Chromatographic materials, their use, and methods for separating adeno-associated capsids

Abstract

This disclosure relates to a chromatographic material comprising a support material in the form of a convection-based membrane structure containing nanofibers, wherein the support material is functionalized with anion-exchange ligands at a ligand density of <300 μmol / mL, and the chromatographic material comprises a linker that links the ligands to the support material, the linker comprising a linear backbone having a length of 2 to 16 atoms. Chromatographic devices comprising a holder containing a chromatographic material such as those disclosed herein are also provided. In addition, this disclosure relates to the use of the chromatographic materials or chromatographic devices disclosed herein for separating adeno-associated virus capsids in which the genetic material is fully packaged from adeno-associated virus capsids in which the genetic material is not fully packaged. Furthermore, the present disclosure provides a method for separating adeno-associated virus capsids in which the genetic material is fully packaged from adeno-associated virus capsids in which the genetic material is not fully packaged, comprising the steps of (a) adding a liquid sample containing adeno-associated virus capsid to a chromatographic material as disclosed herein, wherein the liquid sample has a purity of at least 90% and at least 10 12 The present invention provides a method comprising the steps of: (a) a chromatographic material comprising adeno-associated virus capsid at a concentration of adeno-associated virus capsid / ml, of which at least 10% are adeno-associated virus capsids with fully packaged genetic material; and (b) eluting the adeno-associated virus capsids with fully packaged genetic material from a chromatographic material, wherein the adeno-associated virus capsids eluted in step (b) are eluted into at least one eluate fraction, the eluate fraction comprising at least 50% of the fully packaged adeno-associated virus capsids present in the liquid sample added in step (a), and at least 60% of the adeno-associated virus capsids eluted in step (b) are adeno-associated virus capsids with fully packaged genetic material.

Description

[Technical Field]

[0001] This disclosure relates to the field of isolation of biological target compounds, particularly adeno-associated virus capsids. This disclosure covers chromatographic materials and their use for separating adeno-associated virus capsids with fully packaged genetic material from adeno-associated virus capsids with incompletely packaged genetic material, as well as methods for separating adeno-associated virus capsids with fully packaged genetic material from adeno-associated virus capsids with incompletely packaged genetic material. [Background technology]

[0002] Adeno-associated viruses (AAVs) are non-enveloped viruses that possess 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 a growing demand for the use of viral vectors for gene therapy. AAV vectors are one of the most attractive gene transfer tools for developing novel gene therapies for muscle diseases and other disorders.

[0003] To use AAV particles as vectors in therapy, it is necessary to purify the viral particles from cellular impurities such as DNA after transfection. Furthermore, since the therapeutic efficacy of AAV vectors depends on a high percentage of viral particles with the desired genetic material fully packaged, it is important to separate such fully packaged AAVs from empty and partially packaged AAV particles. WO2023285011A1 describes a method for separating fully packaged AAV particles from fully unpackaged AAV particles. However, there is a continuing need in the art for novel chromatographic materials and purification strategies to increase the speed and cost of the purification process. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] WO2023285011A1 [Patent Document 2] WO2015052460A1 [Patent Document 3] WO2015052465A1 [Non-patent literature]

[0005] [Non-Patent Document 1] www.cytivalifesciences.com [Non-Patent Document 2] 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 [Overview of the project] [Problems that the invention aims to solve]

[0006] The object of this disclosure is to provide a chromatographic material that enables faster separation of fully packaged adeno-associated virus capsids and fully unpackaged adeno-associated virus capsids with similar or improved resolution and purity compared to already known chromatographic materials. This is achieved by providing an optimized convection-based chromatographic material. [Means for solving the problem]

[0007] More specifically, the present disclosure relates to a chromatographic material comprising a support material in the form of a convection-based membrane structure containing nanofibers, wherein the support material is functionalized with anion-exchange ligands at a ligand density of <300 μmol / mL, and the chromatographic material comprises a linker that links the ligands to the support material, the linker comprising a linear backbone having a length of 2 to 16 atoms.

[0008] This disclosure also provides a chromatography device including a holder containing chromatography material as disclosed herein.

[0009] In addition, this disclosure relates to the use of chromatographic materials or chromatographic devices disclosed herein for separating adeno-associated virus capsids in which the genetic material is fully packaged from adeno-associated virus capsids in which the genetic material is not fully packaged.

[0010] Furthermore, this disclosure relates to a method for separating adeno-associated virus capsids in which the genetic material is fully packaged from adeno-associated virus capsids in which the genetic material is not fully packaged. a. A step of adding a liquid sample containing adeno-associated virus capsid to a chromatographic material as disclosed herein, wherein the liquid sample has a purity of at least 90% and at least 10 12 The process involves a concentration of adeno-associated virus capsids at a rate of adeno-associated virus capsid / ml, of which at least 10% are adeno-associated virus capsids in which the genetic material is fully packaged. b. The process of eluting adeno-associated virus capsids, in which the genetic material is fully packaged, from the chromatography material. comprising, in step (b), eluting an adeno-associated virus capsid that elutes in at least one eluate fraction, the eluate fraction comprising at least 50% of the fully packaged adeno-associated virus capsids present in the liquid sample added in step (a), and at least 60% of the adeno-associated virus capsids eluted in step (b) having the genetic material fully packaged, provides a method.

[0011] In particular, the present disclosure is directed to the isolation of adeno-associated virus capsids of adeno-associated virus serotypes 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, and 13 (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, and AAV13) or variants thereof.

[0012] Preferred embodiments of the present disclosure are described below in the detailed description and the dependent claims. It should be noted that the present disclosure relates to all possible combinations of the features recited in the claims.

[0013] These and other aspects of the present disclosure will now be described in more detail with reference to the accompanying drawings that illustrate embodiments of the invention.

Brief Description of the Drawings

[0014] [Figure 1] FIG. schematically shows a cross-section of an exemplary chromatography device comprising a chromatography material according to the present disclosure. [Figure 2] FIG. is a flowchart outlining the steps of a method for separating adeno-associated virus capsids with fully packaged genetic material from adeno-associated virus capsids with incompletely packaged genetic material according to the present disclosure. [Figure 3] FIG. is a chromatogram for the separation of full and empty capsids of AAV8 on different anion exchange prototypes as described in Example 1 herein. [Figure 4]This graph shows the UV260:280 ratio for the separation of complete and empty capsids of AAV8 in different chromatographic material prototypes, such as those described in Example 1 of this specification. [Figure 5] This is a chromatogram of the separation of complete and empty capsids of AAV5 in an anion exchange prototype as described in Example 1 of this specification. [Modes for carrying out the invention]

[0015] As shown in the figures, some features may be exaggerated for illustrative purposes and are therefore provided to illustrate the overall structure of the embodiments of this disclosure.

[0016] This disclosure provides a chromatography material 1 comprising a support material 2 in the form of a convection-based membrane structure containing nanofibers, as shown in Figure 1, wherein the support material is functionalized with an anion-exchange ligand 3 at a ligand density of <300 μmol / mL, and the chromatography material comprises a linker 4 that links the ligand to the support material, the linker comprising a linear backbone having a length of 2 to 16 atoms, thereby solving or at least mitigating problems associated with existing chromatography materials for the separation of fully packaged adeno-associated virus capsids from fully packaged adeno-associated virus capsids.

[0017] A notable advantage of the chromatography material disclosed herein is that it enables faster separation of fully packaged adeno-associated virus capsids and fully unpackaged adeno-associated virus capsids with similar or improved resolution and purity compared to chromatography materials already known.

[0018] The term "chromatographic material" is used herein to describe a type of separation matrix.

[0019] The term “separation matrix” is used herein to describe a material comprising a support material coupled with one or more ligands containing functional groups. The functional groups of the ligands bind to a compound, also referred herein, called the analyte, which is separated from a 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 material. The term “linker” may be used to describe such compounds, as further described below. In this specification, the term “support material” may be used interchangeably with the term “support.”

