EP4694995A1

EP4694995A1 - A chromatography material, use thereof, and a method for separating adeno-associated capsids

Info

Publication number: EP4694995A1
Application number: EP24718746.1A
Authority: EP
Inventors: Jean-Luc Maloisel; Åsa HAGNER MCWHIRTER; Brigitta NÉMETH
Original assignee: Cytiva Bioprocess R&D AB
Current assignee: Cytiva Bioprocess R&D AB
Priority date: 2023-04-12
Filing date: 2024-04-08
Publication date: 2026-02-18
Also published as: WO2024213492A1; CN120936420A; KR20250172635A

Abstract

The present disclosure is directed to a chromatography material comprising a support material in the form of a convection-based membranous structure comprising nanofibres, wherein the support material is functionalised with an anion exchange ligand at a ligand density of <300 µmol/mL, wherein the chromatography material comprises a linker connecting the ligand to the support material, the linker comprising a linear backbone having a length of 2-16 atoms. Also provided is a chromatography device comprising a holder comprising the chromatography material as disclosed herein. Additionally, the present disclosure is directed to use of the herein disclosed chromatography material or chromatography device for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material. Further, the present disclosure provides a method for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material, the method comprising the following steps: (a) adding a liquid sample comprising adeno-associated virus capsids to the chromatography material as disclosed herein, wherein the liquid sample comprises adeno-associated virus capsids of a purity of at least 90% and of a concentration of at least 10¹² adeno-associated virus capsids/ml, of which at least 10% of the adeno-associated virus capsids are adeno-associated virus capsids fully packaged with genetic material; (b) eluting the adeno-associated virus capsids fully packaged with genetic material from the chromatography material; wherein the adeno-associated virus capsids eluted in step (b) are eluted into at least one eluate fraction, which eluate fraction comprises at least 50% of the fully packaged adeno-associated virus capsids present in the liquid sample added in step (a), and wherein at least 60% of the adeno-associated virus capsids eluted in step (b) are fully packaged with genetic material.

Description

A CHROMATOGRAPHY MATERIAL, USE THEREOF, AND A METHOD FOR SEPARATING ADENO- ASSOCIATED CAPSIDS

FIELD OF DISCLOSURE

The present disclosure relates to the field of separation of biological target compounds, in particular adeno-associated capsids. The disclosure is directed to a chromatography material and use thereof for separating adeno-associated virus capsids fully packaged with genetic material from adeno- associated virus capsids not fully packaged with genetic material, as well as a method for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material.

BACKGROUND OF THE DISCLOSURE

Adeno-associated viruses (AAV) are non-enveloped viruses that have linear single-stranded DNA (ssDNA) genome and that can be engineered to deliver DNA to target cells. Recombinant adeno- associated virus (rAAV) vectors have emerged as one of the most versatile and successful gene therapy delivery vehicles. There is an increasing demand to use viral vectors for gene therapy. The AAV vector is one of the most attractive gene transfer tools for developing novel genetic therapies for muscle diseases as well as other disorders.

To use AAV particles as vectors in therapy it is necessary to purify the virus particles from cell impurities like DNA after transfection. Further, since therapeutic efficacy of AAV vectors is dependent on high percentage of virus particles fully packaged with genetic material of interest, it is important to separate such fully packaged AAV from empty and partially packaged AAV particles. WO2023285011 Al describes a method for separating fully packaged AAV particles from not fully packaged AAV particles. However, there is a continuous need in the art for novel chromatography materials and purification strategies to increase the speed and decrease the cost of the purification process.

SUMMARY OF THE INVENTION

The object of the present disclosure is to provide a chromatography material enabling a faster separation of fully packaged adeno-associated virus capsids from not fully packaged adeno- associated virus capsids at similar or improved resolution and purity compared to previously known chromatography materials. This is achieved by providing an optimised convection-based chromatography material. More particularly, the present disclosure is directed to a chromatography material comprising a support material in the form of a convection-based membranous structure comprising nanofibres, wherein the support material is functionalised with an anion exchange ligand at a ligand density of <300 pmol/mL, wherein the chromatography material comprises a linker connecting the ligand to the support material, the linker comprising a linear backbone having a length of 2-16 atoms.

