adeno-associated viral capsids in which genetic material is fully packaged from adeno-associated viral capsids in which genetic material is not fully packaged, the surface extender having formula IV:

[0127] [ka]

[0128] Use of an anion exchange chromatography material defined by a. adding a liquid sample to a chromatographic material, said liquid sample being at least 90% pure and at least 10% pure; 12 comprising adeno-associated virus capsids at a concentration of adeno-associated virus capsids / ml, at least 10% of the adeno-associated virus capsids being adeno-associated virus capsids in which the genetic material is fully packaged; b. Eluting the adeno-associated virus capsids containing fully packaged genetic material from the chromatographic material. wherein the adeno-associated virus capsids eluted in step (b) are eluted in an eluate fraction, the combined eluate fraction comprising at least 50% of the adeno-associated virus capsids of the liquid sample added in step (a), at least 60% of the adeno-associated virus capsids having fully packaged genetic material.

[0129] According to a currently preferred embodiment of said use, the elution buffer of step (b) comprises sodium acetate.

[0130] The adeno-associated virus capsid separated according to said use may be a capsid of adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), 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. In a particularly preferred embodiment of said use, the adeno-associated virus capsid is a capsid of adeno-associated virus serotype 9 (AAV9) or a variant thereof.

[0131] The present disclosure further provides a method for preventing or treating an organ or tissue-related disease or disorder in a subject, comprising administering to the subject a pharmaceutical composition comprising adeno-associated virus capsids obtained by carrying out the above-disclosed isolation method comprising one or more of steps c1-c3 (as described in detail above), wherein in the pharmaceutical composition, the ratio of adeno-associated virus capsids in which genetic material is fully packaged to adeno-associated virus capsids in which genetic material is not fully packaged is at least 3:2, i.e., the number of full capsids is at least 1.5 times the total number of empty and partially filled capsids. Preferably, the ratio of adeno-associated virus capsids in which genetic material is fully packaged to adeno-associated virus capsids in which genetic material is not fully packaged is at least 2:1, e.g., 3:1, 4:1, or 5:1. That is, preferably the number of complete capsids is at least 2-fold, such as 3, 4, or 5-fold, the total number of empty and partially filled capsids.

[0132] Said methods for preventing or treating an organ or tissue associated disease or disorder may suitably include gene therapy, It should be understood that the therapeutically relevant gene or genetic material is present in the fully packaged adeno-associated virus capsid, which genetic material is contained in the pharmaceutical composition administered to the subject.

[0133] In the above disclosed methods for preventing or treating an organ or tissue associated disease or disorder, the adeno-associated virus capsid may advantageously be a capsid of adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 9 (AAV9), or adeno-associated virus serotype 10 (AAV10), or a variant thereof.

[0134] In the above disclosed methods for preventing or treating a disease or disorder associated with an organ or tissue, the organ or tissue may optionally be selected from the central nervous system, heart, kidney, liver, lung, pancreas, photoreceptor cells, retinal pigment epithelium, skeletal muscle, and brain. More specifically, the following combinations of AAV serotypes and organ / tissue types are contemplated: (i) the capsid is selected from an AAV1, AAV2, AAV4, AAV5, AAV8, and AAV10 capsid, and the organ or tissue is the central nervous system; (ii) the capsid is selected from an AAV1 and an AAV8 capsid, and the organ or tissue is the heart; (iii) the capsid is an AAV2 capsid and the organ or tissue is a kidney; (iv) the capsid is selected from AAV7 and AAV8 capsids, and the organ or tissue is liver; (v) the capsid is selected from AAV4, AAV5, and AAV6 capsids, and the organ or tissue is lung; (vi) the capsid is AAV8 and the organ or tissue is the pancreas; (vii) the capsid is selected from AAV2, AAV5, and AAV8 capsids, and the organ or tissue is a photoreceptor cell; (viii) the capsid is selected from AAV1, AAV2, AAV4, AAV5, and AAV8 capsids, and the organ or tissue is retinal pigment epithelium; (ix) the capsid is selected from AAV1, AAV6, AAV7, and AAV8 capsids, and the organ or tissue is skeletal muscle; (x) the capsid is an AAV10 capsid and the organ or tissue is brain; or (xi) the capsid is an AAV9 capsid or a variant thereof, and the organ or tissue is selected from the central nervous system, heart, liver, lung, and skeletal muscle.

[0135] It should be understood that the above mentioned tissue types are non-limiting examples of organ and tissue types that may be amenable to treatment by administration of a pharmaceutical composition comprising adeno-associated virus capsids, in particular capsids of adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 9 (AAV9), or adeno-associated virus serotype 10 (AAV10), or variants thereof.