[0020] In this context, “ligand” is a molecule having known or unknown affinity for a given analyte and containing any functional group or scavenger immobilized on its surface, whereas “analyte” contains any specific binding partner to the ligand. The term “ligand” may be used herein interchangeably with the terms “specific binding molecule,” “specific binding partner,” “scavenger,” and “scavenger.” In this specification, a molecule in a liquid sample that interacts with a ligand is referred to as the “analyte.” The analyte of interest according to this disclosure is an adeno-associated virus capsid, more particularly, an adeno-associated virus capsid in which the genetic material is fully packaged or not fully packaged. Therefore, in this specification, the terms “analyte,” “adeno-associated virus capsid,” and “capsid” may be used interchangeably.

[0021] In this specification, the term "surface" means all external surfaces, and in the case of a porous support, includes the outer surface and the pore surface.

[0022] The chromatographic materials disclosed herein include a linker that links a ligand to a support material; i.e., coupling of the ligand to the support is achieved by introducing a linker between the support material and the ligand. The linker includes a linear backbone having a length of 2 to 16 atoms. The linear backbone includes at least two carbon atoms and may contain up to 16 atoms selected from heteroatoms containing carbon atoms and / or one or more oxygen, nitrogen, and / or sulfur atoms. One or more heteroatoms containing oxygen, nitrogen, and / or sulfur atoms may be incorporated into a functional group. Non-limiting examples of such functional groups are ethers, thioethers, amides, sulfonamides, ketones, and sulfones.

[0023] Optionally, the linker may contain one or more side chains bonded to the linear main chain. Each side chain may contain at most three atoms selected from heteroatoms including a carbon atom and / or one or more oxygen, nitrogen, and / or sulfur atoms. Non-limiting examples of such side chains are C1-C3 alkanes, hydroxyls, ethers, and amides.

[0024] The term “backbone” is intended to mean the main chemical structure of the linker. As mentioned above, a linear backbone has a length of 2 to 16 atoms, and the “length” of the linear backbone extends from the atom at one end of the linear backbone bonded to the supporting material to the atom at the other end of the linear backbone bonded to the ligand. It should be understood that both end atoms are included in the length of 2 to 16 atoms. The linear backbone is composed of carbon atoms and, optionally, heteroatoms. The 2 to 16 atoms of the linear backbone do not include any hydrogen atoms bonded to any carbon and / or any heteroatom of the backbone. Furthermore, the linear backbone is defined as not including side chains. However, one or more side chains may be optionally coupled to the backbone, as described in more detail above.

[0025] The term "heteroatom" has its traditional meaning in the field of chemistry, namely, an atom other than carbon or hydrogen.

[0026] Non-limiting examples of starting chemicals for generating such linkers include diepoxides such as epichlorohydrin, diglycidyl ether or 1,4-butanediol diglycidyl ether, allyl glycidyl ether, or divinyl sulfone. The coupling may be carried out according to any conventional covalent coupling method well known in the art, which can be easily carried out by those skilled in the art.

[0027] A non-limiting example of a suitable linker is one containing vinyl sulfone. Another non-limiting example is one containing vinyl sulfone and glycidol.

[0028] The support material for the chromatography material disclosed herein is in the form of a convection-based membrane structure containing nanofibers. Thus, the support material is a type of convection-based chromatography matrix. A convection-based chromatography matrix includes any matrix in which the application of a water pressure difference between the inflow and outflow of the matrix compels perfusion of the matrix, achieving substantial convective transport of substances into or out of the matrix. The convection-based matrix may be, for example, an adsorbent membrane, and the flow through such a material is convective rather than diffusive. The chromatography material, i.e., the stationary phase, has a high surface area for high binding capacity and macroporosity required for viruses to enter the matrix. When the mobile phase (i.e., a liquid sample or solution containing the biological target compound to be purified) is added to the convective stationary phase, there is convection of the mobile phase in the stationary phase so that the mobile phase comes into direct contact with the ligand in the matrix. Thus, the biological target compound in the mobile phase (here, the AAV vector) does not need to rely on diffusion to reach the ligand.

[0029] Accordingly, this disclosure enables chromatographic materials for the purification of AAV vectors that combine the high binding capacity traditionally associated with porous bead-based materials with the higher flow rates achievable with monolithic / membrane materials. The chromatographic materials can be made sufficiently porous so that the binding region is accessible by larger vectors, and appropriately short residence times can be achieved.

[0030] The diameters of different viruses range from 20 to 300 nm. AAV vectors typically have a diameter of about 25 nm.

[0031] Convection-based membrane structures may include a nonwoven web or matrix of polymer nanofibers. When in use, such nanofibers form a stationary phase containing multiple pores through which the mobile phase can permeate.

[0032] A nonwoven polymer matrix containing nanofibers is a mat of one or more polymer nanofibers, each fiber essentially randomly oriented; that is, it is not fabricated so that one or more fibers take on a specific pattern. Nonwoven polymer matrices are typically provided by known methods. In certain circumstances, a nonwoven matrix may consist of a single polymer nanofiber. Alternatively, a nonwoven matrix may contain two or more polymer nanofibers.

[0033] Polymer nanofibers can be electrospun polymer nanofibers. Such electrospun polymer nanofibers are well known to those skilled in the art.

[0034] Polymer nanofibers typically have an average diameter of 10 nm to 1000 nm. For some applications, polymer nanofibers with an average diameter of 200 nm to 800 nm or 200 nm to 400 nm may be suitable.

[0035] The length of polymer nanofibers is not particularly limited. Therefore, conventional processes, such as electrospinning, can produce polymer nanofibers hundreds of meters or even several kilometers in length. However, typically one or more polymer nanofibers have a length of up to 10 km, preferably 10 m to 10 km.

[0036] Nonwoven matrices typically have a surface area of ​​1–40 g / m², 5–25 g / m², 1–20 g / m², or 5–15 g / m².

[0037] Nonwoven matrices typically have a thickness of 5 to 120 μm.

[0038] Polymer nanofibers can be produced, for example, from cellulosic polymers selected from the group consisting of cellulose and partial derivatives of cellulose, particularly cellulose acetate or other cellulose esters, crosslinked cellulose, grafted cellulose, or ligand-coupled cellulose. In some cases, cellulose and cellulose acetate are preferred. Cellulose acetate can be readily formed into nanofibers, for example, by electrospinning, and can be readily converted to cellulose after electrospinning. Cellulose fiber chromatography (known as Fibro™ chromatography; Cytiva, Sweden) is an ultrafast chromatographic purification method for short process times and high productivity, utilizing high flow rates and high capacity of cellulose fibers.

[0039] Alternatively, polymer nanofibers can be made from synthetic polymers. Non-limiting examples of suitable polymers may be selected from polysulfones (e.g., polyethersulfones), polyamides, nylon, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, polypropylene, and polyethylene oxide, as well as mixtures thereof.

[0040] In some embodiments, the matrix includes one or more nanofibers formed from different polymers. Typical polymers are as defined above. For example, polymer nanofibers may be made from a combination of cellulosic polymers and synthetic polymers.

[0041] Before functionalization with ligands, the nanofibers may be optionally physically modified and fused together at their intersections by thermal or chemical means and / or by pressurizing the polymer nonwoven matrix. This can improve the structural stability of the matrix. The thickness and / or porosity of the resulting matrix can also be altered by varying the pressurizing and heating conditions.

[0042] In a non-limiting example, polymer nanofibers can be made from cellulosic polymers reinforced with synthetic polymers.

[0043] Convection-based membrane structures can include a single membrane, multiple membranes, or filters. The use of multiple nonwoven matrices / sheets allows for the preparation of thicker materials that may have greater adsorption capacity. Thus, functionalized polymer matrices are typically formed by preparing two or more nonwoven matrices, one laminated on top of the other, each matrix containing one or more polymer nanofibers, and simultaneously heating and pressurizing the matrix laminate to fuse the contact points between the nanofibers of adjacent matrices / sheets.

[0044] In the case of cellulose matrices, this is typically formed by preparing two or more nonwoven matrices, one laminated on top of the other, each matrix containing one or more cellulose acetate nanofibers, and simultaneously heating and pressurizing the laminate of sheets to fuse the contact points between the nanofibers of adjacent matrices / sheets. Polymer matrices may consist solely of cellulose. Alternatively, the matrices may contain cellulose combined with one or more polymer nanofibers. Preferred processing conditions for pressurizing and heating the polymer nanofiber / nonwoven sheets can be found, for example, in WO2015052460A1 and WO2015052465A1.

