Method for pre-screening adeno-associated virus capsids and method for isolating same

Abstract

The present disclosure is directed to a method for determining elution conditions suitable for separating adeno-associated virus (AAV) capsids in which genetic material is fully packaged from AAV capsids in which the genetic material is not fully packaged, the method comprising the steps of: (a) applying a liquid sample containing the AAV capsids to a strong or partially strong anion exchange chromatography material comprising a surface extender; (b) eluting the AAV viral capsids from the chromatography material by applying an elution buffer comprising a step gradient of increasing electrical conductivity, the step gradient increasing by about 0.5 to about 3 mS / cm per step; (c) determining, based on the elution profile obtained in step (b), a first value of electrical conductivity or a conductivity-related parameter suitable for eluting the adeno-associated viral capsids in which the genetic material is not fully packaged; and (d) determining, based on the elution profile obtained in step (b), a second value of electrical conductivity or a conductivity-related parameter suitable for eluting the adeno-associated viral capsids in which the genetic material is fully packaged. Further disclosed are methods for separating fully packaged AAV capsids from non-fully packaged AAV capsids based on predetermined first and second values ​​of electrical conductivity or a conductivity-related parameter, as well as the use of an anion exchange chromatography material to separate fully packaged AAV capsids from non-fully packaged AAV capsids.

Description

[Technical field]

[0001] The present disclosure relates to the field of adeno-associated capsid separation and is directed to a method for determining suitable elution conditions 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. Further disclosed is 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, and the use of anion exchange chromatography materials for such separation. The present disclosure is applicable to the separation of capsids of adeno-associated virus serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10) and their variants. [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. 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. Usually, several filtration steps and several chromatography steps are used to separate AAV particles from cell culture (see, for example, Weihong Qu et al.).

[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] Methods for the separation of fully and non-fully packaged capsids have been described previously (see, e.g., Hejmowski et al.), however, further optimization of purification strategies is always desirable to increase the speed and reduce the cost of downstream processing of the various adeno-associated virus serotypes. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] US6,428,707 [Patent Document 2] US6,602,990 [Patent Document 3] WO2018122089 [Patent Document 4] US20140296464A1 [Patent Document 5] US20160288089A1 [Patent Document 6] WO2018011600A1 [Patent Document 7] WO2018037244A1 [Patent Document 8] WO2013068741A1 [Patent Document 9] WO2015052465A1 [Patent Document 10] US7867784B2 [Non-Patent Document]

[0006] [Non-Patent Document 1] S Hjerten: Biochim Biophys Acta 79(2), pp. 393 - 398 (1964) [Non-Patent Document 2] "Styrene based polymer supports developed by suspension polymerization" (R Arshady: Chimica e L'Industria 70(9), pp. 70 - 75 (1988) [Summary of the Invention]

[0007] The objective of the present disclosure is to provide an improved method for the separation of fully packaged and not fully packaged adeno-associated virus capsids. This is accomplished by first performing a method for determining suitable elution conditions for separating fully packaged capsids from not fully packaged capsids. In the present specification, this method is referred to as a pre-screening method. This is followed by performing a method for separating fully packaged capsids from not fully packaged capsids by eluting not fully packaged capsids at a first electrical conductivity value as determined in the pre-screening method, and by eluting fully packaged capsids at a second electrical conductivity value as determined in the pre-screening method. This results in improved resolution of fully packaged and not fully packaged capsids, which will achieve a composition with a higher ratio of fully packaged capsids to not fully packaged capsids. The focus of the present disclosure is the pre-screening method for establishing optimal elution conditions for the polishing step of the separation method, also referred to as secondary or final purification.

[0008] More specifically, a first aspect of the present disclosure is a so-called pre-screening method for determining suitable elution conditions for separating adeno-associated virus (AAV) capsids in which genetic material is fully packaged from AAV capsids in which genetic material is not fully packaged, comprising: (a) applying a liquid sample containing AAV capsids to a strong or partial strong anion exchange chromatography material containing a surface extender; (b) eluting the AAV viral capsids from the chromatographic material by applying an elution buffer containing a step gradient of increasing electrical conductivity, the step gradient increasing by about 0.5 to about 3 mS / cm per step; (c) determining a first value of electrical conductivity or a conductivity-related parameter suitable for eluting adeno-associated virus capsids in which the genetic material is not fully packaged based on the elution profile obtained in step (b); and (d) determining a second value of electrical conductivity or a conductivity-related parameter suitable for eluting adeno-associated virus capsids in which the genetic material is fully packaged based on the elution profile obtained in step (b). The present invention relates to a method comprising:

[0009] Even more particularly, the pre-screening method is a method for determining suitable elution conditions 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: a) applying the liquid sample to a strong or partially strong anion exchange chromatography material comprising a support and a ligand for binding to adeno-associated virus capsids, wherein the liquid sample has a purity of at least 90% and at least 10 adeno-associated virus capsids. 12 adeno-associated virus capsids at a concentration of 5% / ml, at least 5%, e.g., 10% of the adeno-associated virus capsids being fully packaged adeno-associated virus capsids with genetic material; 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 in which the genetic material is not fully packaged and the adeno-associated virus capsids in which the genetic material is fully packaged from the chromatographic material by applying an elution buffer comprising a step gradient of increasing electrical conductivity, the step gradient starting from about 0 to about 5 mS / cm and increasing by about 0.5 to about 3 mS / cm per step, such as about 1 to about 2 mS / cm per step, such as about 1.2 to about 1.5 mS / cm per step, up to and including the electrical conductivity at which the adeno-associated virus capsids in which the genetic material is not fully packaged and the adeno-associated virus capsids in which the genetic material is fully packaged are eluted from the chromatographic material; c) determining a first value of electrical conductivity or a conductivity-related parameter suitable for eluting adeno-associated virus capsids in which genetic material is not fully packaged based on the elution profile obtained in step (b); d) determining a second value of the electrical conductivity or electrical conductivity-related parameter suitable for eluting the adeno-associated virus capsids in which the genetic material is fully packaged based on the elution profile obtained in step (b); The method includes:

[0010] 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, the method comprising steps (a)-(d) of the pre-screening method described above, e. Adding the liquid sample to the chromatographic material defined in step (a), wherein the liquid sample has a purity of at least 90% and at least 10 adeno-associated virus capsids. 12 adeno-associated virus capsids at a concentration of about 10 ... f. eluting adeno-associated virus capsids that are not fully packaged with genetic material by applying an elution buffer having a first value of electrical conductivity or electrical conductivity-related parameter as determined in step (c); g. eluting the adeno-associated virus capsids in which the genetic material is fully packaged by applying an elution buffer having a second value of the electrical conductivity or electrical conductivity-related parameter as determined in step (d); Further comprising: (i) the duration of step (f) is at least three times, such as four times, the duration of step (g); and / or (ii) The method further comprises a step (f') between steps (f) and (g), wherein step (f') comprises applying an additional step elution and / or an increasing conductivity gradient between a first value and a second value of the conductivity or conductivity-related parameter, and wherein the duration of steps (f) and (f') is at least three times, such as four times, the duration of step (g).

[0011] Additionally, 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, the method comprising: (I) applying the liquid sample to a strong or partially strong anion exchange chromatography material comprising a support and a ligand for binding to adeno-associated virus capsids, wherein the liquid sample has a purity of at least 90% and at least 10 adeno-associated virus capsids. 12 adeno-associated virus capsids at a concentration of 5% / ml, at least 5%, e.g., 10% of the adeno-associated virus capsids being fully packaged adeno-associated virus capsids with genetic material; 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: (II) eluting adeno-associated virus capsids in which genetic material is not fully packaged by applying an elution buffer having a predetermined first value of electrical conductivity or an electrical conductivity-related parameter; (III) eluting the adeno-associated virus capsids in which the genetic material is fully packaged by applying an elution buffer having a second predetermined value of electrical conductivity or a conductivity-related parameter; The predetermined first and second values ​​of electrical conductivity or an electrical conductivity-related parameter are determined during separation of the liquid sample, the liquid sample having a purity of at least 90% and a content of at least 10 adeno-associated virus capsids. 12 the liquid sample comprises adeno-associated virus capsids at a concentration of 0.1 to 10 ... (i) the duration of step (II) is at least three times, such as four times, the duration of step (III); and / or (ii) A process comprising a step (II') between steps (II) and (III), wherein step (II') comprises applying a step elution and / or a gradient of increasing conductivity between a first value and a second value of the conductivity or a conductivity-related parameter, and wherein the duration of steps (II) and (II') is at least three times, such as four times, the duration of step (III).