[0136] A non-limiting example of an organ- or tissue-related disease or disorder is spinal muscular atrophy, where a method of treatment or prevention may be suitably carried out by administration of an adeno-associated virus, in particular AAV5, AAV8, or AAV10, or a variant thereof.

[0137] Another non-limiting example of an organ- or tissue-related disease or disorder is an inherited retinal dystrophy, the treatment or prevention of which may be suitably carried out by administration of an adeno-associated virus, in particular AAV2 or a variant thereof.

[0138] Other non-limiting examples of organ- or tissue-related diseases or disorders are pancreatic tumors and metabolic disorders in the liver, such as ornithine transcarbamylase (OTC) deficiency, for which methods of treatment or prevention may be suitably carried out by administration of an adeno-associated virus, in particular AAV8 or a variant thereof.

[0139] Those skilled in the art will understand that to achieve the desired medical effect, the pharmaceutical composition must be administered to the subject in a pharma- ceutical effective amount or dose. The pharma- ceutical effective amount and dose depend on various factors, including, but not limited to, the disease or disorder being treated and the weight and condition of the subject being treated.

[0140] The present disclosure further provides a composition comprising adeno-associated virus capsids obtained by carrying out a method for separating fully packaged adeno-associated virus capsids from not fully packaged adeno-associated virus capsids as described in detail above, wherein the ratio of adeno-associated virus capsids in which genetic material is fully packaged to adeno-associated virus capsids in which genetic material is not fully packaged in the composition is at least 3:2, i.e., the number of full capsids is at least 1.5 times the total number of empty capsids and partially filled capsids. Preferably, the ratio of adeno-associated virus capsids in which genetic material is fully packaged to adeno-associated virus capsids in which genetic material is not fully packaged is at least 2:1, e.g., 3:1, 4:1, or 5:1. That is, preferably, the number of full capsids is at least 2 times, e.g., 3, 4, or 5 times the total number of empty capsids and partially filled capsids.

[0141] The present disclosure also provides a pharmaceutical composition comprising adeno-associated virus capsids obtained by carrying out the above-disclosed separation method comprising one or more of steps c1 to c3 (as described in detail above), in which the ratio of adeno-associated virus capsids in which genetic material is fully packaged to adeno-associated virus capsids in which genetic material is not fully packaged is at least 3:2, i.e., the number of full capsids is at least 1.5 times the total number of empty capsids and partially filled capsids. Preferably, the ratio of adeno-associated virus capsids in which genetic material is fully packaged to adeno-associated virus capsids in which genetic material is not fully packaged is at least 2:1, e.g., 3:1, 4:1, or 5:1. That is, preferably, the number of full capsids is at least 2 times, e.g., 3, 4, or 5 times the total number of empty capsids and partially filled capsids.

[0142] The pharmaceutical compositions described above may be for use in therapy, optionally for use in gene therapy.

[0143] In the above pharmaceutical compositions for use in treatments such as gene therapy, the adeno-associated virus capsid may advantageously be a capsid of adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 9 (AAV9), or adeno-associated virus serotype 10 (AAV10), or a variant thereof.

[0144] Furthermore, the pharmaceutical composition may advantageously be for use in the prevention or treatment of a disease or disorder associated with an organ or tissue selected from the central nervous system, heart, kidney, liver, lung, pancreas, photoreceptor cells, retinal pigment epithelium, skeletal muscle, and brain.

[0145] A device or composition that "comprises" 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 recited components, with no other features or components present. Similarly, a method that "comprises" one or more recited steps may also include other steps not specifically recited.

[0146] The singular forms "a" and "an" are intended to be construed as including the plurals as well. EXAMPLES

[0147] Example 1 Separation of AAV8 capsids on anion exchange chromatography material using magnesium chloride and increasing sodium chloride linear gradients Chromatography Materials The following currently available anion exchange chromatography materials provided improved results, as further described below.

[0148] Capto Q (Cytiva, Sweden): Ligand: Quaternary amine Particle size, d 50V : Approx. 90 μm Matrix: Highly cross-linked agarose with dextran surface extenders Ion capacity: 0.16~0.22mmol Cl - / ml medium pH stability, operable: 2-12

[0149] Capto DEAE (Cytiva, Sweden): Ligand: Diethylaminoethyl, partially quaternized amine group Particle size, d 50V : Approx. 90 μm Matrix: Highly cross-linked agarose with dextran surface extenders Ion capacity: 0.29~0.35mmol Cl - / ml medium pH stability, operable: 2-9.

[0150] Additionally, the currently available Capto Q ImpRes (Cytiva, Sweden) was also tested as follows: The support material of the ImpRes resin consists of substantially spherical particles or beads with a diameter of 40 μm.