[0045] The support material may have an average flow pore diameter of 0.1 to 2.0 μm, for example, 0.1 to 1.8 μm, 0.1 to 1.6 μm, 0.1 to 1.4 μm, 0.1 to 1.2 μm, 0.1 to 1.0 μm, 0.1 to 0.8 μm, 0.1 to 0.6 μm, 0.1 to 0.4 μm, 0.1 to 0.2 μm, 0.2 to 2.0 μm, 0.4 to 2.0 μm, 0.6 to 2.0 μm, 0.8 to 2.0 μm, 1.0 to 2.0 μm, 1.2 to 2.0 μm, 1.4 to 2.0 μm, 1.6 to 2.0 μm, 1.8 to 2.0 μm, or 0.5 to 1.5 μm. The average flow pore (MFP) diameter is an indicator of the flow characteristics of a material and is measured by capillary flow porometry based on the discharge of a wet liquid with known surface tension from sample pores by applying a gas at increasing pressure. A larger MFP diameter indicates a larger flow rate of liquid through the material at a given pressure. The average flow pore diameter is calculated from the point at which 50% of the flow rate passes through the sample. Therefore, the average flow pore diameter corresponds to the pore diameter calculated at the pressure where the wet and semi-dry curves intersect. In an alternative definition, the average flow pore diameter of a supporting material can be considered as the effective pore diameter, defined as the size of the largest sphere that can pass through the pore.

[0046] As described above, the support material is functionalized with an anion exchange ligand.

[0047] The density of anion-exchange ligands is <300 μg of ligand per mL of chromatographic material, for example, about 50–250 μmol / mL, about 70–220 μmol / mL, about 90–200 μmol / mL, or for example, about 250 μmol / mL, about 200 μmol / mL, about 190 μmol / mL, about 180 μmol / mL, about 170 μmol / mL, about 160 μmol / mL, about 150 μmol / mL, about 140 μmol / mL, about 130 μmol / mL, about 120 μmol / mL, about 110 μmol / mL, about 100 μmol / mL, about 90 μmol / mL, about 80 μmol / mL, or less. In this specification, the term "about" is intended to cover a range of densities including ±5% of the specified value. The density of ligands in chromatographic material can be determined by titration methods for determining the number of ligand moieties in the functionalized material. Those skilled in the art know the appropriate method.

[0048] The anion exchange ligand may contain a quaternary amine group. The ligand is of formula I:

[0049] [ka]

[0050] (In the formula, R1 is selected from H and C1-C3 alkyl groups, and R2 and R3 are independently selected from H, C1-C3 alkyl groups, CH2OH, and CH2CHOHCH3.) It can be defined by:

[0051] As a non-restrictive example, R1, R2, and R3 are each CH3.

[0052] In another non-limiting example, R1 and R2 are ethyl, and R3 is methyl.

[0053] In yet another, non-restrictive example, R1 and R2 are methyl, and R3 is CH2CHOHCH3.

[0054] The wavy portion represents the supporting material containing the linker. Ligands can bind to carbon atoms of the linker.

[0055] There are currently available chromatographic materials containing ligands defined by Formula I (wherein R1, R2, and R3 are each CH3), such as the chromatographic material available under the name Capto® Q, provided by Cytiva, Sweden (www.cytivalifesciences.com). Capto® Q further contains dextran as a surface extender and is a chromatographic medium for high-resolution polishing steps in industrial purification processes for the purification of monoclonal antibodies, for example. Recently, it has also been proposed for the separation of fully packaged adeno-associated capsids from fully packaged adeno-associated capsids (see WO2023285011A1). Capto® Q comprises a support material in the form of substantially spherical particles or beads that have homogeneous porosity throughout their overall volume and are at least partially permeable to adeno-associated virus capsids throughout their overall volume.

[0056] In contrast, the present disclosure provides a chromatographic material comprising a convection-based membrane structure containing nanofibers as a support material. As mentioned above, such a convection-based support material enables faster separation of fully packaged adeno-associated virus capsids from fully packaged adeno-associated virus capsids with similar resolution and purity compared to already known chromatographic materials such as Capto® Q.

[0057] A further differentiating feature of the chromatographic material disclosed herein is that it contains a linker comprising a linear backbone having a length of only 2 to 16 atoms. This linker is much shorter than that of a polymer dextran surface extender. The results described in Example 1 herein demonstrate that a convection-based chromatographic material having such a short linker provides the same resolution and purity of isolated adeno-associated virus capsids of serotypes AAV5 and AAV8 as shown in WO2023285011A1 for a resin bead chromatographic material containing a surface extender. This is surprising considering that WO2023285011A1 showed that beads containing a surface extender resulted in improved separation of complete and empty AAV capsids compared to the same chromatographic material without a surface extender.

[0058] A linker is defined as described in detail elsewhere in this specification.

[0059] Non-limiting examples of chromatographic materials provided herein include a support material in the form of a nonwoven web of cellulose acetate nanofibers, a ligand defined by formula I (wherein R1, R2, and R3 are each CH3), and a linker containing a vinyl sulfone. The ligand density may be about 90–100 μmol / mL, for example, about 90, 95, 100, 105, or 110 μmol / mL.

[0060] Another non-limiting example of a chromatography material according to this disclosure includes a support material in the form of a nonwoven web of cellulose acetate nanofibers, a ligand defined by formula I (wherein R1, R2, and R3 each being CH3), and a linker comprising vinyl sulfone and glycidol. The ligand density may be about 110–130 μmol / mL, for example, about 110, 115, 118, 120, 125, or 130 μmol / mL.

[0061] Anion exchange ligands can be substituted with formula II:

[0062] [ka]

[0063] (In the formula, X is independently of H, OH and C for each occurrence) 1~3 Selected from the base, R1, R2, R3, and R4 are independently H and C 1~3 Selected from the base, The C3 group is linear or branched. C 1~3 The base is HR, OC 1~2 SC 1~2 , comprising a group independently selected from NH, NHR, and NR2, R is H and C 1~3 (Selected from the base) It can be defined by:

[0064] More specifically, the ligand defined by formula II may be selected from the group consisting of N,N,N'-triethylethylenediamine, diethylenetriamine, N,N'-dimethylethylenediamine, N-methylethylenediamine, 1,3-diaminopropane, 1,3-diamino-2-hydroxypropane, 2-methyl-1,3-propanediamine, and N,N-diethylethylenediamine.

[0065] As a non-restrictive example, the ligand defined by formula II is N,N-diethylethylenediamine.

[0066] Supporting materials and linkers are defined as described in detail elsewhere in this specification.

[0067] Therefore, non-limiting examples of chromatographic materials according to this disclosure include a support material in the form of a nonwoven web of cellulose acetate nanofibers, a ligand which is N,N-diethylethylenediamine, and a linker which contains vinyl sulfone. The ligand density may be about 150 to 210 μmol / mL, for example, about 150, 160, 162, 165, 170, 175, 180, 185, 190, 195, 200, 203, 205, or 210 μmol / mL.

[0068] Chromatographic materials may be used in a chromatography system or manually with a syringe. All devices may be used with a peristaltic pump, a diaphragm pump, or positive gas pressure. Chromatographic materials may be contained in any type of separation device that allows for a uniform flow distribution on the chromatographic material. The term “separation device” should be understood to have its conventional meaning in the field of bioprocessing and to encompass any type of separation device that can separate and purify compounds from fluids containing by-products from the generation of compounds and is suitable for doing so. Separation devices may include separation matrices as further defined elsewhere in this specification.

[0069] In this specification, the separation device may be alternatively referred to as a chromatography device 5, as schematically shown in Figure 1. More specifically, the chromatography device 5 includes a holder containing a chromatography material, as disclosed herein. Non-limiting examples of the holder are capsules and cartridges. The chromatography device may further include one or more spacers positioned between layers of a convection-based membrane structure. Non-limiting examples of spacer materials are frits, nonwoven materials, and woven materials.