The present disclosure also provides a chromatography device comprising a holder comprising the chromatography material as disclosed herein.

Additionally, the present disclosure is directed to use of the herein disclosed chromatography material or chromatography device for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material.

Further, the present disclosure provides a method for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material, the method comprising the following steps: a. adding a liquid sample comprising adeno-associated virus capsids to the chromatography material as disclosed herein, wherein the liquid sample comprises adeno-associated virus capsids of a purity of at least 90% and of a concentration of at least 10¹² adeno-associated virus capsids/ml, of which at least 10% of the adeno-associated virus capsids are adeno-associated virus capsids fully packaged with genetic material; b. eluting the adeno-associated virus capsids fully packaged with genetic material from the chromatography material; wherein the adeno-associated virus capsids eluted in step (b) are eluted into at least one eluate fraction, which eluate fraction comprises at least 50% of the fully packaged adeno-associated virus capsids present in the liquid sample added in step (a), and wherein at least 60% of the adeno- associated virus capsids eluted in step (b) are fully packaged with genetic material.

In particular, the present disclosure is directed to separation 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 a variant thereof.

Preferred aspects of the present disclosure are described below in the detailed description and in the dependent claims. It is noted that the present disclosure relates to all possible combinations of features recited in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects of the present disclosure will now be described in more detail, with reference to the appended drawings showing embodiment(s) of the invention, in which:

Fig. 1 schematically illustrates in cross-section an exemplary chromatography device comprising a chromatography material according to the present disclosure.

Fig. 2 is a flow chart outlining the steps of a method for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material according to the present disclosure.

Fig. 3 shows chromatograms for separation of AAV8 full and empty capsids on different anion exchange prototypes, as described in Example 1 herein.

Fig. 4 is a graph showing the UV 260:280 ratios for separation of AAV8 full and empty capsids on different chromatography material prototypes, as described in Example 1 herein.

Fig. 5 shows chromatograms for separation of AAV5 full and empty capsids on an anion exchange prototype, as described in Example 1 herein.

As illustrated in the figures, some features may be exaggerated for illustrative purposes and, thus, are provided to illustrate the general structures of embodiments of the present disclosure.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure solves or at least mitigates the problems associated with existing chromatography materials for separation of fully packaged adeno-associated virus capsids from not fully packaged adeno-associated virus capsids by providing, as illustrated in Fig. 1, a chromatography material 1 comprising a support material 2 in the form of a convection-based membranous structure comprising nanofibres, wherein the support material is functionalised with an anion exchange ligand 3 at a ligand density of <300 pmol/mL, wherein the chromatography material comprises a linker 4 connecting the ligand to the support material, the linker comprising a linear backbone having a length of 2-16 atoms.

A significant advantage of the presently disclosed chromatography material is that it enables a faster separation of fully packaged adeno-associated virus capsids from not fully packaged adeno- associated virus capsids at similar or improved resolution and purity compared to previously known chromatography materials.

The term "chromatography material" is used herein to denote a type of separation matrix. The term "separation matrix" is used herein to denote a material comprising a support material to which one or more ligands comprising functional groups have been coupled. The functional groups of the ligand(s) bind compounds herein also called analytes, which are to be separated from a liquid sample and/or which are to be separated from other compounds present in the liquid sample. A separation matrix may further comprise a compound which couples the ligand(s) to the support material. The term "linker" may be used to describe such a compound, as further described below. Herein, the term "support material" may be used interchangeably with the term "support".