[0151] Equipment and Samples Each resin was packed into a Tricorn 5 column (2 mL), 10 cm bead height, and run at a flow rate of 1 mL / min using an Agilent Bio-Inert 1260 system or an AKTA pure P25 system. The applied samples contained approximately 1 × 10 6 elution of affinity purified AAV8, containing both full and empty capsids (>15% full capsids), for all resins. 13 The viral capsid was detected by fluorescence (excitation at 280 nm, emission 348 nm) or UV (280 and 260 nm).

[0152] result Currently available cation resins (SP Sepharose XL, Capto SP ImpRes, Capto SP ImPact and Capto Adhere ImpRes; Cytiva, Sweden) were evaluated in 50 mM acetate pH 4.5 and 5.5 with a salt gradient up to 1 M NaCl. A multimodal anion exchange resin (Currently available Capto Adhere; Cytiva, Sweden) was evaluated in 20 mM Tris pH 6-9.5 and with a salt gradient up to 1 M NaCl. When affinity purified AAV8 was applied, the results for both types of resin tested showed no separation, only a single peak (data not shown).

[0153] Each of the currently available resins Capto Q ImpRes (Figure 4A), Capto Q (Figure 4B) and Capto DEAE (Figure 5) was run in 20 mM Bis-Tris Propane (BTP), pH 9.5 (for Capto Q ImpRes and Capto Q) or pH 7.5 (for Capto DEAE), 2 mM MgCl2, 1% sucrose, 0.1% poloxamer 188 with a linear gradient elution to 400 mM NaCl. Peak 1 area with a UV260:280 ratio less than 1 was defined as empty capsid and peak 2 area with a UV260:280 ratio greater than 1 was defined as full capsid. The % full capsid was calculated based on the peak areas for UV260 and UV280 (Table 1). Peak content in terms of % full and empty capsids was also analyzed by qPCR (viral genome, complete capsid) and ELISA (total capsid) to confirm peak identity (data not shown).

[0154] The chromatograms in Figures 4 and 5 show that the dextran extender (present in Capto Q and Capto DEAE, but not Capto Q ImpRes) dramatically improves the separation of full (F)AAV8 capsids and empty (E)AAV8 capsids. For Capto DEAE, lowering the pH to 7.5 improved the separation compared to higher pHs up to 9.5 (data not shown).

[0155] The UV260:280 ratio is lower for Capto Q ImpRes (without dextran extender), indicating a lower % complete capsid, which is less efficient for enrichment. For Capto Q and Capto DEAE (with dextran extender), the ratio is higher in peak 2, indicating a higher % complete capsid and better separation from empty capsids (Table 1). The calculated % complete capsid based on the area of ​​peak 2 was not significantly different from the UV260:280 ratio, but for Capto Q ImpRes (Figure 4A), the separation of complete and empty capsids is poor, so the determined area for complete capsids is not accurate. The complete and empty peaks overlap, and the later peak is enriched for complete capsids.

[0156] [Table 1]

[0157] Example 2 Isolation of AAV8 capsids under variable conditions The experimental design for separation of fully packaged and empty AAV8 capsids is carried out using the equipment and samples as in Example 1 above, and further using anion exchange chromatography material as in Example 1, with the following variations.

[0158] Regarding Surface Extenders: 1) Dextran of different sizes (kDa) (T10, T70, T250). 2) Different amounts of dextran. 3) Instead of dextran, a glycidol-based polyhydric alcohol is used.

[0159] Regarding ligand density: 1) Capto Q: 60~160, 220~260 μmol / mL. 2) Capto DEAE: 150~290, 350~400 μmol / mL.

[0160] Regarding the support for the chromatographic material: 1)Smaller resin bead size: 35~90μm. 2) Resin beads with larger pore sizes than Capto Q and Capto DEAE.

[0161] Regarding the chemical nature of the ligand: 1) Capto DEAE ligands (ligands according to formula III) with different levels of quaternization. 2) Capto Q Analogues (Capto Q ligands are defined by formula I): a.R1 and R2 are ethyl, and R3 is methyl. b.R1, R2 are methyl and R3 is CH2CHOHCH3. 3) Capto DEAE Analogs (Capto DEAE ligands are defined by formula II or III): am=1, n=1, R1, R2, R3, and R4=methyl, R5=H. bm=1, n=1, R1, R2, R3, and R4=methyl, R5=CH2CHOHCH3. cm=1, the following structures (i) to (iv): (i) the structure in which n is 0, R3 and R4 are methyl, and R5 is hydrogen or CH2CHOHCH3; (ii) the structure in which n is 1, R1, R2, R3, and R4 are methyl, and R5 is hydrogen or CH2CHOHCH3; (iii) the structure, in which n is 2, each of R1 and R2 is methyl, R3 and R4 are methyl, and R5 is hydrogen or CH2CHOHCH3; (iv) the structure in which n is 3, R1 and R2 are methyl, R3 and R4 are methyl, and R5 is hydrogen or CH2CHOHCH3. A combination of two or more of the above.