[0070] This disclosure relates to a method 100 for separating adeno-associated virus capsids in which the genetic material is fully packaged from adeno-associated virus capsids in which the genetic material is not fully packaged, as schematically shown in the flowchart of Figure 2. a. Step 110 of adding a liquid sample containing adeno-associated virus capsid 6 (as shown in Figure 1) to a chromatographic material as disclosed in detail elsewhere in this specification, The liquid sample has a purity of at least 90% and at least 10 12 The process involves a concentration of adeno-associated virus capsids at a rate of adeno-associated virus capsid / ml, of which at least 10% are adeno-associated virus capsids in which the genetic material is fully packaged. b. Step 120: Elution of adeno-associated virus capsids, in which the genetic material is fully packaged, from the chromatography material. The present invention provides a method comprising, wherein the adeno-associated virus capsid eluted in step (b) is eluted into at least one elution fraction, which comprises at least 50% of the fully packaged adeno-associated virus capsid present in the liquid sample added in step (a), and at least 60% of the adeno-associated virus capsid eluted in step (b) is fully packaged genetic material.

[0071] The term "viral particle" is used herein to refer to a complete infectious viral particle. It comprises a core containing the viral genome (i.e., viral genome) in either the form of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), the core being surrounded by a morphologically defined shell. The shell is called a capsid. Together, the capsid and the encapsulated viral genome constitute a so-called nucleocapsid. The nucleocapsid of some viruses is surrounded by a lipoprotein bilayer envelope. In the field of bioprocessing, for the purpose of producing viral vectors for various applications such as therapeutics, the genome of a viral particle is modified to include a gene insert containing the genetic material of interest. The modified viral particle is allowed to infect host cells in a cell culture, the viral particle proliferates within the host cells, and thereafter the viral particle is purified from the cell culture by any means of isolation and purification. In this specification, viral particles isolated from a cell culture by the methods disclosed herein may alternatively be referred to as "target molecules" or "targets." The term "viral particle" is intended to mean a certain type of viral particle, and it should be understood that the singular form of this term may encompass a number of individual viral particles. In this specification, the term "viral particle" may be used interchangeably with the terms "vector" and "capsid," respectively, as further defined below.

[0072] The term “vector” is used herein to refer to a viral particle, usually a recombinant viral particle, intended for use in achieving gene transfer to modify a particular type of cell or tissue. Viral particles may be engineered, for example, to provide a vector that expresses a therapeutic gene. 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 (AAVs), baculoviruses, and herpes simplex viruses. In this specification, the terms “vector” may be used interchangeably with the terms “viral particle” and “capsid,” respectively.

[0073] The term "capsid" refers to the outer shell of a viral particle. The capsid surrounds the core of the viral particle and should typically contain the viral genome. Modified (recombinant) capsids, such as those produced in upstream manufacturing processes, are expected to contain the complete viral genome, which contains the genetic material intended for one or more uses, e.g., for various therapeutic applications. However, due to low packaging efficiency, constructed capsids may not necessarily contain genetic material, or they may simply encase cleaved gene fragments, resulting in so-called empty capsids and partially filled capsids, respectively. These capsids lack therapeutic function but still compete for binding to receptors during cell-mediated processes. This can reduce overall therapeutic efficacy and trigger undesirable immune responses. As a result, tracking these capsids throughout the generation process is crucial to ensure consistent product quality and appropriate administration response (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). In up to 20-30% of artificially generated virus particle populations in cell cultures, the capsids are only partially filled with genetic material. Furthermore, in as much as 98% of artificially generated virus particles, the capsids contain no part of the viral genome at all, i.e., they are empty. However, generally between 80% and 90% of artificially generated virus particles have empty capsids, and in the best-case scenario, currently only 50% empty capsids are achieved.

[0074] In this specification, 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.

[0075] The term "target genetic material" is intended to mean, in the field of bioprocessing, genetic material that is suitable and valuable for purification so that it can be used for various applications, including therapeutic purposes, but is not limited to, that can be produced by viral replication. In a non-limiting example, target genetic material may include therapeutically appropriate genetic material such as therapeutically appropriate nucleotide sequences.

[0076] The term "capsid in which genetic material is completely packaged" refers precisely to a capsid that is produced (by the host cell), or in other words, A capsid containing the complete viral genome, or in other words, A capsid containing 100% of the viral genome, or in other words, Capsid containing a functional viral genome This term is used herein to represent.

[0077] The viral genome includes a gene insert containing the genetic material of interest as defined elsewhere in this specification.

[0078] A capsid containing the complete viral genome may, for the purposes of this specification, be referred to as a “complete capsid” or “fully packaged capsid.” The terms “complete capsid,” “fully packaged capsid,” and “capsid with genetic material fully packaged” may be used interchangeably throughout this document.

[0079] The term "capsid in which genetic material is not fully packaged" refers to a capsid that was not properly produced (by the host cell), or in other words, A capsid that does not contain the complete viral genome, or in other words, The capsid contains less than 100% of the viral genome. This term is used herein to represent.

[0080] Capsids that do not completely contain genetic material are either partially filled with genetic material or not filled at all with genetic material.

[0081] The term "capsid in which genetic material is not fully packaged" encompasses the terms "partially filled capsid" and "empty capsid," as defined below.

[0082] A "partially filled capsid" is 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, A capsid containing an incomplete viral genome, or in other words, A capsid containing a missing viral genome, or in other words, Capsids containing more than 0% and less than 100% of the complete viral genome, for example, about 1% to about 99% of the complete viral genome, 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%. As defined herein, partially filled capsids are improperly produced capsids, and therefore it is desirable to separate and remove as many partially filled capsids as possible from the capsid population before using the population for its intended purpose, such as therapeutic use. In this specification, partially filled capsids may be alternatively referred to as “intermediate capsids.”

[0083] An "empty capsid" is a capsid that does not contain any part of the viral genome, that is, a capsid containing 0% of the viral genome, or in other words, An empty capsid is defined herein as a capsid that is not filled with any genetic material. Therefore, an empty capsid does not contain the genetic material of interest. For this reason, it is desirable to enrich the capsid population with complete capsids, i.e., to increase the percentage of complete capsids at the expense of the percentage of partially filled and empty capsids, by separating and removing as many empty capsids as possible from the capsid population before using the capsid population for its intended purpose, e.g., therapeutic use (this may be required, e.g., by clinical regulations).

[0084] The percentage of complete and empty capsids in a capsid population can be estimated or analyzed by several methods known in the art. Some of these methods are briefly described below.

[0085] 1: The A260:280 ratio in the chromatogram will give an estimate of the percentage of complete capsid present at the peak (a ratio of 1–1.5 indicates enrichment of complete capsid, while a ratio of 0.5–0.7 indicates mainly empty capsid).

[0086] 2. qPCR:ELISA ratio. qPCR quantifies the viral genome, while ELISA quantifies total viral particles. The ratio of two assays with variability will likely be less precise and uncertain. Orthogonal analysis is necessary for confirmation (see 3, 4, or 5 below).

[0087] 3. Analytical anion exchange to separate complete and empty capsids (A260:280 ratio and peak area to calculate percentage). Accuracy depends on peak definition.

[0088] 4. Analytical ultracentrifugation (AUC). Detects and quantifies particles of different densities (corresponding to complete, partially filled, and empty capsids). This is currently known as the "optimal criterion" in the art. However, ultracentrifugation is not scalable and is therefore unsuitable for the analysis of large batches of capsids.

[0089] 5. Transmission electron microscopy (TEM). Image analysis to count particles (complete, partially filled, and empty capsids). May introduce artifacts from sample preparation.

[0090] Some methods for estimating or analyzing the percentage of complete and empty capsids in a capsid population are described in more detail in Xiaotong Fu et al., *Analytical Strategies for Quantification of Adeno-Associated Virus Empty Capsids to Support Process Development*, *Human gene therapy methods*, 2019, 30(4):144–152, which are incorporated herein by reference.

[0091] As used herein, the term “liquid sample” 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 purified at least partially by any means of separation and purification.

[0092] The term "eluate" is used in its conventional sense in this field, namely, the portion of a liquid sample that is loaded onto a chromatographic material, bound to the chromatographic material, and recovered by elution from the chromatographic material.

[0093] In the method shown in Figure 2, the flow rate used depends on the dimensions of the stationary phase (i.e., the chromatographic material or the chromatographic device containing the chromatographic material) and the selected residence time. The feasible residence time for adeno-associated virus capsids with fully packaged genetic material in the convection-based chromatographic material disclosed herein is approximately 0.1 seconds to 2 minutes. The equivalent flow rate in a 0.4 ml laboratory-scale unit is approximately 3000 to 2 cm / h. In a 2.4 liter unit, the maximum feasible flow rate on the stationary phase is approximately 850 cm / h.