In this context, "ligand" is a molecule that has a known or unknown affinity for a given analyte and includes any functional group, or capturing agent, immobilized on its surface, whereas "analyte" includes any specific binding partner to the ligand. The term "ligand" may herein be used interchangeably with the terms "specific binding molecule", "specific binding partner", "capturing molecule" and "capturing agent". Herein, the molecules in a liquid sample which interact with a ligand are referred to as "analyte". The analytes of interest according to the present disclosure are adeno-associated virus capsids, more particularly adeno-associated virus capsids either fully packaged or not fully packaged with genetic material. Consequently, herein the terms "analyte", "adeno-associated virus capsid" and "capsid" may be used interchangeably.

The term "surface" herein means all external surfaces and includes in the case of a porous support outer surfaces as well as pore surfaces.

The herein disclosed chromatography material comprises a linker connecting the ligand to the support material, i.e., the coupling of the ligand to the support is provided by introducing a linker between the support material and ligand. The linker comprises a linear backbone having a length of 2-16 atoms. The linear backbone includes at least 2 C atoms and may contain at most 16 atoms selected from C atoms and/or heteroatoms including one or more O, N, and/or S atoms. The one or more heteroatoms, including O atoms, N atoms, and/or S atoms, may be integrated in functional groups. Non-limiting examples of such functional groups are ether, thioether, amide, sulphonamide, ketone, and sulphone.

Optionally, the linker may comprise one or more side chains attached to the linear backbone. Each side chain may contain at most 3 atoms selected from C atoms and/or heteroatoms including one or more O, N, and/or S atoms. Non-limiting examples of such side chains are C1-C3 alkanes, hydroxyl, ether, and amide.

The term "backbone" is intended to mean the main chemical structure of the linker. As mentioned above, the linear backbone has a length of 2-16 atoms, wherein the "length" of the linear backbone extends from the atom at one end of the linear backbone, said atom being attached to the support material, to the atom at the other end of the linear backbone, said atom being attached to the ligand. It is to be understood that both end atoms are included in the length of 2-16 atoms. The linear backbone is composed of carbon atoms and optionally also of heteroatoms. The 2-16 atoms of the linear backbone do not include any hydrogen atom bound to any carbon and/or to any heteroatom of the backbone. Further, the linear backbone is defined as not including any side chains. One or more side chains may however optionally be coupled to the backbone, as described in more detail above.

The term "heteroatom" has its conventional meaning in the art of chemistry, i.e., an atom other than carbon or hydrogen.

Non-limiting examples of start chemicals to generate such a linker include epichlorohydrin, diepoxide such as diglycidyl ether or 1,4-butanediol diglycidyl ether, allyl glycidyl ether or divinyl sulfone. The coupling may be carried out following any conventional covalent coupling methodology well known in the art and easily carried out by the skilled person.

A non-limiting example of a suitable linker is a linker comprising vinyl sulfone. Another non-limiting example is a linker comprising vinyl sulfone and glycidol.

The support material of the presently disclosed chromatography material is in the form of a convection-based membranous structure comprising nanofibres. Accordingly, the support material is a type of convection-based chromatography matrix. A convection-based chromatography matrix includes any matrix in which application of a hydraulic pressure difference between the inflow and outflow of the matrix forces perfusion of the matrix, achieving substantially convective transport of substance(s) into the matrix or out of the matrix. A convection-based matrix can be for example an adsorptive membrane where a flow through such materials is convective rather than diffusional. The present chromatography material, i.e., the stationary phase, has a high surface area for high binding capacity and a macroporosity needed for viruses to enter the matrix. When adding the mobile phase (i.e., a liquid sample or solution comprising the biological target compounds to be purified) to the convective stationary phase, there is a convective flow of the mobile phase in the stationary phase, such that the mobile phase is directly in contact with the ligands in the matrix. Thus, the biological target compounds (here, AAV vectors) in the mobile phase do not have to rely on diffusion to reach the ligands.

Hence, the present disclosure enables chromatography materials for purification of AAV vectors which combine the high binding capacity traditionally associated with porous bead-based materials, with the higher flowrates that are achievable with monolith/membrane materials. The chromatography material can be made sufficiently porous so that the binding area is accessible to the large vectors, and suitably short residence times may be achieved.