[0162] Regarding buffers and elution conditions: 1) Different concentrations of MgCl2 between 1 and 20 mM. 2) Different NaCl linear gradients 0.1-1M, with or without MgCl2 as in 1). 3) Different pH linear gradients pH 4-10, with or without MgCl2 as in 1). 4) Different buffer solutions: a. Tris bN-Methyldiethanolamine 5) Stepwise elution with pH, ​​NaCl, and MgCl2 as in 1), 2), and 3). 6) Conditions suitable for flow-through separation, where some analytes bind to the chromatographic material but others do not (e.g., empty capsids bind but full capsids do not bind). 7) All of the above, with or without additives such as sucrose (0.1-5%) and poloxamer 188 surfactant (0.01-1%).

[0163] Example 3 Isolation of capsids from various adeno-associated virus serotypes under variable conditions. The experimental design for separation of full and empty capsids of adeno-associated virus serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV9, and AAV10 is carried out according to the variables in Example 1 and Example 2 above.

[0164] Example 4 Separation of capsids of serotypes AAV2, AAV5, AAV8, and AAV9 on anion exchange chromatography material using magnesium chloride and increasing sodium acetate step gradients Chromatography Materials The same anion exchange chromatography materials (i.e., Capto Q, Capto DEAE and Capto Q ImpRes) as described in Example 1 were used for the separation of full and empty capsids of AAV2, AAV5, AAV8 and AAV9.

[0165] Equipment and Samples Each resin was packed into a Tricorn 5 column (2 mL) according to the packing instructions. Runs were performed using an Akta Pure P25 system (P25-20031) at a flow rate of 1 CV / min (i.e., 2 mL / min) with the system mixer disconnected to minimize dead volume and obtain a sharp conductivity step. Samples were applied to the pre-equilibrated column using a capillary loop. Typically, samples applied to each resin contained a mixture of full and empty capsids (>5% full capsids, as follows: AAV2 7-10%, AAV5 47%, AAV8 11-35%, AAV9 40%), approximately 5 × 10 affinity-purified or affinity and size-exclusion purified AAV2, AAV5, AAV8 or AAV9, respectively. 12 The material should have a low electrical conductivity (1-3 mS / cm) to ensure binding of AAV to the anion exchange ligand.

[0166] 280 and 260 nm UV absorbance was monitored during the run, and the 260 / 280 ratio was used as a diagnostic tool to navigate and distinguish between full and empty capsid populations in the chromatograms. The chromatograms were analyzed using the Unicorn evaluation package. A 260 / 280 ratio above 1.2 is considered to indicate 100% full capsids, and a 260 / 280 ratio of approximately 0.6-0.7 or less than 0.6-0.7 is considered to indicate 100% empty capsids. A blank run with buffer without AAV was performed to subtract any background signal if necessary and ensure removal of potential UV signal from the buffer.

[0167] Process Conditions and Results The currently available cation exchange resin Capto S, and the prototype cation exchange resin Capto CM Dx ImpRes were evaluated by applying isocratic or continuous gradient elution, respectively, with various elution salts (NaCl, NaOAc, NH4Cl or NH4SO4 up to 500 mM) and additional salts (MgCl2 and MgSO4 up to 20 mM), with and without additives (0.1% poloxamer 188, 1% sucrose) in acetate buffers of pH 4.5 and 5. None of the above mentioned conditions or resins provided good baseline separation of full and empty capsids (data not shown).

[0168] Currently available anion exchange resins with dextran extenders, Capto DEAE (strong or partial strong anion exchange) and Capto Q (strong anion exchange), were each evaluated by applying a two-step elution method using a buffer system comprising Buffer A and Buffer B, both containing 20 mM Bis-Tris Propane (BTP) pH 9.0 and 2 mM MgCl2, with Buffer B further comprising 250 mM sodium acetate (NaOAc) as an elution salt.

[0169] In Figures 6-7, the y-axis on the right side of each chromatogram represents the percentage of Buffer B contained in the elution buffer obtained during elution from the chromatographic material (the remainder being Buffer A).

[0170] Equilibration: 5 CV of Buffer A Injection: empty loop w 3 ml of buffer A Wash: 5 CV of Buffer A Two-step elution: conditions applicable for each serotype are specified below Re-equilibration: 5 CV of Buffer A

[0171] Capto Q AAV2: Stage 1 40%B, 20CV Stage 2 100%B, 5CV AAV5: stage 1 35%B, 20CV Stage 2 100%B, 5CV AAV8: Stage 1 30% B, 20 CV Stage 2 100%B, 5CV AAV9: stage 1 5% B, 20 CV Stage 2: 30%B, 5CV

[0172] When using Capto Q resin, all tested serotypes (AAV2, AAV5, AAV8, and AAV9) gave good separation of full and empty capsids (Figures 6A-6D). Elution of full (F) and empty (E) capsids, as well as UV260:280 ratios, are shown in Figures 6A-6D.