[0012] 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:

[0013] [ka]

[0014] Use of an anion exchange chromatography material defined by determining suitable elution conditions to separate 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; The elution conditions are a. adding a liquid sample to a chromatographic material, said liquid sample being at least 90% pure and containing at least 10 adeno-associated virus capsids; 12 adeno-associated virus capsids at a concentration of 50 / ml, of which at least 5%, e.g., 10%, of the adeno-associated virus capsids are adeno-associated virus capsids in which the genetic material is fully packaged; b. eluting the adeno-associated virus capsids in which the genetic material is not fully packaged and the adeno-associated virus capsids in which the genetic material is fully packaged from the chromatographic material by applying an elution buffer comprising a step gradient of increasing electrical conductivity, the step gradient starting from about 0 to about 5 mS / cm and increasing by about 0.5 to about 3 mS / cm per step, e.g., about 1 to about 2 mS / cm per step, e.g., about 1.2 to about 1.5 mS / cm per step, up to and including the electrical conductivity at which the adeno-associated virus capsids in which the genetic material is not fully packaged and the adeno-associated virus capsids in which the genetic material is fully packaged were eluted from the chromatographic material; c. determining a first value of electrical conductivity or an electrical conductivity-related parameter suitable for eluting adeno-associated virus capsids in which genetic material is not fully packaged based on the elution profile obtained in step (b); d. determining a second value of electrical conductivity or electrical conductivity-related parameter suitable for eluting adeno-associated virus capsids in which genetic material is fully packaged based on the elution profile obtained in step (b); It also provides for use, as determined by carrying out the.

[0015] Preferred aspects of the present disclosure are set out below in the detailed description and dependent claims. [Brief description of the drawings]

[0016] [Figure 1] 1 is a flow chart of a pre-screening method, i.e., a method for determining suitable elution conditions for isolating adeno-associated virus capsids according to the present disclosure. [Diagram 2] 2 is a flow chart of a method for isolating adeno-associated virus capsids, comprising steps (a)-(d) according to the pre-screening method of FIG. 1 and further comprising steps (e)-(g) according to the present disclosure. [Diagram 3] 1 is a flow chart of a method for isolating adeno-associated virus capsids comprising applying a predetermined value of electrical conductivity in accordance with the present disclosure. [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] 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 7]1 is a graph showing elution curves for fully packaged and empty capsids following the separation method as described in Example 2 below. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present disclosure provides a pre-screening method, as illustrated in FIG. 1, for determining suitable elution conditions 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: a) applying the liquid sample to a strong or partially strong anion exchange chromatography material comprising a support and a ligand for binding to adeno-associated virus capsids, wherein the liquid sample has a purity of at least 90% and at least 10 adeno-associated virus capsids. 12 adeno-associated virus capsids at a concentration of 5% / ml, at least 5%, e.g., 10% of the adeno-associated virus capsids being fully packaged adeno-associated virus capsids with genetic material; 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 in which the genetic material is not fully packaged and the adeno-associated virus capsids in which the genetic material is fully packaged from the chromatographic material by applying an elution buffer comprising a step gradient of increasing electrical conductivity, the step gradient starting from about 0 to about 5 mS / cm and increasing by about 0.5 to about 3 mS / cm per step, such as about 1 to about 2 mS / cm per step, such as about 1.2 to about 1.5 mS / cm per step, up to and including the electrical conductivity at which the adeno-associated virus capsids in which the genetic material is not fully packaged and the adeno-associated virus capsids in which the genetic material is fully packaged are eluted from the chromatographic material; c) determining a first value of electrical conductivity or a conductivity-related parameter suitable for eluting adeno-associated virus capsids in which genetic material is not fully packaged based on the elution profile obtained in step (b); d) determining a second value of the electrical conductivity or electrical conductivity-related parameter suitable for eluting the adeno-associated virus capsids in which the genetic material is fully packaged based on the elution profile obtained in step (b); The present invention solves or at least alleviates problems associated with existing methods for separating fully packaged adeno-associated virus capsids from non-fully packaged adeno-associated virus capsids by providing a method comprising:

[0018] The presently disclosed pre-screening method is shown herein to be a universal method that addresses the weaknesses of previously applied methods, where differences in ionic capacity, anion exchange ligand density, amount of surface extender, and support material between different lots of chromatographic materials, as well as variations in feed amounts and variations in buffer preparations, make it nearly impossible to predict the amount of electrical conductivity required to achieve baseline separation of full and empty capsids.

[0019] "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.

[0020] 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.

[0021] 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 manufacturing, are expected to contain the complete viral genome, which contains the genetic material of interest for one or more applications, for example, 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 capsids 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 response (Xiaotong Fu et al.). 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.

[0022] 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.

[0023] 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.

[0024] 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

[0025] The viral genome comprises a genetic insert which contains the genetic material of interest as defined elsewhere herein.

[0026] 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.

[0027] 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

[0028] 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.

[0029] The term "capsid in which genetic material is not completely packaged" encompasses the terms "partially filled capsid" and "empty capsid" as defined below.

[0030] 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 a defective viral genome, 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."

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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).

[0035] 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).

[0036] 3. Analytical anion exchange to separate full and empty capsids (A260:280 ratio and peak area to calculate percentages). Precision dependent on peak definition.

[0037] 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.

[0038] 5. Transmission Electron Microscopy (TEM). Image analysis to count particles (full, partially filled and empty capsids). Can introduce artifacts from sample preparation.

[0039] 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., which is hereby incorporated by reference.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] The separation matrix may be contained in any type of separation device as further defined elsewhere herein. As a non-limiting example, the chromatographic material may be packed into a chromatographic column, and then the liquid sample may be added to the chromatographic material contained in the chromatographic column. As another example, for the present pre-screening method, the chromatographic material may be provided in a multi-well format, for example, in the form of a multi-well plate having wells that contain the chromatographic material (e.g., PreDictor Capto Q plate, Cytiva, Sweden).

[0046] 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.

[0047] 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.

[0048] The ligand is 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 6,428,707, which is hereby incorporated 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 may be 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. Extenders can be linear and non-linear (branched) polymers, such as brush polymers, which are long linear structures with functional appendages along their length. The results described in the Examples 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.

[0049] 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.

[0050] 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.

[0051] As mentioned above, in the method for determining suitable elution conditions for separating fully packaged capsids from not fully packaged capsids, the liquid sample applied to the chromatographic material in step (a) is at least 90%, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure and at least 10% adeno-associated virus capsid. 12 / ml, e.g., adeno-associated virus capsid 10 13 , 10 14 , or 10 15 adeno-associated viral capsids at a concentration of 0.01% / ml, of which at least 5%, e.g., 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% are adeno-associated viral capsids in which the genetic material is fully packaged. With respect to the purity of adeno-associated 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 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.

[0052] As specified in step (b) of the pre-screening method above, the step gradient elution is designed in the form of stepwise slowly increasing electrical conductivity starting from about 0 to about 5 mS / cm, for example, about 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mS / cm. To identify which values ​​of electrical conductivity are required to wash out empty and complete capsids, respectively, the step gradient increases in increments of approximately 0.5 to 3 mS / cm per step, such as about 1 to about 2 mS / cm per step, such as about 1.2 to about 1.5 mS / cm per step, or about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 mS / cm per step, up to and including the conductivity at which capsids not fully packaged in genetic material and capsids fully packaged in genetic material elute from the chromatographic material. It should be understood that the conductivity step gradient may optionally be increased above the conductivity at which both empty and intact capsids are eluted.

[0053] As specified in steps (c) and (d) of the pre-screening method above, the first and second values ​​of the electrical conductivity or electrical conductivity-related parameter are determined based on the elution profile obtained in step (b). The elution profile may be in the form of a chromatogram, or a table or graph comprising the elution-related values.