The diameter of different viruses ranges from 20-300 nm. AAV vectors typically have a diameter of about 25 nm.

The convection-based membranous structure may comprise a non-woven web or matrix of polymer nanofibres. When in use, such nanofibers form a stationary phase comprising a plurality of pores through which a mobile phase can permeate.

The non-woven polymer matrix comprising nanofibres is a mat of one or more polymer nanofibres with each fibre oriented essentially randomly, i.e., it has not been fabricated so that the fibre or fibres adopts a particular pattern. The non-woven polymer matrix is typically provided by known methods. The non-woven matrix may, in certain circumstances, consist of a single polymer nanofibre. Alternatively, the non-woven matrix may comprise two or more polymer nanofibres.

The polymer nanofibres may be electrospun polymer nanofibres. Such electrospun polymer nanofibres are well known to the person skilled in the art.

The polymer nanofibres typically have mean diameters from 10 nm to 1000 nm. For some applications, polymer nanofibres having mean diameters from 200 nm to 800 nm or 200 nm to 400 nm may be appropriate.

The length of polymer nanofibres is not particularly limited. Thus, conventional processes e.g,. electrospinning can produce polymer nanofibres many hundreds of metres or even kilometres in length. Typically, though, the one or more polymer nanofibres have a length up to 10 km, preferably from 10 m to 10 km.

The non-woven matrix typically has a surface area from 1 to 40 g/m2, from 5 to 25 g/m2, from 1 to 20 or 5 to 15 g/m2.

The non-woven matrix typically has a thickness from 5 to 120 pm.

The polymer nanofibres may be made of a cellulosic polymer, such as selected from a group consisting of cellulose and a partial derivative of cellulose, particularly cellulose acetate or other cellulose ester, cross-linked cellulose, grafted cellulose, or ligand-coupled cellulose. In some cases, cellulose and cellulose acetate are preferred. Cellulose acetate is readily formed into nanofibres, e.g., by electrospinning and can readily be transformed into cellulose after electrospinning. Cellulose fiber chromatography (known as Fibro™ chromatography; Cytiva, Sweden) is an ultrafast chromatography purification for short process times and high productivity, which utilizes the high flow rates and high capacities of cellulose fiber.

Alternatively, the polymer nanofibres may be made of a synthetic polymer. Non-limiting examples of suitable polymers may be selected from polysulfones (e.g., polyethersulfone), polyamides, nylon, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, polypropylene, and polyethylene oxide, and mixtures thereof.

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

Prior to functionalising with the ligand, the nanofibres may optionally be physically modified, fused together at points where nanofibers intersect one another, by thermal or chemical methods and/or by pressing the polymer non-woven matrix. This may improve the structural stability of the matrix. The pressing and heating conditions may also be varied to alter the thickness and/or porosity of the resultant matrix.

According to a non-limiting example, the polymer nanofibres may be made of cellulosic polymer reinforced with synthetic polymer.

The convection-based membranous structure may comprise a single membrane, a pile of membranes or a filter. Use of multiple non-woven matrices/sheets enables a thicker material to be prepared, which may have a greater capacity for adsorbance. The functionalised polymer matrix is typically therefore formed by providing two or more non-woven matrices stacked one on top of the other, each matrix comprising one or more polymer nanofibres, and simultaneously heating and pressing the stack of matrices to fuse points of contact between the nanofibres of adjacent matrices/sheets.

In the case of a cellulose matrix, this is typically formed by providing two or more non-woven matrices stacked one on top of the other, each said matrix comprising one or more cellulose acetate nanofibres, and simultaneously heating and pressing the stack of sheets to fuse points of contact between the nanofibres of adjacent matrices/sheets. The polymer matrix may consist of cellulose only. Alternatively, the matrix may comprise cellulose in combination with one or more polymer nanofibers. Preferred processing conditions for pressing and heating of polymer nanofibres/non- woven sheets can be found, e.g., in W02015052460 Al and WO2015052465 Al.