[0173] FIG. 6A shows a chromatogram for the NaOAc two-step elution of AAV2.

[0174] FIG. 6B shows a chromatogram for the NaOAc two-step elution of AAV5.

[0175] FIG. 6C shows a chromatogram for the NaOAc two-step elution of AAV8.

[0176] FIG. 6D shows a chromatogram for the NaOAc two-step elution of AAV9.

[0177] Percent full capsids were calculated based on peak areas for UV260 and UV280. Peak contents for percent full and empty capsids were also analyzed by qPCR (viral genome (VG), full capsid) and ELISA (total capsid). Results are summarized in Table 2.

[0178] [Table 2]

[0179] Capto DEAE When using Capto DEAE resin, all tested AAV serotypes (AAV2, AAV5, AAV8, and AAV9) gave good separation of full and empty capsids, however, they eluted with a lower percentage of Buffer B compared to when separated with Capto Q resin.

[0180] Figure 7 shows the results of AAV9 elution on Capto DEAE resin. Using the same buffer system described above for Capto Q (Buffer A: 20 mM BTP pH 9.0, 2 mM MgCl2; Buffer B: Buffer A + 250 mM NaOAc), AAV9 empty capsids eluted in the flow-through and AAV9 full capsids eluted when 4% Buffer B was applied. The UV260:280 ratio was 0.76 in the first peak (i.e., the flow-through peak), suggesting mainly empty AAV9 capsids, but also small amounts of full capsids. The UV260:280 ratio was 1.3 in the second peak, indicating high purity of AAV9 full capsids (Figure 7).

[0181] Capto Q ImpRes (without dextran extender) Capto Q ImpRes resin was evaluated for the separation of AAV9 and AAV5, respectively, under identical conditions as described above, except that a flow rate of 1 ml / min was applied due to the high delta column pressure. The resin did not work for AAV5, but worked well for a two-step elution for the separation of AAV9 full capsids and AAV9 empty capsids (results not shown). However, Capto Q ImpRes (without extenders) does not bind AAV9 empty capsids (thereby eluting in the flow-through), and only weakly binds AAV9 full capsids, thus providing a less robust separation method than Capto Q (with extenders).

[0182] Example 5 Separation of serotype AAV5 capsids on anion exchange chromatography material using an increasing magnesium chloride step gradient Capto Q resin was evaluated for separation of AAV5 by applying a two-step elution as described in Example 4, with the difference that the buffer system Buffer A and Buffer B both contained 20 mM Bis-Tris Propane (BTP) pH 7.0, 1% sucrose and 0.1% Pluronic, with Buffer B additionally containing 20 mM MgCl2.

[0183] Equilibration: 5 CV of Buffer A Injection: empty loop w 3 ml of buffer A Wash: 5 CV of Buffer A Step elution: step 1, 50% buffer B, 20 CV; step 2, 70% buffer B, 20 CV Re-equilibration: 5 CV of Buffer A

[0184] Figure 8 shows the results of a two-step elution method for AAV5 on Capto Q resin, applying 50% Buffer B in the first step and 70% Buffer B in the second step. Full (F) and empty (E) capsids and the UV260:280 ratio are shown in Figure 8.

[0185] Example 6 Separation of serotype AAV5 capsids on anion exchange chromatography material using magnesium chloride and increasing sodium chloride step gradients Capto Q resin was evaluated for separation of AAV5 by applying a two-step elution as described in Example 4 and Example 5, with the difference that the buffer system Buffer A and Buffer B both contained 20 mM Bis-Tris Propane (BTP) pH 9.5, 18 mM MgCl2, 1% sucrose and 0.1% Pluronic, with Buffer B additionally containing 400 mM NaCl.

[0186] Equilibration: 5 CV of Buffer A Injection: empty loop w 3 ml of buffer A Wash: 5 CV of Buffer A Two-step elution: step 1, 2.5% buffer B, 6 CV; step 2, 5% buffer B, 6 CV Re-equilibration: 5 CV of Buffer A

[0187] Figure 9 shows the results of a two-step elution method for AAV5 on Capto Q resin, applying 2.5% Buffer B in the first step and 5% Buffer B in the second step. Full (F) and empty (E) capsids and the UV260:280 ratio are shown in Figure 9. It is shown that the above conditions resulted in successful baseline separation of AAV5 capsids.