[0054] The term "electrical conductivity-related parameter" as used herein is intended to mean a parameter that affects the electrical conductivity of a solution. Electrical conductivity-related parameters may, for example, be directly or inversely correlated with electrical conductivity. Non-limiting examples of electrical conductivity-related parameters that may be relevant in this context are salt concentration and pH, as well as the presence / concentration of compounds that improve separation of capsids in which genetic material is fully packaged and capsids in which genetic material is not fully packaged. Compounds that improve separation may be selected from, for example, carbohydrates, divalent metal ions, and surfactants, as described in more detail below.

[0055] The first value of the determined electrical conductivity or electrical conductivity-related parameter is suitable for eluting adeno-associated virus capsids in which genetic material is not fully packaged, and the second value of the determined electrical conductivity or electrical conductivity-related parameter is suitable for eluting adeno-associated virus capsids in which genetic material is fully packaged. In a pre-screening method in which empty capsids are eluted before full capsids, the first value of electrical conductivity is usually determined to be the value of electrical conductivity applied when eluting the first peak containing empty and / or full capsids. If empty capsids do not bind to the column and end up in the flow-through, the first value of electrical conductivity is the same as the baseline electrical conductivity value, i.e., the electrical conductivity before the step gradient of electrical conductivity is applied. Thus, in some cases, the first value of electrical conductivity can be determined to be as low as 0 mS / cm.

[0056] In pre-screening methods where empty capsids are eluted before full capsids, the second value of electrical conductivity is typically determined to be a conductivity value equal to or higher than the conductivity value applied when eluting the last peak containing empty and / or full capsids, e.g., if the last peak is eluted at a conductivity value of 5 mS / cm, the second value of electrical conductivity is determined to be ≧5 mS / cm.

[0057] The elution buffer applied in step (b) of the pre-screening method may contain a salt. The step gradient of increasing electrical conductivity in step (b) of the pre-screening method may be a step gradient of increasing salt concentration. In this context, the electrical conductivity-related parameter referred to in steps (c) and (d) of the pre-screening method may be salt concentration. The salt may be a kosmotropic salt. A salt in an aqueous medium is defined as kosmotropic (order-creating) if it contributes to the stability and structure of water-water interactions. In contrast, chaotropic (disorder-creating) salts have the opposite effect of disrupting the water structure, increasing the solubility of non-polar solvent particles, and destabilizing solute aggregates. Kosmotropes make water molecules interact well, which in fact stabilizes intramolecular interactions in macromolecules such as proteins (Moelbert S et al.). For example, a scale may be established by referring to the Hofmeister series or lyotropic permutation, which is a classification of ions in order of their ability to salt out or salt out proteins (Hyde A et al.).

[0058] More specifically, the 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 of such other salts 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, e.g., 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.

[0059] Step (b) of the pre-screening method may comprise adding a volume of elution buffer corresponding to about 1 to about 10 volumes, such as about 2 to about 8 volumes, such as about 5 volumes, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 volumes of chromatographic material per step of the step gradient.

[0060] The above pre-screening method is followed by a two-step elution method designed based on information provided by the elution profile (e.g., chromatogram) obtained in the pre-screening method. More specifically, the present disclosure provides a method for separating fully packaged adeno-associated virus capsids from non-fully packaged adeno-associated virus capsids, as illustrated in FIG. 2, comprising carrying out steps (a)-(d) of the pre-screening method as described in detail above, and further comprising steps (e)-(g) as follows: e. Adding the liquid sample to the chromatographic material defined in step (a), wherein the liquid sample has a purity of at least 90% and at least 10 adeno-associated virus capsids.12 adeno-associated virus capsids at a concentration of at least 100000000000000000 / ml, of which at least 5%, e.g., 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% are adeno-associated virus capsids with fully packaged genetic material, and the liquid sample is derived from the cell culture harvest from which the liquid sample of step (a) was derived; f. eluting adeno-associated virus capsids that are not fully packaged with genetic material by applying an elution buffer having a first value of electrical conductivity or electrical conductivity-related parameter as determined in step (c); g. eluting the adeno-associated virus capsids in which the genetic material is fully packaged by applying an elution buffer having a second value of the electrical conductivity or electrical conductivity-related parameter as determined in step (d); Further comprising: (i) the duration of step (f) is at least three times, such as four times, the duration of step (g); and / or (ii) The method further comprises a step (f') between steps (f) and (g), wherein step (f') comprises applying an additional step elution and / or an increasing conductivity gradient between a first value and a second value of the conductivity or conductivity-related parameter, and wherein the duration of steps (f) and (f') is at least three times, such as four times, the duration of step (g).

[0061] To ensure that the elution conditions determined in the pre-screening method are also applicable to the liquid sample added in step (e), the liquid sample added in step (e) should be derived from the same cell culture harvest as the liquid sample in step (a) of the pre-screening method.

[0062] The objective of steps (f) and (g) of the method disclosed above is to obtain fully packaged capsids of the highest possible purity. The skilled artisan will readily appreciate that this can be achieved by applying a variety of different separation conditions. A non-limiting example of a separation condition to obtain fully packaged capsids of the highest possible purity would be one that allows binding of not fully packaged capsids to a chromatographic material, but does not allow binding of fully 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.

[0063] 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 practice, 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 added to the chromatographic material in step (e). More specifically, the adeno-associated virus capsids eluted in step (g), i.e., adeno-associated virus capsids with fully packaged genetic material, may be 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 (e), 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 (e), of which at least 60% are intact capsids, such as 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 80% recovery of viral genomes from the harvest, of which at least 70% are intact capsids.

[0064] It has been found to be advantageous to carry out step (f) for a duration that is at least three times, e.g., four times, five times, or more, longer than the duration of step (g). Without wishing to be bound by theory, it is believed that the relatively longer duration of step (f) is beneficial or even important for eluting substantially all or nearly all empty capsids present in the liquid sample.

[0065] Instead of carrying out step (f) for a duration at least three times the duration of step (g), the method may comprise applying an additional step (f') between steps (f) and (g), the duration of steps (f) and (f') being at least three times, for example four times, five times or more, compared to the duration of step (g). (i) an elution step carried out at a value of the electrical conductivity or electrical conductivity-related parameter between a first and a second value of the electrical conductivity or electrical conductivity-related parameter; (ii) a linear slope of increasing electrical conductivity between a first and a second value of the electrical conductivity or electrical conductivity-related parameter; or (iii) A combination of (i) and (ii) may include.

[0066] The difference in duration between step (f) and step (g) can be achieved by adding a volume of elution buffer in step (f) that is at least 3 times, e.g., 4 times or more, the volume of elution buffer added in step (g). As a non-limiting example, in step (f), a volume of elution buffer corresponding to about 3 to about 30 volumes, e.g., about 6 to about 24 volumes, e.g., about 15 volumes, or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 volumes of chromatographic material is added, whereas in step (g), a volume of elution buffer corresponding to about 1 to about 10 volumes, e.g., about 2 to about 8 volumes, e.g., about 5 volumes, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 volumes of chromatographic material is added. Similarly, if the method includes step (f'), the difference in duration of step (f)+(f') compared to step (g) may be achieved by adding a volume of elution buffer in step (f)+(f') that is, in total, at least three times, such as four times or more, the volume of elution buffer added in step (g).

[0067] The elution buffer applied in steps (b), (f), optionally (f') and (g) of the above method for separating capsids (as illustrated in FIG. 2) may comprise a salt. The step gradient of increasing electrical conductivity as referred to in step (b) of the method may be a step gradient of increasing salt concentration. In this case, the electrical conductivity related parameter as referred to in steps (c), (f) and (g) of the method may be salt concentration. The salt may be a cosmotropic salt as defined elsewhere herein.

[0068] More specifically, the salt is: (i) CO3 2- , SO4 2- , S2O3 2- , H2PO4 - , HPO4 2- , Acetate - , Citrate - , 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. However, it should be understood that other salts consisting of combinations of anions such as those listed in (i) and cations such as those listed in (ii) may alternatively be used to elute the capsids.

[0069] Step (b) of the method may comprise adding a volume of elution buffer corresponding to about 1 to about 10 volumes, such as about 2 to about 8 volumes, such as about 5 volumes, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 volumes of chromatographic material per step of the step gradient.