The support material may have a mean flow pore size of 0.1-2.0 pm, such as 0.1-1.8 pm, 0.1-1.6 pm, 0.1-1.4 pm, 0.1-1.2 pm, 0.1-1.0 pm, 0.1-0.8 pm, 0.1-0.6 pm, 0.1-0.4 pm, 0.1-0.2 pm, 0.2-2.0 pm, 0.4- 2.0 pm, 0.6-2.0 pm, 0.8-2.0 pm, 1.0-2.0 pm, 1.2-2.0 pm, 1.4-2.0 pm, 1.6-2.0 pm, 1.8-2.0 pm, or 0.5- 1.5 pm. Mean flow pore (MFP) size is an indicator of material flow characteristics, and is measured by capillary flow porometry, based on the displacement of a wetting liquid with a known surface tension from the sample pores by applying a gas at increasing pressure. The higher the MFP size, the larger the flow of liquid through the material at a given pressure. The mean flow pore size is calculated from the point at which 50 % of the flow goes through a sample. Mean flow pore size thus corresponds to the pore size calculated at the pressure where the wet curve and the half-dry curve meet. In an alternative definition, the mean flow pore size of a support material may be seen as an effective pore size defined as the size of the largest sphere that is able to pass through the pore.

As described above, the support material is functionalised with an anion exchange ligand.

The density of the anion exchange ligand is <300 pg of ligand per mL of the chromatography material, such as about 50-250 pmol/mL, about 70-220 pmol/mL, about 90-200 pmol/mL, or such as about 250 pmol/mL, about 200 pmol/mL, about 190 pmol/mL, about 180 pmol/mL, about 170 pmol/mL, about 160 pmol/mL, about 150 pmol/mL, about 140 pmol/mL, about 130 pmol/mL, about 120 pmol/mL, about 110 pmol/mL, about 100 pmol/mL, about 90 pmol/mL, about 80 pmol/mL, or lower. Herein, the term "about" is intended to cover a range of densities including the value specified ± 5%. The density of ligand in the chromatography material may be determined by a titration method to determine the number of ligand moieties in the functionalised material. A skilled person will be aware of suitable methods.

The anion exchange ligand may comprise a quaternary amine group. The ligand may be defined by Formula I: wherein Ri is selected from H and C1-C3 alkyl, and R? and R3 are independently selected from H, Cl- C3 alkyl, CH2OH, and CH2CHOHCH3.

As a non-limiting example, each of Ri, R?, and R3 is CH3.

According to another non-limiting example, Riand R? are ethyl, and R3 is methyl.

According to yet another non-limiting example, Riand R? are methyl, and R3 is CH2CHOHCH3.

The wavy moiety represents the support material, including the linker. The ligand may be attached to a carbon atom of the linker. There are currently available chromatography materials comprising a ligand defined by Formula I, wherein each of Ri, R?, and R3 is CH3; e.g., a chromatography material made available under the name Capto™ Q, provided by Cytiva, Sweden (www.cytivalifesciences.com). Capto™ Q. further comprises dextran as surface extender and is a chromatography medium for high-resolution polishing steps in industrial purification processes, e.g., for purification of monoclonal antibodies. Recently, it has also been proposed for separation of fully packaged adeno-associated capsids from not fully packaged adeno-associated capsids (see WO2023285011 Al). Capto™ Q. comprises a support material in the form of substantially spherical particles or beads, which have a homogeneous porosity throughout its entire volume, and which are at least partly permeable to adeno-associated virus capsids throughout their entire volume.

In contrast, the present disclosure provides a chromatography material comprising a convectionbased membranous structure comprising nanofibres as support material. As mentioned above, such a convection-based support material enables a faster separation of fully packaged adeno-associated virus capsids from not fully packaged adeno-associated virus capsids at similar resolution and purity compared to previously known chromatography materials, such as Capto™ Q.