[0094] As will be understood by those skilled in the art, the objective is to bind adeno-associated virus capsids with fully packaged genetic material to an anion-exchange ligand; therefore, step (a) of the method, i.e., the step of adding the liquid sample to the chromatographic material, is performed under conditions that enable such binding. In contrast, adeno-associated virus capsids that are not fully packaged genetic material either do not bind to the anion-exchange ligand or bind to the anion-exchange ligand to a much lesser extent than fully packaged capsids. Thus, the unpackaged capsids exit the chromatographic material before the fully packaged capsids, typically during flow-through, i.e., without the application of elution conditions.

[0095] Steps (a) and (b) of the methods disclosed above may include applying a buffer having a pH of about 6.0 to about 10.5, for example, about 7.0 to about 10.0, for example, 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. In a non-limiting example, as described in Example 1 below, a pH of about 9 may be applied to a chromatographic material containing a ligand defined by formula I or formula II.

[0096] The buffer solution is appropriately selected from buffer solutions commonly recommended for anion exchange chromatography and may include, for example, tris(hydroxymethyl)aminomethane (i.e., Tris), 1,3-bis(tris(hydroxymethyl)methylamino)propane (i.e., bis-trispropane), triethanolamine, N-methyldiethanolamine, diethanolamine, 1,3-diaminopropane, or ethanolamine. Those skilled in the art can select a suitable concentration for any one of the buffer solutions listed above.

[0097] In the method disclosed above, step (b) may include applying a buffer solution, optionally one of the buffer solutions mentioned above, which buffer solution includes a compound that improves the separation between capsids in which the genetic material is fully packaged and capsids in which the genetic material is not fully packaged. This compound may or may not be present in the buffer solution applied in step (a). Without being bound by theory, such a compound may improve the separation, for example, by affecting the interaction between the capsid and the ligand or the interaction between capsids. The compound that improves the separation may be selected, for example, from carbohydrates, divalent metal ions, and surfactants.

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

[0099] When the compound that improves the separation is a divalent metal ion, it may be selected, for example, from Mg 2+ 、Fe 2+ 、and Mn 2+ and may be present in the form of a salt, optionally in combination with, for example, chloride ions or sulfate ions. A non-limiting example of a metal salt suitable for inclusion in the buffer solution of step (b) is MgCl2. Non-limiting examples of suitable concentrations of MgCl2 are from about 0.5 to about 30 mM of MgCl2, such as about 1 to about 20 mM, such as about 2 to about 10 mM, or about 0.5, 1.0, 1.5, 2.0, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 mM of MgCl2.

[0100] When the compound that improves the separation is a surfactant, it may be selected, for example, from poloxamers such as poloxamer 188 or Pluronic (trademark) F68, and polysorbates such as Tween 20 or Tween 80.

[0101] In the above method, step (b) may include the step of applying a buffer, optionally one of the buffers mentioned above, the buffer containing a compound that can help elute the capsid bound to the chromatographic material. This compound is not present in the buffer applied in step (a). Non-limiting examples of such compounds are salts such as salts of monovalent metal ions. More specifically, the salt may be a cosmotropic salt. A salt in an aqueous solvent is defined as cosmotropic (order-making) if it contributes to the stability and structure of water-water interactions. In contrast, chaotropic (disorder-making) salts have the opposite effect of disrupting the water structure, increasing the solubility of nonpolar solvent particles, and destabilizing solute aggregates. Cosmotropes allow water molecules to interact well, which in fact stabilizes intramolecular interactions in macromolecules such as proteins (Moelbert S et al.). For example, the scale can be established by referring to the Hofmeister series or synergistic permutations, which is a classification of ions in order of their ability to salt out or dissolve proteins (Hyde A et al.).

[0102] More specifically, cosmotropic salts are (i)CO3 2- SO4 2- , S2O3 2- H2PO4 - HPO4 2- acetic acid - , citric acid - , and Cl - An anion selected from the group consisting of (ii) NH4 + , K + na + , and Li +It may contain a cation selected from the group consisting of the following. 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, for example, 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 enumerated in (i) and cations as enumerated in (ii) may be used alternatively to elute the capsid. Non-limiting examples are NaCl, LiCl, KCl, or other equivalent metal salts suitable for use in salt elution, as are well known in the art. A non-limiting example of a suitable concentration of NaCl includes NaCl of about 5 mM to about 2 M, for example, NaCl of 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. Furthermore, step (b) may include applying a gradient of such compounds to improve the elution of adeno-associated virus capsids, in which the genetic material is fully packaged, from the chromatographic material. Such a gradient may be a linear gradient, a stepped gradient, or a combination thereof.

[0103] A non-limiting example of a suitable buffer solution to be applied in step (b) may include 20 mM bis-trispropane (BTP), pH 9, 2 mM MgCl2, and 250 mM sodium acetate.

[0104] In the methods disclosed herein, the chromatographic materials referred to in steps (a) and (b) of the method may, advantageously, be polishing chromatographic materials, meaning that the chromatographic materials are applied in the polishing step.

[0105] In the context of liquid chromatography, the term "polishing step" refers to the final purification step in which trace impurities are removed, leaving an active and safe product. The impurities removed during the polishing step are often conformational isomers of the target molecule, i.e., forms of the target molecule with a specific molecular conformation, or pseudo-leakage products. The polishing step may alternatively be called a "secondary purification step."

[0106] Furthermore, the liquid sample added in step (a) of the method disclosed herein for separating adeno-associated virus capsids with fully packaged genetic material from adeno-associated virus capsids with unpacked genetic material may, advantageously, be a pre-purified liquid sample.

[0107] The method disclosed herein may include a step (a1) of pre-purifying the adeno-associated virus capsid by separating it from adeno-associated virus capsid-containing cell culture harvest products, thereby obtaining a pre-purified liquid sample containing the adeno-associated virus capsid, and then adding the pre-purified liquid sample containing the adeno-associated virus capsid to a chromatography material according to step (a) of the method.

[0108] Such a pre-purification step (a1) may alternatively be called a “capture step,” and in the context of liquid chromatography, it refers to the initial step of the separation procedure. Most commonly, the capture step includes clarification (e.g., by filtration, centrifugation, or precipitation), and usually also includes concentration and / or stabilization of the sample, as well as significant purification from soluble impurities by applying chromatography, for example, after clarification, concentration, and stabilization of the sample. Intermediate purification may follow the capture step, which further reduces the residual amounts of impurities such as host cell proteins, DNA, viruses, endotoxins, nutrients, antifoaming agents, and antibiotics, as well as product-related impurities such as aggregates, misfolded species, and aggregates.

[0109] Such pre-purification steps may include subjecting adeno-associated virus capsid-containing cell culture harvests 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, using Capto® Core 400 chromatography material (Cytiva, Sweden), which combines capsid flow-through and binding of impurities to the chromatography material. (iv) TFF, followed by ion exchange chromatography, and (v) TFF, followed by ion exchange chromatography and Capto Core.

[0110] Non-limiting examples of chromatographic materials suitable for application in the pre-purification process include affinity chromatography materials, ion exchange chromatography materials, and size exclusion chromatography materials, respectively. 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. Chromatographic materials may also be functionalized with ion exchange groups, affinity peptide / protein-based ligands, hydrophobic interaction ligands, IMAC ligands, or DNA-based ligands such as Oligo dT.

[0111] In this specification, the term “cell culture” means a culture of cells or a group of cultured cells, and the cells may be any type of cell, such as bacterial cells, viral cells, fungal cells, insect cells, or mammalian cells. A cell culture may not be clarified, i.e., it may contain cells, or it may be cell-depleted, i.e., it may contain few or no cells, but may contain biomolecules released from the cells before the cells were removed. Furthermore, an unclarified cell culture may contain intact cells, destroyed cells, cell homogenates, and / or cell lysates.

[0112] The term “cell culture harvest” is used herein to refer to a cell culture harvested and removed from a container or apparatus in which cells were cultured.

[0113] Non-limiting examples of separation devices suitable for use in capture or pre-purification steps as described herein include filtration devices, chromatography columns, and membrane devices. Chromatography columns suitable for use in capture steps may, for example, be packed with affinity chromatography materials, ion exchange chromatography materials, mixed-mode chromatography materials, or hydrophobic interaction chromatography materials.