[0070] As illustrated in FIG. 3, 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: (I) applying the liquid sample to a strong or partially strong anion exchange chromatography material comprising a support and a ligand for binding to adeno-associated virus capsids, wherein the liquid sample has a purity of at least 90% and at least 10 adeno-associated virus capsids. 12 adeno-associated virus capsids at a concentration of 1000 / ml, at least 5% of which are adeno-associated virus capsids in which the genetic material is fully packaged; 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: (II) eluting adeno-associated virus capsids in which genetic material is not fully packaged by applying an elution buffer having a predetermined first value of electrical conductivity or an electrical conductivity-related parameter; (III) eluting the adeno-associated virus capsids in which the genetic material is fully packaged by applying an elution buffer having a second predetermined value of electrical conductivity or a conductivity-related parameter. Including, The predetermined first and second values ​​of electrical conductivity or an electrical conductivity-related parameter are determined during separation of the liquid sample, the liquid sample having a purity of at least 90% and a content of at least 10 adeno-associated virus capsids. 12 adeno-associated virus capsids at a concentration of 10 ... (i) the duration of step (II) is at least three times, e.g., four times, five times or more, the duration of step (III); and / or (ii) The method further comprises a step (II') between steps (II) and (III), wherein step (II') comprises applying a step elution and / or a gradient of increasing conductivity between a first value and a second value of the conductivity or a conductivity-related parameter, and wherein the duration of steps (II) and (II') is at least three times, such as four times, five times or more, the duration of step (III).

[0071] The method as illustrated in Figure 3 is identical to the method as illustrated in Figure 2, except that the method of Figure 3 does not include a step for determining the first and second values ​​of the electrical conductivity or electrical conductivity-related parameter. Instead, the method of Figure 3 uses predetermined first and second values ​​of the electrical conductivity or electrical conductivity-related parameter. Optionally, the first and second values ​​of the electrical conductivity or electrical conductivity-related parameter may be predetermined (i.e., previously determined) by performing steps (a)-(d) of the pre-screening method disclosed herein, as further described in detail above.

[0072] It should be understood that the above embodiments and details for steps (e), (f), optionally (f'), and (g), respectively, of the method of FIG. 2 may be similarly applied to steps (I), (II), optionally (II'), and (III), respectively, of the method of FIG. 3.

[0073] As mentioned above, the chromatographic material applicable to any of the methods disclosed herein, as described in detail above and illustrated in Figures 1, 2, and 3, respectively, includes a strong or partial strong anion exchange chromatographic material comprising a support, a ligand for binding to the adeno-associated virus capsid, and a surface extender.

[0074] The strong anion exchange chromatography material has the following formula I:

[0075] [ka]

[0076] (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:

[0077] As a non-limiting example, each of R1, R2, and R3 is CH3.

[0078] There are currently available chromatography materials comprising ligands defined by formula I, where each of R1, R2, and R3 is CH3, such as the chromatography 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 chromatography medium for high-resolution polishing steps in industrial purification processes, such as for the purification of monoclonal antibodies.

[0079] According to another non-limiting example, R1 and R2 are ethyl and R3 is methyl.

[0080] By yet another non-limiting example, R1 and R2 are methyl and R3 is CH2CHOHCH3.

[0081] 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.

[0082] Alternatively, the strong or partially strong anion exchange chromatography material may be of the following formula II:

[0083] [ka]

[0084] (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 strong or partially strong anion exchange chromatography material can be defined by the following formula III:

[0085] [ka]

[0086] (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:

[0087] 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:

[0088] 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), which 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.

[0089] 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.

[0090] 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.

[0091] 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:

[0092] 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 anion exchange chromatography material.

[0093] 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.

[0094] 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.

[0095] Steps (a) and (b) of the pre-screening method (FIG. 1) and separation method (FIG. 2) disclosed above, as well as steps (e), (f), (f'), (g) of the separation method of FIG. 2, and steps (I), (II), (II'), and (III) of the separation method of FIG. 3 may include 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, a pH of about 7.0, 9.0, or 9.5 may be applied to a chromatographic material comprising a ligand defined by formula I, as described in Examples 1-3 below. Additionally, a pH of about 9.0 may be applied to a chromatographic material comprising a ligand defined by formula II or formula III, as described in Example 1 below.

[0096] 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.

[0097] In the pre-screening method disclosed above, step (b) may comprise the step of 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. In that case, in the separation method (FIG. 2), the buffer should comprise the same compound in steps (b), (f), (f'), and (g). Similarly, steps (II), (II'), and (III) should comprise the step of applying the same buffer, optionally one of the buffers mentioned above, as the buffer applied when pre-determining the first and second values ​​of the electrical conductivity or electrical conductivity-related parameter, which comprises the same compound that improves the separation of fully packaged and not fully packaged capsids. This compound may or may not be present in the buffer applied in steps (a), (e), and (I), respectively, of the method disclosed above. Without being bound by theory, such compounds may improve separation, for example, by affecting capsid-ligand or capsid-capsid interactions. The compounds that improve separation may be selected from, for example, carbohydrates, divalent metal ions, and surfactants.

[0098] When the compound that improves separation is a carbohydrate, it may be selected, for example, from sucrose, sorbitol, and polysaccharides.

[0099] 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)

[0100] 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.

[0101] As described in Example 1 below, a non-limiting example of a suitable buffer system applied in the method disclosed above includes Buffer A and Buffer B, both containing 20 mM Bis-Tris Propane (BTP) pH 9.0 and 2 mM MgCl2, and Buffer B further containing 250 mM sodium acetate (NaOAc) as an elution salt. Buffer A is applied in steps (a), (e), and (I), respectively. A step gradient of Buffer B is applied in step (b) of the pre-screening method. The first and second values ​​of electrical conductivity or electrical conductivity-related parameters are obtained by applying a mixture of Buffer A and Buffer B in appropriate ratios in steps (f) and (g) of the method of FIG. 2 or in steps (II) and (III) of the method of FIG. 3, respectively.

[0102] As described in Example 2 below, another non-limiting example of a suitable buffer system to be applied in the methods disclosed above includes Buffer A and Buffer B, both containing 20 mM Bis-Tris Propane (BTP) pH 7.0, 1% sucrose and 0.1% Pluronic, with Buffer B further containing 20 mM MgCl2.

[0103] As described in Example 3 below, another non-limiting example of a suitable buffer system to be applied in the methods disclosed above includes Buffer A and Buffer B, both containing 20 mM Bis-Tris Propane (BTP) pH 7.0 or 9.5, 18 mM MgCl, 1% sucrose, and 0.1% Pluronic, respectively, with Buffer B further containing 400 mM NaCl.

[0104] 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 (S Hjerten: Biochim Biophys Acta 79(2), pp. 393-398 (1964)). 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, e.g., US 6,602,990 (Berg). In an alternative embodiment, the support is prepared from a synthetic polymer or copolymer, such as a cross-linked synthetic polymer, for example, styrene or a styrene derivative, divinylbenzene, acrylamide, acrylate esters, methacrylate esters, vinyl esters, vinyl amides, etc. Such synthetic polymers are easily prepared and optionally cross-linked according to standard methods, see for example "Styrene based polymer supports developed by suspension polymerization" (R Arshady: Chimica e L'Industria 70(9), pp. 70-75 (1988)). Natural or synthetic polymer supports are also available from commercial sources, for example in the form of porous particles, such as Cytiva, Sweden. In yet another 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] The chromatography materials Capto Q and Capto DEAE advantageously used in Examples 1 to 3 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).

[0112] 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.

[0113] 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.

[0114] The chromatographic material may be in the form of magnetic particles, i.e. magnetic adsorptive beads. The term "magnetic particles" is defined herein as particles that can be attracted by a magnetic field. At the same time, magnetic particles for use in the methods disclosed herein do not aggregate in the absence of a magnetic field. In other words, the magnetic particles behave like superparamagnetic particles. The particles may have any symmetrical shape, such as a sphere or a cube, or any asymmetrical shape. Spherical magnetic particles are often referred to as magnetic beads. It should be understood that the terms "magnetic particles", "magnetic beads", "Mag particles", "Mag beads", "magparticles" and "magbeads" may be used interchangeably herein without limiting the scope to magnetic particles having a spherical shape. The separation of biomolecules by the use of magnetic adsorptive beads is known in the art. Magnetic particles suitable for use in the methods disclosed herein are described in WO2018122089, which is incorporated herein by reference in its entirety. A non-limiting example of a magnetic particle that can be used in the methods disclosed herein is Mag Sepharose™ Prism A (Cytiva, Sweden).