A further differentiating feature of the presently disclosed chromatography material is that it comprises a linker comprising a linear backbone having a length of only 2-16 atoms. This linker is much shorter than a polymeric dextran surface extender. The results described in Example 1 herein show that convection-based chromatography materials having such a short linker provide a similar resolution and purity of separated adeno-associated virus capsids of serotypes AAV5 and AAV8 as the results shown in WO2023285011 Al for a resin bead chromatography material including a surface extender. This is surprising considering that WO2023285011 Al showed that beads comprising a surface extender provided an improved separation of full AAV capsids from empty AAV capsids compared to the same chromatography material not including a surface extender.

The linker is defined as described in detail elsewhere herein.

A non-limiting example of a chromatography material according to the present disclosure comprises a support material in the form of a non-woven web of cellulose acetate nanofibres, a ligand defined by Formula I, wherein each of Rl, R2, and R3 is CH3, and a linker comprising vinyl sulfone. The ligand density may be about 90-100 pmol/mL, such as about 90, 95, 100, 105, or 110 pmol/mL.

Another non-limiting example of a chromatography material according to the present disclosure comprises a support material in the form of a non-woven web of cellulose acetate nanofibres, a ligand defined by Formula I, wherein each of Rl, R2, and R3 is CH3, and a linker comprising vinyl sulfone and glycidol. The ligand density may be about 110-130 pmol/mL, such as about 110, 115, 118, 120, 125, or 130 pmol/mL.

The anion exchange ligand may alternatively be defined by Formula II: wherein X, for each occurrence independently, is selected from H, OH and a C1-3 group, and Ri, R2, R3 and R₄ are independently selected from H and a C1-3 group, wherein a C3 group is straight or branched, wherein a C1-3 group comprises groups independently selected from OH, O-C1.2, S-C1.2, NH, NHR, and NR₂, wherein R is selected from H and a C1-3 group.

More particularly, the ligand defined by Formula II may be selected from a group consisting of N,N,N'-triethylethylenediamine, diethylenetriamine, N,N'-dimethylethylenediamine, N- methylethylenediamine, 1,3-diaminopropane, l,3-diamino-2-hydroxypropane, 2-methyl-l,3- propanediamine and N,N-diethylethylenediamine.

As a non-limiting example, the ligand defined by Formula II is N,N-diethylethylenediamine.

The support material and the linker are defined as described in detail elsewhere herein.

Accordingly, a non-limiting example of a chromatography material according to the present disclosure comprises a support material in the form of a non-woven web of cellulose acetate nanofibres, a ligand being N,N-diethylethylenediamine, and a linker comprising vinyl sulfone. The ligand density may be about 150-210 pmol/mL, such as about 150, 160, 162, 165, 170, 175, 180, 185, 190, 195, 200, 203, 205, or 210 pmol/mL.

The chromatography material may be used in a chromatography system or used manually with syringes. All devices could be used with peristaltic pump, diaphragm pump, or positive gas pressure. The chromatography material may be contained in any type of separation device that allows an even flow distribution over the chromatography material. The term "separation device" has its conventional meaning in the field of bioprocessing and is to be understood as encompassing any type of separation device which is capable of and suitable for separating and purifying compounds from a fluid containing by-products from the production of the compounds. A separation device may comprise a separation matrix, as further defined elsewhere herein.

Herein, a separation device may alternatively be referred to as a chromatography device 5, as schematically illustrated in Fig. 1. More particularly, the chromatography device 5 comprises a holder comprising the chromatography material as disclosed herein. Non-limiting examples of holders are capsules and cartridges. The chromatography device may further comprise one or more spacers, being located between layers of the convection-based membranous structure. Non-limiting examples of spacer materials are frits, non-woven materials, and woven materials.