[0114] The methods disclosed herein may include the step of isolating the adeno-associated virus 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 10 (AAV10), adeno-associated virus serotype 11 (AAV11), adeno-associated virus serotype 12 (AAV12), or adeno-associated virus serotype 13 (AAV13), or variants thereof.

[0115] The term “variant” in relation to adeno-associated virus (AAV) serotypes 1, 2, 3, 4, 5, 6, 7, 8, or 10 listed above is intended to mean a modified or engineered AAV whose capsid structure has been altered, for example, toward a specific target organ, in order to improve clinical performance. As a non-limiting example, an AAV8 variant would include the capsid portion of AAV8 and may further include the capsid portions of other AAV serotypes other than AAV8, such as AAV5. However, an AAV8 variant as referred to herein must retain a significant structural similarity to an unmodified AAV8 capsid, for example, retaining at least 50%, e.g., 60%, 70%, 80%, or 90% of the outer surface structure of an unmodified AAV8 capsid. This also applies to variants of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, or 13, compared to, respectively, unmodified AAV serotypes 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, or 13. Furthermore, as a non-limiting example, in the context of the purification or isolation of AAV8 variants, “variant” is defined herein as an adeno-associated virus having functionally equivalent binding ability to the ligand of a specified chromatographic material compared to the binding ability of the original AAV8 to the specified chromatographic material. This also applies to variants of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, or 13, compared to, respectively, the original AAV serotypes 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, or 13. The specified chromatographic material may be a potent or partially potent anion exchange chromatographic material, for example, as disclosed in more detail elsewhere herein. Adeno-associated virus variants can be obtained, for example, through spontaneous mutations in one or more nucleotides of the adeno-associated virus genome, or through engineered modifications (i.e., obtained through human interaction).

[0116] In particular, the adeno-associated virus capsid may be a capsid selected from the group consisting of serotypes AAV2, AAV5, and AAV8, or any one variant of the said serotypes.

[0117] A non-limiting example of the method disclosed herein includes adding the liquid sample in step (a) to a chromatographic material comprising a linker comprising a ligand defined by formula I (wherein R1, R2, and R3 each being CH3) and a vinyl sulfone, and optionally further comprising glycidol, and eluting the capsid in step (b) using an elution buffer comprising magnesium chloride and / or sodium acetate. Optionally, this non-limiting example of the method is for separating the capsids of serotypes AAV2, AAV5, AAV8 or their variants.

[0118] Another non-limiting example of the method disclosed herein includes adding the liquid sample in step (a) to a chromatographic material comprising a ligand containing N,N-diethylethylenediamine and a linker containing vinyl sulfone, and eluting the capsid in step (b) using an elution buffer containing magnesium chloride and / or sodium acetate. Optionally, this non-limiting example of the method is for separating capsids of serotypes AAV2, AAV5, AAV8 or their variants.

[0119] This disclosure further provides the use of chromatographic materials as disclosed elsewhere herein, or chromatographic devices as disclosed elsewhere herein, for separating adeno-associated virus capsids in which the genetic material is fully packaged from adeno-associated virus capsids in which the genetic material is not fully packaged.

[0120] In a non-limiting example, the present disclosure provides the use of a chromatographic material comprising a ligand defined by formula I (wherein each of R1, R2, and R3 is CH3) and a linker comprising a vinyl sulfone, and optionally further comprising glycidol. The aforementioned use may be intended for the isolation of adeno-associated virus capsids selected from 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 10 (AAV10), adeno-associated virus serotype 11 (AAV11), adeno-associated virus serotype 12 (AAV12), and adeno-associated virus serotype 13 (AAV13), or their variants. Optionally, this non-limiting example of use is for separating capsids of serotypes AAV2, AAV5, AAV8, or their variants. Optionally, such use may include applying an elution buffer containing magnesium chloride and / or sodium acetate.

[0121] To give another non-limiting example, the present disclosure provides the use of a chromatography material comprising a ligand containing N,N-diethylethylenediamine and a linker containing vinyl sulfone. The aforementioned use may be intended for the isolation of adeno-associated virus capsids selected from 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 10 (AAV10), adeno-associated virus serotype 11 (AAV11), adeno-associated virus serotype 12 (AAV12), and adeno-associated virus serotype 13 (AAV13), or their variants. Optionally, this non-limiting example of use is for separating capsids of serotypes AAV2, AAV5, AAV8, or their variants. Optionally, such use may include applying an elution buffer containing magnesium chloride and / or sodium acetate.

[0122] Adeno-associated virus capsids, fully packaged with genetic material obtained by the methods disclosed herein and by the use of chromatography materials or chromatography devices disclosed herein, may be subjected to subsequent steps of concentration to a pharmaceutically appropriate dose, replacement of elution buffer with a pharmaceutically acceptable buffer, and / or sterilization, thereby obtaining a pharmaceutical composition comprising adeno-associated virus capsid. Such pharmaceutical compositions may be for therapeutic use, optionally, for gene therapy use. The pharmaceutical compositions may be administered to a subject in a manner for preventing or treating organ- or tissue-related diseases or disorders in the subject.

[0123] Those skilled in the art will understand that the pharmaceutically appropriate dose depends on various factors, including, but not limited to, the disease or disorder being treated and the body weight and condition of the subject being treated with the pharmaceutical composition. Pharmaceutically acceptable buffers are well known in the art and can be readily selected by those skilled in the art.

[0124] This disclosure is not limited to the exemplary embodiments described below, and it should be understood that several possible modifications of this disclosure are possible within the scope of the following claims. In the claims, any reference numerals placed between parentheses shall not be construed as limiting the claims. The use of the verb "to comprise" and its conjugations shall not exclude the existence of elements or processes other than those described. The articles "a" or "an" preceding an element shall not exclude the existence of multiple such elements. [Examples]

[0125] (Example 1) Chromatographic material prototypes were prepared by functionalizing a support material containing a nonwoven web of cellulose acetate nanofibers with anionic ligands containing an N,N-diethylethylenediamine group (hereinafter referred to as "DAX") or a quaternary amine group (hereinafter referred to as "Q"), using an activated linker containing or not containing glycidol ("G") (hereinafter referred to as "non-glycidol" or "NG") (Table 1).

[0126] [Table 1]

[0127] Preparation of Fibro-NG-DAX(a): Fibro-VS(NG) preparation: A fibrous cellulose acetate (CA) sheet was inserted into the reactor with gauze in between. The sheet was washed with MQ-water (3 × 5 L, 15 minutes per wash). The reactor was drained, the temperature was raised to 30°C, and KOH reaction solution (309 g in 33% EtOH in 5500 mL) was added. The solution was circulated for 2 hours. The reactor was drained, and the sheet was washed with MQ-water (4 × 5 L) and acetone (2 × 5 L). The reactor was drained, and the sheet was dried overnight. The reactor was then filled with 3 L of sodium carbonate buffer (306.6 g of Na2CO3 in 2960 mL of MQ-water) and acetonitrile (1224 mL). The solution was cooled to 15°C and then pumped into the reaction chamber. Divinyl sulfone (DVS) (945 mL) was added directly to the reaction chamber. Circulation was restarted, and the reaction was allowed to proceed at room temperature for 6 hours. The reaction mixture was drained, and the sheet was washed with 1:1 acetone / MQ-water (4 x 5 L), followed by MQ-water (4 x 5 L).

[0128] DAX coupling: The sheets were placed in food boxes and washed with MQ-water (4 × 100 mL, 20 minutes) on a shaking table (84 rpm). 25 mL of MQ-water and 2 mL of DAX were added to each box. The boxes were placed on a heated shaking table (45°C, 75 rpm) for 23 hours. The reaction solution was decanted and the sheets were washed with MQ-water (6 × 100 mL, 20 minutes).

[0129] Inactivation: Thioglycerol (15.2 mL) was added to Tris buffer (600 mL, Tris 0.1 M, EDTA 0.001 M, pH 10) to adjust the pH to 8.6. This solution (153 mL) was added to a food box, the reaction mixture was sealed, and the box was placed on a shaking table (84 rpm) at room temperature for 16 hours. The reaction solution was decanted, and the sheet was washed with 20% EtOH (3 × 100 mL, 20 min) and MQ-water (3 × 100 mL, 20 min). The titration showed an ion volume / membrane mL of 162 μmol.