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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 adsorber are described, for example, in US20140296464A1, US20160288089A1, WO2018011600A1, WO2018037244A1, WO2013068741A1, WO2015052465A1, US7867784B2, which are incorporated by reference in their entireties.

[0122] In the pre-screening methods disclosed herein, the methods for separating fully packaged capsids from non-fully packaged adeno-associated virus capsids, and the use of chromatographic materials for separating full capsids from empty capsids, the chromatographic materials referred to may advantageously be polishing chromatographic materials, meaning that the chromatographic materials are applied in a polishing step.

[0123] 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 suspected leakage products. The polishing step may alternatively be called a "secondary purification step".

[0124] Furthermore, the liquid sample added in each of steps (a), (e) and (I) of the method disclosed herein may advantageously be a pre-purified liquid sample.

[0125] 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 steps (a)-(g) or alternatively steps (I)-(III) as 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, 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), step (e), or step (I), respectively, of the above method.

[0126] 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.

[0127] 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.

[0128] Non-limiting examples of chromatographic materials suitable for application in the pre-purification step 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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 anion exchange chromatographic material comprising a ligand as defined by Formula I, II or III, as described in detail elsewhere herein.

[0133] 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.

[0134] The method disclosed herein for separating fully packaged adeno-associated virus capsids from non-fully packaged adeno-associated virus capsids comprises separating an eluate fraction containing adeno-associated virus capsids in which genetic material is fully packaged, which is eluted in step (g) or step (III), respectively, of the method as described above, by: h1) concentrating the adeno-associated virus capsid to a pharma- ceutical suitable dose; h2) replacing the buffer applied in steps (b) / (g) of the process with a pharma- ceutically acceptable buffer, and / or h3) Sterilizing the eluate fraction containing the adeno-associated virus capsid to obtain a pharmaceutical composition comprising the adeno-associated virus capsid.

[0135] 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.

[0136] Pharmaceutically acceptable buffers are well known in the art and can be readily selected by one of ordinary skill in the art.

[0137] In order for the resulting composition to meet all regulatory requirements for pharmaceutical compositions, usually all three steps h1 to h3 listed above must be carried out.

[0138] In the methods disclosed above, the adeno-associated virus capsid may advantageously be a capsid of adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 9 (AAV9), or adeno-associated virus serotype 10 (AAV10), or a variant thereof.

[0139] The term "variant" in relation to adeno-associated virus (AAV) serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 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 is equally true for variants of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 9, or 10 compared to unmodified AAV serotypes 1, 2, 3, 4, 5, 6, 7, 9, or 10, respectively. Furthermore, as a 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 is equally true for variants of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 9, or 10 compared to the original AAV serotypes 1, 2, 3, 4, 5, 6, 7, 9, or 10, respectively. The designated chromatographic material may be, for example, a 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.

[0140] According to a currently preferred embodiment, in the separation method as illustrated in FIG. 2, the chromatographic material is represented by formula IV:

[0141] [ka]

[0142] and the elution buffers of steps (b), (f), and optionally (f'), and (g) comprise sodium acetate. The method may be applied to the separation of AAV capsids of any serotype or variant as described above. In particular, the capsids to be separated may be capsids of the AAV9 serotype or variants thereof.

[0143] According to another currently preferred embodiment, in the separation method as illustrated in FIG. 3, the chromatographic material is represented by formula IV:

[0144] [ka]

[0145] and the elution buffer of steps (II), optionally (II'), and (III) comprises sodium acetate. The method may be applied to the separation of AAV capsids of any serotype or variant as described above. In particular, the separated capsid may be a capsid of the AAV9 serotype or a variant thereof.

[0146] 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:

[0147] [ka]

[0148] 2. Use of an anion exchange chromatography material as defined by the formula: The elution conditions are a. adding a liquid sample to a chromatographic material, said liquid sample being at least 90% pure and containing at least 10 adeno-associated virus capsids; 12 adeno-associated virus capsids at a concentration of 50 / ml, of which at least 5%, e.g., 10%, of the adeno-associated virus capsids are adeno-associated virus capsids in which the genetic material is fully packaged; b. eluting the adeno-associated virus capsids in which the genetic material is not fully packaged and the adeno-associated virus capsids in which the genetic material is fully packaged from the chromatographic material by applying an elution buffer comprising a step gradient of increasing electrical conductivity, the step gradient starting from about 0 to about 5 mS / cm and increasing by about 0.5 to about 3 mS / cm per step, e.g., about 1 to about 2 mS / cm per step, e.g., about 1.2 to about 1.5 mS / cm per step, up to and including the electrical conductivity at which the adeno-associated virus capsids in which the genetic material is not fully packaged and the adeno-associated virus capsids in which the genetic material is fully packaged were eluted from the chromatographic material; c. determining a first value of electrical conductivity or an electrical conductivity-related parameter suitable for eluting adeno-associated virus capsids in which genetic material is not fully packaged based on the elution profile obtained in step (b); d. determining a second value of electrical conductivity or electrical conductivity-related parameter suitable for eluting adeno-associated virus capsids in which genetic material is fully packaged based on the elution profile obtained in step (b); The present invention further provides a use, which is determined by performing

[0149] In the use disclosed herein, by carrying out the above steps (a) to (d), e. adding the liquid sample to a chromatographic material, wherein the liquid sample has a purity of at least 90% and at least 10 adeno-associated virus capsids; 12adeno-associated virus capsids at a concentration of about 10 ... f. eluting adeno-associated virus capsids that are not fully packaged with genetic material by applying an elution buffer having a first value of electrical conductivity or electrical conductivity-related parameter as determined in step (c); g. eluting the adeno-associated virus capsids in which the genetic material is fully packaged by applying an elution buffer having a second value of the electrical conductivity or electrical conductivity-related parameter as determined in step (d); by carrying out the steps of: separating adeno-associated viral capsids in which genetic material has been fully packaged from adeno-associated viral capsids in which genetic material has not been fully packaged; (i) the duration of step (f) is at least three times, such as four times, the duration of step (g); and / or (ii) the method comprises a step (f') between steps (f) and (g), step (f') comprising applying an additional step elution and / or an increasing conductivity gradient between a first value and a second value of the conductivity or conductivity-related parameter, and the duration of steps (f) and (f') is at least three times, such as four times, the duration of step (g).

[0150] Preferably, the elution buffer of step (b) of the above use, and, if applicable, the elution buffer of steps (f) and (g) of the above use, also comprises sodium acetate. The above use may be applied to the separation of AAV capsids of any serotype or variant as described above. In particular, the separated capsid may be a capsid of the AAV9 serotype or a variant thereof.

[0151] 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.

[0152] The singular forms "a" and "an" are intended to be construed as including the plurals as well. EXAMPLES

[0153] Example 1 Separation of capsids of serotypes AAV2, AAV5, AAV8, and AAV9 on anion exchange chromatography material using an increasing sodium acetate step gradient for elution Chromatography Materials The following currently available anion exchange chromatography materials provided improved results, as further described below.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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) with a flow rate of 1 CV / min (i.e., 2 mL / min) and 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 approximately 5 × 10 affinity-purified or affinity- and size-exclusion-purified AAV2, AAV5, AAV8, or AAV9, respectively, containing a mixture of full and empty capsids (>5% full capsids, as follows: AAV2 7-10%, AAV5 47%, AAV8 11-35%, AAV9 40%). 12 The material should have a low electrical conductivity (1-3 mS / cm) to ensure binding of AAV to the anion exchange ligand.

[0158] 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.

[0159] 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 elution or continuous gradient elution, respectively, with and without additives (0.1% poloxamer 188, 1% sucrose) and with various elution salts (NaCl, NaOAc, NH4Cl or NH4SO4 up to 500 mM) and additional salts (MgCl2 and MgSO4 up to 20 mM) in acetate buffers of pH 4.5 and 5. None of the above mentioned conditions or resins resulted in good baseline separation of full and empty capsids (data not shown).

[0160] Currently available anion exchange resins with dextran extenders, Capto DEAE (partial strong anion exchange) and Capto Q (strong anion exchange), were each evaluated 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 containing 250 mM sodium acetate (NaOAc) as an elution salt. In Figures 4-7, 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).