The present disclosure further provides, as schematically illustrated in the flow chart of Fig. 2, a method 100 for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material, the method comprising the following steps: a. adding 110 a liquid sample comprising adeno-associated virus capsids 6 (as illustrated in Fig. 1) to the chromatography material as disclosed in detail elsewhere herein, wherein the liquid sample comprises adeno-associated virus capsids of a purity of at least 90% and of a concentration of at least 10¹² adeno-associated virus capsids/ml, of which at least 10% of the adeno-associated virus capsids are adeno-associated virus capsids fully packaged with genetic material; b. eluting 120 the adeno-associated virus capsids fully packaged with genetic material from the chromatography material; wherein the adeno-associated virus capsids eluted in step (b) are eluted into at least one eluate fraction, which eluate fraction comprises at least 50% of the fully packaged adeno-associated virus capsids present in the liquid sample added in step (a), and wherein at least 60% of the adeno- associated virus capsids eluted in step (b) are fully packaged with genetic material.

A "virus particle" is herein used to denote a complete infectious virus particle. It includes a core, comprising the genome of the virus (i.e., the viral genome), either in the form of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and the core is surrounded by a morphologically defined shell. The shell is called a capsid. The capsid and the enclosed viral genome together constitute the so- called nucleocapsid. The nucleocapsid of some viruses is surrounded by a lipoprotein bilayer envelope. In the field of bioprocessing, for the purpose of producing viral vectors for various applications such as therapy, the genome of a virus particle is modified to include a genetic insert, comprising genetic material of interest. Modified virus particles are allowed to infect host cells in a cell culture and the virus particles are propagated in said host cells, after which the virus particles are purified from the cell culture by any means of separation and purification. Herein, a virus particle to be separated from a cell culture by the presently disclosed method may alternatively be referred to as a "target molecule" or "target". It is to be understood that "a virus particle" is intended to mean a type of virus particle and that the singular form of the term may encompass a large number of individual virus particles. Herein, the term "virus particle" may be used interchangeably with the terms "vector" and "capsid", respectively, as further defined below.

The term "vector" is herein used to denote a virus particle, normally a recombinant virus particle, which is intended for use to achieve gene transfer to modify specific cell type or tissue. A virus particle can for example be engineered to provide a vector expressing therapeutic genes. Several virus types are currently being investigated for use to deliver genetic material (e.g., genes) to cells to provide either transient or permanent transgene expression. These include adenoviruses, retroviruses (y-retroviruses and lentiviruses), poxviruses, adeno-associated viruses (AAV), baculoviruses, and herpes simplex viruses. Herein, the term "vector" may be used interchangeably with the terms "virus particle" and "capsid", respectively.

The term "capsid" means the shell of a virus particle. The capsid surrounds the core of the virus particle, and normally should comprise a viral genome. A modified (recombinant) capsid, as produced in an upstream process of manufacturing, is supposed to comprise a complete viral genome, which genome includes genetic material of interest for one or more applications, for example of interest for various therapeutic applications. However, owing to low packaging efficiency, assembled capsids do not always contain any genetic material or only encapsidate truncated genetic fragments, resulting in so-called empty capsids and partially filled capsids, respectively. These capsids possess no therapeutic function, yet they compete for binding receptors during the cell-mediated processes. This may diminish the overall therapeutic efficacy and trigger undesirable immune responses. As a result, tracking these capsids throughout the production process is crucial to ensure consistent product quality and a proper dosing response (Xiaotong Fu et al, 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 a population of virus particles artificially produced in a cell culture, the capsid is only partially filled with genetic material. Further, in up to as much as 98% of artificially produced virus particles, the capsid does not comprise any part of the viral genome at all, i.e., it is empty. However, generally between 80% to 90% of artificially produced virus particles have empty capsids, and best cases currently achieve as little as 50% empty capsids. Herein, the term "capsid" may be used interchangeably with the terms "vector" and "virus particle", respectively. In the context of the present disclosure, a capsid may or may not comprise genetic material.

The term "genetic material of interest" is intended to mean genetic material which in the field of bioprocessing is considered relevant and valuable to get produced by viral replication and to purify such that it can be used in various applications, such as, but not limited to, therapeutic applications. As a non-limiting example, genetic material of interest may comprise a therapeutically relevant genetic material, such as a therapeutically relevant nucleotide sequence.