[0130] Preparation of Fibro-NG-DAX(b): Fibro-NG-DAX(b) was prepared in the same manner as Fibro-NG-DAX(a), except that coupling was performed at 2.25 mL per membrane sheet, resulting in a material with an ion capacity of 203 μmol / molecular-weight.

[0131] Preparation of Fibro-GQ: Preparation of Fibro-Glycidol-Allyl-VS: Fibro-CA laser-cut discs (32 mm, 12 pieces) were washed with MQ-water (4 × 100 mL, 10 min) in a food box on a shaking table. The discs were placed between two chemically inert nets and wound onto a chemically inert plastic roll. The two rolls were placed in a beaker (600 mL) and a magnetic stirring bar was added to the center. A glass stopper was used to stop the rolls from rotating during stirring. KOH (0.5 M, 150 mL) was added to the beaker and the solution was stirred for 10 minutes. Glycidol (40 mL) was added to the beaker and the reaction mixture was stirred for 3 hours. The reaction solution was decanted and the rolls were washed with MQ-water (4 × 200 mL, 10 min). KOH (1 M, 172 mL) was added and the reaction mixture was stirred for 10 minutes. Allyl glycidyl ether (AGE) (28 mL) was added to the reaction mixture and the reaction mixture was stirred overnight.

[0132] Next, the reaction solution was decanted. The rolls were washed with acetone / MQ-water (1:1, 2 × 300 mL, 10 min), followed by MQ-water (3 × 300 mL, 10 min). A solution of Na2CO3 (0.28 M) in 25% v / v MeCN in MQ-water was added to the rolls (250 mL per beaker). Divinyl sulfone (40 mL per beaker) was added, and the reaction mixture was stirred for 16 hours. The reaction solution was decanted. The rolls were washed with acetone / MQ-water (1:1, 2 × 300 mL, 10 min), followed by MQ-water (3 × 300 mL, 10 min).

[0133] Coupling of triethylamine ammonium chloride (TMAC): In a beaker containing the functionalized membrane from above, N-bromosuccinimide (NBS) (7 g) was added to H2O:MeCN (250 mL, 75% v / v), and the reaction mixture was stirred for 4 hours. The reaction solution was decanted, and the membrane was washed with MQ-water (6 × 300 mL). Phosphate buffer (pH 12.01, 250 mL) was added, and the beaker was placed in a water bath (30°C). TMAC (40 mL) was added, and the pH was adjusted to 11.3 with 50% KOH. The reaction mixture was stirred overnight.

[0134] The reaction solution was then decanted, and the disc was washed with water (6 × 300 mL, 10 minutes). The titration showed an ion capacity of 118 μmol / molecular weight.

[0135] Preparation of Fibro-NG-Q: Preparation of Fibro-Allyl-VS: Fibro-CA laser-cut discs (32 mm, 12 pieces) were washed with MQ-water (4 x 100 mL, 10 mins) in a food box on a shaking table. The discs were placed between two chemically inert nets and wound onto a chemically inert plastic roll. The two rolls were placed in a beaker (600 mL) and a magnetic stirring bar was added to the center. A glass stopper was used to stop the rolls from rotating during stirring. KOH (1 M, 172 mL) was added and the reaction mixture was stirred for 10 minutes. AGE (28 mL) was added and the reaction mixture was stirred overnight.

[0136] The reaction solution was decanted. The rolls were washed with acetone / MQ-water (1:1, 2 x 300 mL, 10 min), followed by washing with MQ-water (3 x 300 mL, 10 min). A solution of Na2CO3 (0.28 M) in 25% v / v MeCN in MQ-water was added to the rolls (250 mL per beaker). Divinyl sulfone (40 mL per beaker) was added, and the reaction mixture was stirred for 16 hours.

[0137] Next, the reaction solution was decanted. The rolls were washed with acetone / MQ-water (1:1, 2 x 300 mL, 10 min), followed by MQ-water (3 x 300 mL, 10 min). NBS (7 g) in H2O:MeCN (250 mL, 75% v / v) was added to the beaker and stirred for 4 hours. The reaction solution was decanted and washed with MQ-water (6 x 300 mL).

[0138] Coupling with triethylamine ammonium chloride (TMAC): Phosphate buffer (pH 12.01, 250 mL) was added to the functionalized membrane from above, and the beaker was placed in a water bath (30°C). TMAC (40 mL) was added, and the pH was adjusted to 11.3 with 50% KOH. The reaction mixture was stirred overnight.

[0139] The reaction solution was then decanted, and the disc was washed with water (6 × 300 mL, 10 minutes). The titration yielded a value of 100 μmol ion volume / membrane mL.

[0140] Preparation of chromatography devices: Each of the Fibro chromatography material prototypes was mounted in a 0.4 mL HiTrap Fibro® (Cytiva, Sweden) chromatography device, which is hereafter referred to herein as a Fibro unit.

[0141] Complete and empty capsid isolation of AAV: The prototype was tested using the AKTA pure P25 system at a flow rate of 10 mL / min, and peaks were detected using UV280 and 260 nm. Fibro units were equilibrated with 30 membrane units (MV) of 20 mM BTP pH9 and 2 mM MgCl2 (buffer A).

[0142] Affinity-purified samples of AAV5 and AAV8 containing both complete and empty capsids (approximately 30-40% complete capsids) were neutralized and diluted to approximately 1 × 10⁶ in equilibration buffer (Buffer A). 12The virus was diluted to viral capsid / mL and loaded into Fibro units. After loading, the units were washed with 20 MV and then eluted sequentially with 20 mM BTP pH 9, 2 mM MgCl2, and 250 mM sodium acetate (buffer B).

[0143] For the separation of complete and empty capsids in AAV8, elution using 5% increments of buffer B (25 MV each) resulted in empty capsids entering the flow-through (FT) and complete capsids eluting into 5% buffer B for all Fibro prototype units tested (Figure 3).

[0144] More specifically, non-glycidol Q (NG-Q, Figure 3A) with an IC50 of 100 μmol / mL, glycidol Q (GQ, Figure 3B) with an IC50 of 118 μmol / mL, non-glycidol DAX(a) (NG-DAX, Figure 3C) with an IC50 of 162 μmol / mL, and non-glycidol DAX(b) (NG-DAX, Figure 3D) with an IC50 of 203 μmol / mL showed similar separation results.

[0145] The UV260:280 ratio in the flow-through peak (empty capsid, peak 1, ratio approximately 0.5–0.7) and the bound and eluted peak (complete capsid, peak 2, ratio approximately 1.1–1.3) was calculated and plotted for two different Fibro anion exchange ligand prototype runs. The results were similar for Q and DAX anion exchange ligands with different ligand densities (Figure 4).

[0146] For the separation of complete and empty capsids of AAV5, a Fibro non-glycidol DAX prototype, NG-DAX(a), with an IC50 of 162 μmol / mL was used. Optimal elution conditions were determined using pre-screening with 1% increasing elution increments in buffer B, i.e., identifying the percentage of buffer B required to elute empty capsids before complete capsids begin to elute (before the critical point where UV260:280 is 1) (Figure 5A). The UV260:280 ratio of 1 (critical point) is indicated by an arrow in 3% buffer B. For this AAV serotype, empty capsids may also bind to the Fibro anion exchange prototype. To allow empty capsids to enter the flow-through without binding, the electrical conductivity during loading and washing can be adjusted (increased, but complete capsids still bind).

[0147] Based on the pre-screening results, 2.5% buffer B was used in step 1 to elute empty capsids, followed by step 2 using 15% buffer B to elute complete capsids (Figure 5B). The UV260:280 ratios obtained for peaks 1 and 2 correspond to typical empty and complete capsid values. The calculated UV260:280 ratios for peak 1 (empty capsids) and peak 2 (complete capsids) are shown in Figure 5B.

[0148] (Example 2) Separation of AAV8 capsid under variable conditions The experimental design for separating fully packaged AAV8 capsids from empty AAV8 capsids is carried out using the same equipment and samples as in Example 1 above, and further using the same anion exchange chromatography material as in Example 1, with the following variations.