[0161] Pre-screening method The step gradient applied in the pre-screening method included short steps (3 CV) of increasing concentrations of NaOAc, more specifically, a 12.5 mM increment per step, i.e., a 5% concentration increment per step of 250 mM NaOAc in buffer B, corresponding to an approximate conductivity increase of 1.2-1.5 mS / cm per step.

[0162] Equilibration: 5 CV of Buffer A Injection: empty loop / w 3 ml of buffer A Wash: 5 CV of Buffer A Step gradient: 5% increase in buffer B, 3 CV each Wash: 100% Buffer B, 5 CV Re-equilibration: 5 CV of Buffer A

[0163] Capto Q Figure 4A shows the results of the pre-screening method for AAV9 separated on Capto Q resin. Multiple peaks close to each other can be seen in the chromatogram (peak numbers 1, 2, and 3 in Figure 4A). Peak numbers 1, 2, and 3 in Figure 4A contained different ratios of full and empty capsids. Peak number 1 contained mainly empty capsids, while peak numbers 2 and 3 contained mainly full capsids. The UV260 / 280 ratios were as follows: peak 1, 0.62, peak 2, 1.21, and peak 3, 1.26.

[0164] Based on the chromatogram in FIG. 4A, a first value of electrical conductivity was determined suitable for elution of empty capsids. The determined first value of electrical conductivity corresponded to the concentration of NaOAc applied when eluting the first peak containing empty and / or full capsids, i.e., peak number 1. The first value of electrical conductivity selected was approximately 1.2-1.5 mS / cm, corresponding to 5% buffer B, i.e., 12.5 mM NaOAc.

[0165] Also based on the chromatogram in FIG. 4A, a second value of conductivity was determined to be suitable for elution of intact capsids. The selected second value of conductivity corresponded to a higher concentration of NaOAc than that applied when eluting the last peak containing empty and / or intact capsids, i.e., peak number 3. The selected second value of conductivity was approximately 7.2-9.0 mS / cm, corresponding to 30% buffer B, i.e., 75 mM NaOAc.

[0166] It is noted here that the second value of electrical conductivity could alternatively have been selected to correspond to the concentration of NaOAc applied when eluting the last peak containing empty and / or full capsids or any higher value. Thus, the second value of electrical conductivity could have been approximately 3.6-4.5 mS / cm, corresponding to 15% buffer B, i.e., 37.5 mM NaOAc as applied when eluting peak number 3, or any higher value, i.e., ≧3.6 mS / cm, corresponding to ≧37.5 mM NaOAc.

[0167] Figure 5A shows the results of the pre-screening method for AAV8 separated on Capto Q resin. Multiple peaks close to each other can be seen in the chromatogram (peak numbers 1, 2, and 3 in Figure 5A). Peak numbers 1, 2, and 3 in Figure 5A contained different ratios of full and empty capsids. Peak numbers 1 and 2 contained mainly empty capsids, while peak number 3 contained mainly full capsids. The UV260 / 280 ratios were as follows: peak 1 was 0.6, peak 2 was 0.66, and peak 3 was 1.3.

[0168] Based on the chromatogram in FIG. 5A, a first value of electrical conductivity was determined suitable for elution of empty capsids. The determined first value of electrical conductivity corresponded to the concentration of NaOAc applied when eluting the first peak containing empty and / or full capsids, i.e., peak number 1. The first value of electrical conductivity selected was approximately 7.2-9.0 mS / cm, corresponding to 30% buffer B, i.e., 75 mM NaOAc.

[0169] Also based on the chromatogram in Figure 5A, a second value of conductivity was determined to be suitable for elution of intact capsids. The selected second value of conductivity corresponded to a higher concentration of NaOAc than that applied when eluting the last peak containing empty and / or intact capsids, i.e., peak number 3. The selected second value of conductivity was approximately 24-30 mS / cm, which corresponds to 100% buffer B, i.e., 250 mM NaOAc.

[0170] It is noted here that the second value of electrical conductivity could alternatively have been selected to correspond to the NaOAc concentration applied when eluting the last peak containing empty and / or full capsids or any higher value. Thus, the second value of electrical conductivity could have been approximately 9.6-12.0 mS / cm, corresponding to 40% buffer B, i.e. 100 mM NaOAc applied when eluting peak number 3, or any higher value, i.e. ≧9.6 mS / cm, corresponding to ≧100 mM NaOAc.

[0171] Capto DEAE Figure 6 shows the results of a pre-screening method for the elution of AAV9 on Capto DEAE resin. In the pre-screening method, using the same conditions as above for Capto Q (Buffer A: 20 mM BTP pH 9.0, 2 mM MgCl2; Buffer B: Buffer A + 250 mM NaOAc), but with 4% increasing elution steps of Buffer B (3 CV each), AAV9 empty capsids eluted in the flow-through and AAV9 full capsids eluted in the first step of the step gradient. The UV260:280 ratio was 0.76 in the first peak (i.e., the flow-through peak), suggesting mainly empty AAV9 capsids, but also a small amount of full capsids. The UV260:280 ratio was 1.3 in the second peak, indicating high purity of AAV9 full capsids (Figure 6).

[0172] Based on the chromatograms in FIG. 6, a first conductivity value suitable for elution of AAV9 empty capsids was determined to be approximately 0 mS / cm, corresponding to 0% buffer B, and a second conductivity value suitable for elution of AAV9 complete capsids was determined to be approximately 1 mS / cm, corresponding to 4% buffer B, i.e., 10 mM NaOAc.

[0173] Two-step elution method The first and second values ​​of electrical conductivity as determined in the pre-screening method above were applied in a subsequent two-step elution method to separate full capsids from empty capsids.

[0174] 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, first value of conductivity, 20 CV; step 2, second value of conductivity, 5 CV Re-equilibration: 5 CV of Buffer A

[0175] Capto Q When using Capto Q resin, all tested serotypes (AAV2, AAV5, AAV8, and AAV9) gave good separation of full and empty capsids.

[0176] Figure 4B shows the results of a two-step elution method for AAV9 on Capto Q resin, as described above, applying in a first step a first value of conductivity corresponding to 5% buffer B and in a second step a second value of conductivity corresponding to 30% buffer B. The full (F) and empty (E) capsids and the UV260:280 ratio are shown in Figure 4B.

[0177] Figure 5B shows the results of a two-step elution method for AAV8 on Capto Q resin, applying 30% Buffer B in the first step and 100% Buffer B in the second step, as described above. Full (F) and empty (E) capsids, as well as the UV260:280 ratio, are shown in Figure 5B.

[0178] In the AAV9 samples tested, peaks containing empty and / or complete capsids were also analyzed by qPCR, showing very low amounts (0.3%) of complete capsids in the empty peak and an overall recovery of 91% of the viral genome in the complete peak.

[0179] Capto DEAE AAV9 capsids were separated on Capto DEAE resin by applying the conditions established in the pre-screening method above, i.e., a first value of conductivity corresponding to 0% buffer B and a second value of conductivity corresponding to 4% buffer B. The resulting separation of full and empty capsids was as shown in Figure 6 (above).

[0180] 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 pre-screening and 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).

[0181] Example 2 Separation of capsids of serotypes AAV5 and AAV9 on anion exchange chromatography material using an increasing magnesium chloride step gradient for elution Capto Q resin was evaluated for the separation of AAV9 and AAV5, respectively, by applying the pre-screening method followed by a two-step elution as described in Example 1, with the difference that the buffer systems Buffer A and Buffer B both contained 20 mM Bis-Tris Propane (BTP) pH 7.0 (AAV5) or pH 9.5 (AAV9), 1% sucrose and 0.1% Pluronic, with Buffer B additionally containing 20 mM MgCl2.

[0182] Pre-screening method Equilibration: 5 CV of Buffer A Injection: empty loop / w 3 ml of buffer A Wash: 5 CV of Buffer A Step gradient elution: 5% increments of buffer B, 3 CV each Wash: 5 CV of 100% Buffer B Re-equilibration: 5 CV of Buffer A

[0183] Figure 7A shows the results of the pre-screening method for AAV5 separated on Capto Q resin. Multiple peaks close to each other are seen in the chromatogram (peak numbers 1, 2, 3, and 4 in Figure 7A). Peak numbers 1, 2, 3, 4, and 5 contained different ratios of full and empty capsids. Peak numbers 1 and 2 contained mainly empty capsids, while peak numbers 3, 4, and 5 contained mainly full capsids. The UV260:280 ratios were as follows: peak 1 was 0.68, peak 2 was 0.87, peak 3 was 1.14, peak 4 was 1.1, and peak 5 was 1.