[0188] It should be understood that the present disclosure is not limited to its above-described exemplary embodiments, and that several possible modifications of the present disclosure are possible within the scope of the following claims. (References) Weihong Qu et al., Scalable Downstream Strategies for Purification of Recombinant Adeno-Associated Virus Vectors in Light of the Properties, Current Pharmaceutical Biotechnology August 2015;16(8):684-695 Xiaotong Fu et al., Analytical Strategies for Quantification of Adeno-Associated Virus Empty Capsids to Support Process Development, Human gene therapy methods, 2019, 30(4):144-152 Moelbert Susanne et al., Kosmotropes and chaotropes: modeling preferential exclusion, binding and aggregate stability, Biophysical Chemistry, December 2004, 112(1): 45-57. Hyde Adam M et al., General Principles and Strategies for Salting-Out Informed by the Hofmeister Series, Organic Process Research & Development, 2017, 21(9): 1355 - 1370.

Claims

1. 1. A method for separating adeno-associated virus capsids in which genetic material has been fully packaged from adeno-associated virus capsids in which genetic material has not been fully packaged, comprising: a. applying a liquid sample containing adeno-associated virus capsids to a chromatographic material, The liquid sample has a purity of at least 90% and 12 adeno-associated virus capsids at a concentration of adeno-associated virus capsids / ml, at least 10% of the adeno-associated virus capsids being fully packaged adeno-associated virus capsids in which the genetic material is; the chromatographic material comprises a strong or partial strong anion exchange chromatographic material comprising a support and a ligand for binding to the adeno-associated virus capsid; The chromatographic material comprises a surface extender that links the ligand to the support, the surface extender being a polymer, the polymer comprising: (i) polymers with naturally occurring backbones, such as polysaccharides, such as starch, cellulose, dextran, or agarose, and (ii) Polymers with synthetic backbones, such as polyvinyl alcohol, polyacrylamide, polymethacrylamide, or polyvinyl ether. and b. eluting the adeno-associated virus capsids containing fully packaged genetic material from the chromatographic material; wherein the adeno-associated virus capsids eluted in step (b) are eluted in an eluate fraction, the combined eluate fraction comprising at least 50% of the adeno-associated virus capsids of the liquid sample added in step (a), at least 60% of the adeno-associated virus capsids being fully packaged with genetic material.

2. The ligand of the strong anion exchange chromatography material has the following formula I: 【Chemistry 1】 (In the formula, R 1 is selected from C1-C3 alkyl, R 2 and R 3 are independently selected from C1-C3 alkyl, CH2OH, and CH2CHOHCH3. The method of claim 1, wherein said first and second inputs are defined by:

3. R 1 , R 2 , and R 3 3. The method of claim 2, wherein each of is CH3.

4. The ligand of the strong or partially strong anion exchange chromatography material has the following formula II: 【Chemistry 2】 (In the formula, m is an integer from 1 to 3; R 1 and R 2 is independently selected from C1-C3 alkyl; R 3 and R 4 is independently selected from C1-C3 alkyl and CH2CHOHCH3; R 5 is selected from hydrogen, C1-C3 alkyl, and CH2CHOHCH3 is defined by With the proviso that when m is 1, the ligand of the strong or partially strong anion exchange chromatography material has the following formula III: 【Chemistry 3】 (wherein n is an integer from 0 to 3, However, if n is 0, R 3 and R 4 is independently selected from C1-C3 alkyl; R 5 is hydrogen or CH2CHOHCH3) The method of claim 1, wherein said first and second inputs are defined by:

5. The ligand is defined by formula III and has the following structures (i) to (iv): (i) n is 0, and R 3 and R 4 is ethyl, and R 5 is hydrogen or CH2CHOHCH3; (ii) n is 1 and R 1 , R 2 , R 3 , R 4 is ethyl, and R 5 is hydrogen or CH2CHOHCH3; (iii) n is 2, and each R 1 and R 2 is ethyl, and R 3 and R 4 is ethyl, and R 5 is hydrogen or CH2CHOHCH3; (iv) n is 3, and each R 1 and R 2 is ethyl, and R 3 and R 4 is ethyl, and R 5 is hydrogen or CH2CHOHCH3 The method of claim 4, comprising a combination of two or more of:

6. The method of claim 1, wherein the surface extender is dextran.

7. The method of claim 6, wherein the dextran has a molecular weight of about 10 to about 2000 kDa, for example about 40 kDa.

8. 8. The method of claim 6 or 7, wherein the density of the dextran is from about 5 to about 30 mg of dextran per ml of strong or partially strong anion exchange chromatography material.

9. 2. The method of claim 1, wherein steps (a) and (b) comprise applying a buffer having a pH of about 6.0 to about 10.5, such as about 7.5 to about 9.5, and optionally the buffer comprises tris(hydroxymethyl)amino-methane (i.e. Tris), 1,3-bis(tris(hydroxymethyl)methylamino)propane (i.e. Bis-Trispropane), triethanolamine, N-methyldiethanolamine, diethanolamine, 1,3-diaminopropane, or ethanolamine.