[0149] Regarding the chemical properties of ligands: 1) Fibro Q analogues whose ligand is defined by formula I: a. R1 and R2 are ethyl, and R3 is methyl. b. R1 and R2 are methyl groups, and R3 is CH2CHOHCH3. 2) Fibro DAX analogs whose ligand is defined by formula II: a. In the formula, X is independently of H, OH, and C for each occurrence. 1~3 Selected from the base, R1, R2, R3, and R4 are independently H and C 1~3 Selected from the base, b. The C3 group is linear or branched. cC 1~3 The base is HR, OC 1~2 SC 1~2 , comprising a group independently selected from NH, NHR, and NR2, dR is H and C 1~3 Selected from the base.

[0150] Regarding buffer solutions and elution conditions: 1) MgCl2 at different concentrations between 1 and 20 mM. 2) Different NaCl linear gradients of 0.1~1M, with or without MgCl2 as in 1). 3) Different pH linear gradients: pH 4-10, with or without MgCl2, as in 1). 4) Different buffers: a. Tris bN-methyldiethanolamine 5) Stepwise elution using pH, NaCl, and MgCl2, as in 1), 2), and 3). 6) Conditions suitable for binding and elution of both empty and complete capsids. 7) All of the above, with or without additives such as sucrose (0.1-5%) and poloxamer 188 surfactant (0.01-1%).

[0151] (Example 3) Isolation of capsids of various adeno-associated virus serotypes under variable conditions The experimental design for the isolation of complete and empty capsids of adeno-associated virus serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV10, AAV11, AAV12, and AAV13 is carried out according to the variable conditions of Examples 1 and 2 described above.

[0152] (References) TIFF2026515772000005.tif111154 [Explanation of symbols]

[0153] 1. Chromatographic materials 2 Supporting material 3 Anion exchange ligands 4 Linker 5 Chromatography devices 6. Adeno-associated virus capsid 100 Methods for separating adeno-associated virus capsids with fully packaged genetic material from adeno-associated virus capsids with unpackaged genetic material. 110 Steps to add a liquid sample containing adeno-associated virus capsid to a chromatography material. 120 Steps to elute adeno-associated virus capsids, in which the genetic material is fully packaged, from chromatography material.

Claims

1. A chromatography material (1) comprising a support material (2) in the form of a convection-based membrane structure containing nanofibers, wherein the support material is functionalized with an anion-exchange ligand (3) at a ligand density of <300 μmol / mL, and the chromatography material comprises a linker (4) linking the ligand to the support material, the linker comprising a linear backbone having a length of 2 to 16 atoms.

2. The chromatography material according to claim 1, wherein the support material has an average flow pore diameter of 0.1 to 2.0 μm.

3. Anion exchange ligand is formula I: 【Chemistry 1】 (In the formula, R 1 R is selected from H and C1-C3 alkyl groups. 2 and R 3 The group is independently selected from H, C1-C3 alkyl, CH2OH, and CH2CHOHCH3, preferably R 1 , R 2 , and R 3 Each of them is CH3. A chromatography material according to claim 1 or 2, as defined by...

4. The chromatography material according to claim 3, wherein the ligand is defined by formula I (wherein each of R1, R2, and R3 is CH3), and the linker comprises a vinyl sulfone and optionally further comprises glycidol.

5. The anion exchange ligand is given by formula II: 【Chemistry 2】 (In the formula, X is independently of H, OH and C for each occurrence) 1～3 Selected from the base, R 1 、R 2 、R 3 and R 4 are each independently selected from H and C 1～3 groups, C 3 The base is linear or branched, C 1～3 The base is HR, OC 1～2 SC 1～2 NH, NHR, and NR 2 Includes a base that is independently selected from, R is H and C 1～3 (Selected from the base) A chromatography material according to claim 1 or 2, as defined by...

6. The chromatography material according to claim 5, wherein the anion exchange ligand is selected from N,N,N'-triethylethylenediamine, diethylenetriamine, N,N'-dimethylethylenediamine, N-methylethylenediamine, 1,3-diaminopropane, 1,3-diamino-2-hydroxypropane, 2-methyl-1,3-propanediamine and N,N-diethylethylenediamine, and is preferably N,N-diethylethylenediamine.

7. The chromatography material according to claim 6, wherein the ligand comprises N,N-diethylethylenediamine and the linker comprises vinyl sulfone.

8. The chromatography material according to any one of claims 1 to 7, wherein the convection-based membrane structure comprises a nonwoven web of polymer nanofibers, and optionally the polymer is a cellulosic polymer such as cellulose acetate, a synthetic polymer, or a combination thereof.

9. The chromatography material according to any one of claims 1 to 8, wherein the convection-based membrane structure comprises a single membrane, a number of membranes, or a filter.

10. A chromatography device (5) comprising a holder containing a chromatography material (1) according to any one of claims 1 to 9.

11. A method (100) for separating adeno-associated virus capsids in which the genetic material is fully packaged from adeno-associated virus capsids in which the genetic material is not fully packaged, a. A step (110) of adding a liquid sample containing adeno-associated virus capsid (6) to the chromatography material according to any one of claims 1 to 9, The liquid sample has a purity of at least 90% and at least 10 12 The process involves a concentration of adeno-associated virus capsids at a rate of adeno-associated virus capsid / ml, of which at least 10% are adeno-associated virus capsids in which the genetic material is fully packaged. b. Step (120) to elute adeno-associated virus capsids, in which the genetic material is fully packaged, from the chromatography material. A method comprising, wherein the adeno-associated virus capsid eluted in step (b) is eluted into at least one eluate fraction, which comprises at least 50% of the fully packaged adeno-associated virus capsid present in the liquid sample added in step (a), and at least 60% of the adeno-associated virus capsid eluted in step (b) is fully packaged genetic material.

12. The method according to claim 11, wherein the residence time of the adeno-associated virus capsid, in which the genetic material is completely packaged, within the chromatographic material is approximately 0.1 seconds to approximately 2 minutes.

13. The method according to claim 11 or 12, wherein steps (a) and (b) include a step of applying a buffer having a pH of about 6.0 to about 10.5, for example, about 7.5 to about 9.5, and optionally the buffer comprises tris(hydroxymethyl)aminomethane (i.e., Tris), 1,3-bis(tris(hydroxymethyl)methylamino)propane, triethanolamine, N-methyldiethanolamine, diethanolamine, 1,3-diaminopropane, or ethanolamine.

14. The method according to any one of claims 11 to 13, wherein step (b) comprises applying a buffer containing a compound that improves the separation of capsids in which the genetic material is fully packaged from capsids in which the genetic material is not fully packaged, wherein the compound is optionally selected from carbohydrates, divalent metal ions, surfactants, and / or salts of monovalent metal ions such as cosmotropic salts.

15. The method according to any one of claims 11 to 14, wherein the liquid sample added in step (a) is a pre-purified liquid sample.

16. Adeno-associated virus capsids are found in 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), and adeno-associated virus serotype 8. The method according to any one of claims 11 to 15, wherein the adeno-associated virus capsid is a capsid of adeno-associated virus serotype 10 (AAV10), adeno-associated virus serotype 11 (AAV11), adeno-associated virus serotype 12 (AAV12), or adeno-associated virus serotype 13 (AAV13), or a variant thereof, and optionally the adeno-associated virus capsid is a capsid of AAV2, AAV5, or AAV8.

17. The chromatography material is as described in claim 4 or 7, and the elution buffer of step (b) comprises magnesium chloride and / or sodium acetate. The method according to any one of claims 11 to 16, wherein the adeno-associated virus capsid is optionally the capsid of AAV2, AAV5, AAV8, or a variant thereof.

18. Use of a chromatography material according to any one of claims 1 to 9 or a chromatography device according to claim 10 for separating adeno-associated virus capsids in which the genetic material is fully packaged from adeno-associated virus capsids in which the genetic material is not fully packaged.

19. The chromatography material is as described in claim 4 or 7, and the adeno-associated virus capsid is 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 The use according to claim 18, wherein the capsid is an adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 10 (AAV10), adeno-associated virus serotype 11 (AAV11), adeno-associated virus serotype 12 (AAV12), or adeno-associated virus serotype 13 (AAV13), or a variant thereof, and optionally the adeno-associated virus capsid is an adeno-associated virus capsid of AAV2, AAV5, AAV8, or a variant thereof.

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