[0184] Based on the chromatogram in Figure 7A, a first value of electrical conductivity was determined to be suitable for eluting empty capsids. The determined first value of electrical conductivity corresponded to the concentration of MgCl2 applied when eluting the first peak containing empty and / or full capsids, i.e., peak number 1. The first value of electrical conductivity selected was approximately 4.6 mS / cm, which corresponds to 50% buffer B, i.e., 10 mM MgCl2.

[0185] Further, based on the chromatogram in Figure 7A, a second value of conductivity was determined to be suitable for elution of intact capsids. The selected second value of conductivity corresponded to a concentration of MgCl2 identical to the concentration of MgCl2 applied when eluting the last peak containing empty and / or intact capsids, i.e., peak number 5. The selected second value of conductivity was approximately 5.3 mS / cm, which corresponds to 70% buffer B, i.e., 14 mM MgCl2.

[0186] Alternatively, the second value of electrical conductivity could have been chosen to be higher than the concentration of MgCl2 applied when eluting the last peak containing empty and / or complete capsids, i.e. peak number 5.

[0187] Two-step elution method The first and second values ​​of electrical conductivity as determined in the pre-screening method above were applied in a subsequent two-step elution method to separate full capsids from empty capsids.

[0188] 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

[0189] Figure 7B 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, as described above. The intact (F) and empty (E) capsids, as well as the UV260:280 ratio, are shown in Figure 7B. It should be noted here that, given that the second peak (intact capsid) is very sharp (almost no tailing), the two-step elution method would have worked equally well by applying a shorter duration of step 2, corresponding to 5 CV (as in the previous example).

[0190] Alternative Buffer Systems An alternative buffer system was also tested, including Buffer A and Buffer B with higher pH, both containing 20 mM Bis-Tris Propane (BTP) pH 9.5, 1% sucrose and 0.1% Pluronic, with Buffer B further containing 30 mM MgCl2, i.e., a higher concentration of MgCl2. Here, in the pre-screening method, increasing MgCl2 in 3.5% increments were applied. The first and second conductivity values ​​determined corresponded to 68% and 90% Buffer B, respectively. The conductivity values ​​were then applied in a two-step elution method.

[0191] The above conditions resulted in successful baseline separation of AAV5 (results for alternative buffer systems not shown). AAV9 binds more weakly to anion exchange, but conditions including higher pH (e.g., 9.5) and application of a small conductivity increase elution step resulted in acceptable performance (results not shown).

[0192] Example 3 Isolation of AAV9 capsids under variable conditions The experimental design for separation of fully packaged, empty and partially packaged AAV9 capsids is carried out using the equipment and samples as in Examples 1 and 2 above, and further using anion exchange chromatography material as in Examples 1 and 2, with the following variations.

[0193] Regarding the capsid: 1. AAV9-based engineered capsid variants 2. An AAV9 capsid with a longer insert than the insert contained in the capsids of Examples 1 and 2, which was green fluorescent protein (GFP).

[0194] Regarding Surface Extenders: 1) Dextran of different sizes (kDa) (T10, T70, T250). 2) Different amounts of dextran. 3) Alternatives to dextran, such as the use of polyhydric alcohols based on glycidol.

[0195] Regarding ligand density: 1) Capto Q: 60~160, 220~260 μmol / mL. 2) Capto DEAE: 150~290, 350~400 μmol / mL.

[0196] 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. 3) Magnetic beads in batch form.

[0197] Regarding the chemical nature of the ligand: 1) Capto DEAE ligands (ligands according to formula III) with different levels of quaternization. 2) Capto Q Analogs (Ligands According to 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 (ligands according to formula II): am=1, n=1, R1, R2, R3, and R4=methyl, R5=H. bm=1, n=1, R1, R2, R3, and R4=methyl, R5=CH2CHOHCH3. 4) Capto DEAE Analogues (ligands according to formula III): an=1, R1, R2, R3, and R4=methyl, R5=H or absent.

[0198] Regarding buffers and elution conditions: 1) Different concentrations of MgCl2 between 1 and 20 mM. 2) Different cosmotropic salts for elution, step gradient 0.1-1M, with or without MgCl2 as in 1). 3) Different pH step gradients pH 4-10, with or without MgCl2 as in 1). 4) Different buffer solutions: a. Tris bN-Methyldiethanolamine 5) Linear gradient elution with pH, ​​cosmotropic salt for elution, MgCl2, as in 1), 2), 3) and 4). 6) An additional step, such as a third elution step between the two elution steps in the two-step elution protocol according to Example 1 or a linear elution gradient. 7) Conditions suitable for flow-through separation, where some analytes bind to the chromatographic material while others do not (anion exchange resins bind full capsids more strongly than empty capsids, and therefore empty capsids may elute with the flow-through). 8) All of the above, with or without additives such as sucrose (0.1-5%) and poloxamer 188 surfactant (0.01-1%).

[0199] Example 4 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, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV10, and their variants, is carried out according to the variables in Examples 1-4 above.

[0200] 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.

[0201] References Weihong Qu et al, Scalable Downstream Strategies for Purification of Recombinant Adeno-Associated Virus Vectors in Light of the Properties, Current Pharmaceutical Biotechnology 2015 Aug; 16(8): 684-695 Hejmowski Adam L et al, Novel anion exchange membrane chromatography method for the separation of empty and full adeno-associated virus, Biotechnol. J. 2021;2100219. https: / / doi.org / 10.1002 / biot.202100219 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, 2004 Dec, 112(1): 45-57 Hyde A 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 determining suitable elution conditions 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: a. adding the liquid sample to a strong or partially strong anion exchange chromatography material comprising a support and a ligand for binding to adeno-associated virus capsids, wherein the liquid sample is at least 90% pure and contains at least 10 adeno-associated virus capsids; 12 adeno-associated virus capsids at a concentration of 1000 / ml, at least 5% of which are adeno-associated virus capsids in which the genetic material is fully packaged; 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 viral capsids in which the genetic material is not fully packaged and the adeno-associated viral capsids in which the genetic material is fully packaged from the chromatographic material by applying an elution buffer comprising a step gradient of increasing electrical conductivity, the step gradient starting from about 0 to about 5 mS / cm and increasing by about 0.5 to about 3 mS / cm per step up to and including the electrical conductivity at which the adeno-associated viral capsids in which the genetic material is not fully packaged and the adeno-associated viral capsids in which the genetic material is fully packaged eluted from the chromatographic material; c. determining a first value of electrical conductivity or an electrical conductivity-related parameter suitable for eluting adeno-associated virus capsids in which genetic material is not fully packaged based on the elution profile obtained in step (b); d. determining a second value of electrical conductivity or electrical conductivity-related parameter suitable for eluting adeno-associated virus capsids in which genetic material is fully packaged based on the elution profile obtained in step (b); The method includes:

2. 2. The method of claim 1, wherein the step gradient of increasing electrical conductivity in step (b) is a step gradient of increasing salt concentration, and optionally the electrical conductivity related parameter in steps (c) and (d) is salt concentration.

3. The method of claim 2, wherein the salt is a cosmotropic salt.

4. The salt is: (i) CO 3 2- , S.O. 4 2- , S 2 O 3 2- , H 2 PO 4 - , H.P.O. 4 2- , Acetate - , Citrate - , 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.

5. 5. The method of claim 1, wherein step (b) comprises adding a volume of elution buffer corresponding to about 1 to about 10 volumes of chromatographic material per step of the step gradient.