10. step (b) comprises applying a buffer containing a compound that improves separation between capsids in which the genetic material is fully packaged and capsids in which the genetic material is not fully packaged, optionally wherein the compound is selected from a carbohydrate, a divalent metal ion, and a detergent; Optionally, the carbohydrate is selected from sucrose, sorbitol, and polysaccharides; Optionally, the divalent metal ion is Mg 2+ , Fe 2+ , and Mn 2+ wherein optionally a divalent metal ion is present in the form of a salt, optionally in combination with chloride or sulfate ions; 2. The method of claim 1, wherein the surfactant is optionally selected from a poloxamer and a polysorbate.

11. the support of the chromatographic material comprises a particle, nanofiber, monolith, or membrane structure; Optionally, the particles are of homogenous porosity and at least partially permeable to the adeno-associated virus capsid; Optionally, the particles are substantially spherical particles; Optionally, the nanofibers comprise electrospun polymer nanofibers; 10. The method of claim 1, optionally wherein the membrane structure comprises a nonwoven web of polymeric nanofibers.

12. The method of claim 1, wherein the chromatographic material is a polishing chromatographic material.

13. The method according to claim 1, wherein the liquid sample added in step (a) defined in claim 1 is a pre-purified liquid sample.

14. The method of claim 1, further comprising the step of (a1) pre-purifying the adeno-associated virus capsid by isolating the adeno-associated virus capsid from an adeno-associated virus capsid-containing cell culture harvest, thereby obtaining a pre-purified liquid sample comprising the adeno-associated virus capsid, and then adding said pre-purified liquid sample comprising the adeno-associated virus capsid to a chromatography material according to step (a) defined in claim 1, and optionally, said pre-purifying step comprises subjecting the adeno-associated virus capsid-containing cell culture harvest to chromatography or to chromatography after clarification.

15. The eluate fraction containing the adeno-associated virus capsids in which the genetic material is fully packaged, which is eluted in step (b) as defined in claim 1, c1) concentrating the adeno-associated virus capsid to a pharma- ceutical suitable dose; c2) replacing the buffer applied in step (b) as defined in claim 1 with a pharma- ceutically acceptable buffer, and / or c3) sterilizing the eluate fraction containing the adeno-associated virus capsids 4. The method of claim 1, further comprising subjecting the adeno-associated virus capsid to one or more of the following steps to obtain a pharmaceutical composition comprising the adeno-associated virus capsid:

16. The method of claim 1, wherein the adeno-associated virus capsid is a capsid of adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 9 (AAV9), or adeno-associated virus serotype 10 (AAV10), or a variant thereof.

17. The elution buffer of step (b) comprises a kosmotropic salt, the salt being: (i) CO 3 2- , S.O. 4 2- , S 2 O 3 2- , H 2 PO 4 - , H.P.O. 4 2- , acetic acid - , Citric Acid - , and Cl - and (ii) an anion selected from the group consisting of NH 4 + , K + , Na + , and Li + and optionally the salt is sodium acetate.

18. 4. The chromatographic material has formula IV: 【Chemistry 4】 and the elution buffer of step (b) comprises sodium acetate; 10. The method of claim 1 or 6, wherein the adeno-associated virus capsid is an adeno-associated virus serotype 9 (AAV9) or variant thereof capsid.

19. 16. A method for preventing or treating an organ or tissue associated disease or disorder in a subject, optionally by gene therapy, comprising administering to the subject a pharmaceutical composition comprising adeno-associated virus capsids obtained by carrying out the method of claim 15, wherein the ratio of adeno-associated virus capsids in which genetic material is fully packaged to adeno-associated virus capsids in which genetic material is not fully packaged is at least 3:2, preferably at least 4:1, and optionally (i) the capsid is selected from AAV1, AAV2, AAV4, AAV5, AAV8, and AAV10 capsids, or variants thereof, and the organ or tissue is the central nervous system; (ii) the capsid is selected from AAV1 and AAV8 capsids, or variants thereof, and the organ or tissue is the heart; (iii) the capsid is an AAV2 capsid or a variant thereof, and the organ or tissue is the kidney; (iv) the capsid is selected from AAV7 and AAV8 capsids, or variants thereof, and the organ or tissue is the liver; (v) the capsid is selected from AAV4, AAV5, and AAV6 capsids, or variants thereof, and the organ or tissue is lung; (vi) the capsid is AAV8 or a variant thereof and the organ or tissue is the pancreas; (vii) the capsid is selected from AAV2, AAV5, and AAV8 capsids, or variants thereof, and the organ or tissue is a photoreceptor cell; (viii) the capsid is selected from AAV1, AAV2, AAV4, AAV5, and AAV8 capsids, or variants thereof, and the organ or tissue is retinal pigment epithelium; (ix) the capsid is selected from AAV1, AAV6, AAV7, and AAV8 capsids, or variants thereof, and the organ or tissue is skeletal muscle; (x) the capsid is an AAV10 capsid or a variant thereof and the organ or tissue is brain; or (xi) The method, wherein the capsid is an AAV9 capsid or a variant thereof, and the organ or tissue is selected from the central nervous system, heart, liver, lung, and skeletal muscle.