6. 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 steps (a) to (d) as defined in claim 1, e. Adding the liquid sample to the chromatographic material defined in step (a), wherein the liquid sample has a purity of at least 90% and at least 10 adeno-associated virus capsids. 12 adeno-associated virus capsids at a concentration of 1000000000000000 / ml, at least 5% of the adeno-associated virus capsids being fully packaged adeno-associated virus capsids with genetic material, the liquid sample being derived from the cell culture harvest from which the liquid sample of step (a) was derived; f. eluting adeno-associated virus capsids in which genetic material is not fully packaged by applying an elution buffer having a first value of electrical conductivity or electrical conductivity-related parameter determined in step (c); g. eluting the adeno-associated virus capsids in which the genetic material is fully packaged by applying an elution buffer having a second value of the electrical conductivity or electrical conductivity-related parameter determined in step (d); Further comprising: (i) the duration of step (f) is at least three times the duration of step (g); and / or (ii) A method comprising a step (f') between steps (f) and (g), wherein step (f') comprises applying an additional step elution and / or an increasing conductivity gradient between a first value and a second value of the conductivity or conductivity-related parameter, and wherein the duration of steps (f) and (f') is at least three times the duration of step (g).

7. 7. The method of claim 6, wherein the elution buffer comprises a salt, and wherein the step gradient of increasing conductivity in step (b) is a step gradient of increasing salt concentration, and optionally the conductivity-related parameter in steps (c), (f) and (g) is salt concentration.

8. 8. The method of claim 7, wherein the salt is a cosmotropic salt.

9. The salt is: (i) CO 3 2- , S.O. 4 2- , S 2 O 3 2- , H 2 PO 4 - , H.P.O. 4 2- , Acetate - , Citrate - , and Cl - and (ii) an anion selected from the group consisting of NH 4 + , K + , Na + , and Li + 9. The method of claim 7 or 8, comprising a cation selected from the group consisting of:

10. 10. The method of any one of claims 6 to 9, wherein step (b) comprises adding a volume of elution buffer corresponding to about 1 to about 10 volumes of chromatographic material per step of the step gradient.

11. 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: (I) applying the liquid sample to a strong or partially strong anion exchange chromatography material comprising a support and a ligand for binding to adeno-associated virus capsids, wherein the liquid sample has a purity of at least 90% and at least 10 adeno-associated virus capsids. 12 adeno-associated virus capsids at a concentration of 1000 / ml, at least 5% of which are adeno-associated virus capsids in which the genetic material is fully packaged; 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: (II) eluting adeno-associated virus capsids in which genetic material is not fully packaged by applying an elution buffer having a predetermined first value of electrical conductivity or an electrical conductivity-related parameter; (III) eluting the adeno-associated virus capsids in which the genetic material is fully packaged by applying an elution buffer having a second predetermined value of electrical conductivity or a conductivity-related parameter. Including, The predetermined first and second values ​​of electrical conductivity or an electrical conductivity-related parameter are determined during separation of the liquid sample, the liquid sample having a purity of at least 90% and a content of at least 10 adeno-associated virus capsids. 12 a liquid sample comprising adeno-associated virus capsids at a concentration of 0.01 to 0.15% / ml, at least 5% of the adeno-associated virus capsids being adeno-associated virus capsids in which the genetic material is fully packaged, the liquid sample being derived from the cell culture harvest from which the liquid sample of step (I) was derived, and the optionally predetermined first and second values ​​being determined by carrying out the method of steps (a) to (d) of claim 1; (i) the duration of step (II) is at least three times the duration of step (III); and / or (ii) A method comprising a step (II') between steps (II) and (III), wherein step (II') comprises applying a step elution and / or a gradient of increasing electrical conductivity between a first value and a second value of electrical conductivity or a conductivity-related parameter, and wherein the duration of steps (II) and (II') is at least three times the duration of step (III).

12. The chromatographic material is i) Formula I: 【Chemistry 1】 (In the formula, R 1 is selected from C1-C3 alkyl, R 2 and R 3 is independently selected from C1-C3 alkyl, CH2OH, and CH2CHOHCH3; Optionally, R 1 , R 2 , and R 3 Each of these is CH3) or ii) 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 chromatographic material has 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) is defined by Optionally, the chromatographic material 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 any one of claims 1 to 11, comprising a combination of two or more of:

13. the surface extender is dextran; Optionally, the dextran has a molecular weight of about 10 to about 2000 kDa, for example about 40 kDa; and / or Optionally, the density of the dextran is from about 5 to about 30 mg of dextran per ml of strong anion exchange chromatography material.

14. 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 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 9 (AAV9), or adeno-associated virus serotype 10 (AAV10), or a variant thereof; Optionally, the adeno-associated virus capsid is a capsid of adeno-associated virus serotype 9 (AAV9) or a variant thereof.

15. 4. The chromatographic material has formula IV: 【Chemistry 4】 wherein the elution buffer of step (b) as defined in claim 1 and the elution buffers of steps (f) and (g) as defined in claim 6 comprise 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 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 9 (AAV9), or adeno-associated virus serotype 10 (AAV10), or a variant thereof; Optionally, the adeno-associated virus capsid is a capsid of adeno-associated virus serotype 9 (AAV9) or a variant thereof.

16. 4. The chromatographic material has formula IV: 【Chemistry 5】 and wherein the elution buffer of steps (II) and (III) as defined in claim 11 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 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 9 (AAV9), or adeno-associated virus serotype 10 (AAV10), or a variant thereof; Optionally, the adeno-associated virus capsid is a capsid of adeno-associated virus serotype 9 (AAV9) or a variant thereof.

17. 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 linking the ligand to the support, the surface extender comprising: 【Chemistry 6】 2. Use of an anion exchange chromatography material as defined by the formula: The elution conditions are a. adding a liquid sample to a chromatographic material, said liquid sample being at least 90% pure and containing at least 10 adeno-associated virus capsids; 12 adeno-associated virus capsids at a concentration of 50 / ml, of which at least 5%, e.g., 10%, of the adeno-associated virus capsids are adeno-associated virus capsids in which the genetic material is fully packaged; b. eluting the adeno-associated viral capsids in which the genetic material is not fully packaged and the adeno-associated viral capsids in which the genetic material is fully packaged from the chromatographic material by applying an elution buffer comprising a step gradient of increasing electrical conductivity, the step gradient starting from about 0 to about 5 mS / cm and increasing by about 0.5 to about 3 mS / cm per step, e.g., about 1 to about 2 mS / cm per step, e.g., about 1.2 to about 1.5 mS / cm per step, at least up to and including the electrical conductivity at which the adeno-associated viral capsids in which the genetic material is not fully packaged and the adeno-associated viral capsids in which the genetic material is fully packaged eluted from the chromatographic material; c. determining a first value of electrical conductivity or an electrical conductivity-related parameter suitable for eluting adeno-associated virus capsids in which genetic material is not fully packaged based on the elution profile obtained in step (b); d. determining a second value of electrical conductivity or electrical conductivity-related parameter suitable for eluting adeno-associated virus capsids in which genetic material is fully packaged based on the elution profile obtained in step (b); Use, as determined by performing

18. By carrying out steps (a) to (d) as defined in claim 17, e. adding the liquid sample to a chromatographic material, wherein the liquid sample has a purity of at least 90% and at least 10 adeno-associated virus capsids; 12 adeno-associated virus capsids at a concentration of about 10 ... f. eluting adeno-associated virus capsids in which genetic material is not fully packaged by applying an elution buffer having a first value of electrical conductivity or electrical conductivity-related parameter determined in step (c); g. eluting the adeno-associated virus capsids in which the genetic material is fully packaged by applying an elution buffer having a second value of the electrical conductivity or electrical conductivity-related parameter determined in step (d); by carrying out the steps of: separating adeno-associated viral capsids in which genetic material has been fully packaged from adeno-associated viral capsids in which genetic material has not been fully packaged; (i) the duration of step (f) is at least three times, such as four times, the duration of step (g); and / or (ii) The use according to claim 17, wherein the method comprises a step (f') between steps (f) and (g), wherein step (f') comprises applying an additional step elution and / or an increasing conductivity gradient between a first value and a second value of the conductivity or conductivity-related parameter, and wherein the duration of steps (f) and (f') is at least three times, such as four times, the duration of step (g).

19. the elution buffer of step (b) as defined in claim 17 and, when referring to claim 18, the elution buffers of steps (f) and (g) as defined in claim 18 also comprise 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 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 9 (AAV9), or adeno-associated virus serotype 10 (AAV10), or a variant thereof; Optionally, the adeno-associated virus capsid is a capsid of adeno-associated virus serotype 9 (AAV9) or a variant thereof.

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