20. 20. The method of claim 1 or 19, wherein the incompletely packaged adeno-associated virus capsid is an empty adeno-associated virus capsid and / or a partially packaged adeno-associated virus capsid.

21. A composition comprising adeno-associated virus capsids obtained by carrying out the method of claim 1, wherein the ratio of adeno-associated virus capsids in which genetic material is fully packaged to adeno-associated virus capsids in which the genetic material is not fully packaged in the composition is at least 3:2, preferably at least 4:

1.

22. A pharmaceutical composition comprising adeno-associated virus capsids obtained by carrying out the method of claim 15, wherein the ratio of adeno-associated virus capsids in which genetic material is fully packaged to adeno-associated virus capsids in which the genetic material is not fully packaged in the pharmaceutical composition is at least 3:2, preferably 4:

1.

23. 23. A pharmaceutical composition according to claim 22 for use in therapy, optionally for use in gene therapy.

24. the adeno-associated virus capsid is a capsid of adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 9 (AAV9), or adeno-associated virus serotype 10 (AAV10), or a variant thereof; the composition is for use in gene therapy, Optionally, the pharmaceutical composition is for use in the prevention or treatment of a disease or disorder associated with an organ or tissue; (i) the capsid is selected from AAV1, AAV2, AAV4, AAV5, AAV8, and AAV10 capsids, and the organ or tissue is the central nervous system; (ii) the capsid is selected from AAV1 and AAV8 capsids, and the organ or tissue is the heart; (iii) the capsid is an AAV2 capsid and the organ or tissue is the kidney; (iv) the capsid is selected from AAV7 and AAV8 capsids and the organ or tissue is the liver; (v) the capsid is selected from AAV4, AAV5, and AAV6 capsids and the organ or tissue is lung; (vi) the capsid is AAV8 and the organ or tissue is the pancreas; (vii) the capsid is selected from AAV2, AAV5, and AAV8 capsids, and the organ or tissue is a photoreceptor cell; (viii) the capsid is selected from AAV1, AAV2, AAV4, AAV5, and AAV8 capsids, and the organ or tissue is retinal pigment epithelium; (ix) the capsid is selected from AAV1, AAV6, AAV7, and AAV8 capsids, and the organ or tissue is skeletal muscle; (x) the capsid is an AAV10 capsid and the organ or tissue is brain, or (xi) The pharmaceutical composition for use according to claim 23, wherein the capsid is an AAV9 capsid or a variant thereof, and the organ or tissue is selected from the central nervous system, heart, liver, lung, and skeletal muscle.

25. 25. The composition of claim 21, or the pharmaceutical composition of claim 22, or the pharmaceutical composition for use of claim 23 or 24, wherein the not fully packaged adeno-associated virus capsid is an empty adeno-associated virus capsid and / or a partially packaged adeno-associated virus capsid.

26. 1. A method for separating adeno-associated viral capsids in which genetic material is fully packaged from adeno-associated viral capsids in which genetic material is not fully packaged, comprising: a support; a ligand; and a surface extender connecting the ligand to the support, the surface extender having formula IV: 【Chemistry 5】 Use of an anion exchange chromatography material defined by a. adding a liquid sample to a chromatographic material, said liquid sample having a purity of at least 90% and a purity of at least 10 12 adeno-associated virus capsids at a concentration of adeno-associated virus capsids / ml, at least 10% of the adeno-associated virus capsids being adeno-associated virus capsids in which the genetic material is fully packaged; b. eluting the adeno-associated virus capsids containing fully packaged genetic material from the chromatographic material; [0023] The adeno-associated virus capsids eluted in step (b) are eluted in an eluate fraction, the combined eluate fraction comprising at least 50% of the adeno-associated virus capsids of the liquid sample added in step (a), at least 60% of the adeno-associated virus capsids having fully packaged genetic material.

27. the elution buffer of step (b) comprises sodium acetate; the adeno-associated virus capsid is a capsid of adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), 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; 27. The use of claim 26, optionally wherein the adeno-associated virus capsid is a capsid of adeno-associated virus serotype 9 (AAV9) or a variant thereof.

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