The term "capsid fully packaged with genetic material" is herein used to denote a capsid which has been correctly produced (by the host cell), or in other words, a capsid which comprises a complete viral genome, or in other words, a capsid comprising 100% of its viral genome, or in other words, a capsid comprising a functional viral genome.

The viral genome includes a genetic insert, comprising genetic material of interest, as defined elsewhere herein.

A capsid which comprises a complete viral genome may herein alternatively be called a "full capsid" or a "fully packaged capsid". The terms "full capsid", "fully packaged capsid", and "capsid fully packaged with genetic material" may be used interchangeably throughout this text.

The term "capsid not fully packaged with genetic material" is herein used to denote a capsid which has not been correctly produced (by the host cell), or in other words, a capsid which does not comprise a complete viral genome, or in other words, a capsid which comprises less than 100% of its viral genome.

A capsid which is not fully packaged with genetic material is either partially filled with genetic material or is not filled with any genetic material at all.

The term "capsid not fully packaged with genetic material" encompasses the terms "partially filled capsid" and "empty capsid", as defined below.

A "partially filled capsid" is herein defined as a capsid which comprises parts of its viral genome, such as defective parts of its viral genome, or in other words, a capsid which comprises a partial viral genome, or in other words, a capsid which comprises a non-complete viral genome, or in other words, a capsid which comprises a defective viral genome, or in other words, a capsid which comprises more than 0% and less than 100% of the complete viral genome, such as from about 1% to about 99%, such as from about 5% to about 95%, such as from about 10% to about 90%, or such as 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. Since a partially filled capsid is an incorrectly produced capsid, it is desirable to separate and remove as many as possible of the partially filled capsids from a population of capsids, before putting the population of capsids to use in its intended application, e.g., a therapeutic application. Herein, a partially filled capsid may alternatively be called an "intermediate capsid".

An "empty capsid" is herein defined as a capsid which does not comprise any part of its viral genome, i.e., which comprises 0% of its viral genome, or in other words, a capsid which is not filled with any genetic material at all. Thus, an empty capsid does not comprise any genetic material of interest. Consequently, it is desirable (and sometimes required, e.g., due to clinical regulations) to enrich the full capsids, i.e., to increase the percentage of full capsids at the expense of the percentage of partially filled capsids and empty capsids, by separating and remove as many as possible of the empty capsids from a population of capsids, before putting the population of capsids to use in its intended application, e.g., a therapeutic application.

The percentage of full capsids and empty capsids in a population of capsids can be estimated or analyzed with several methods known in the art. Some of these methods are briefly described below:

1: A260:280 in chromatogram will give an estimation of percentage full capsids present in peaks (ratio 1-1.5 indicate enriched in full capsids, ratio 0.5-0.7 is containing mainly empty capsids).

2. qPCR:ELISA ratio. qPCR quantifies viral genomes and ELISA quantifies total viral particles. A ratio of 2 assays with variation is less accurate and will be uncertain. Requires orthogonal analysis for confirmation (see below, 3,4 or 5).

3. Analytical anion exchange separating full and empty capsids (A260:280 ratio and peak area to calculate the percentage). Accuracy dependent of peak definition.

4. Analytical ultracentrifugation (AUC). Detects and quantifies particles of different density (corresponding to full, partially filled, and empty capsids). This is currently known as the "golden standard" in the art. However, ultracentrifugation is not scalable and thus is not suitable for analysis of large-scale batches of capsids.

5. Transmission electron microscopy (TEM). Image analysis counting particles (full, partially filled, and empty capsids). May introduce artifacts from sample preparation. Some methods for estimating or analyzing the percentage of full capsids and empty capsids in a population of capsids are described in more detail in Xiaotong Fu et al, Analytical Strategies for Quantification of Adeno-Associated Virus Empty Capsids to Support Process Development, Human gene therapy methods, 2019, 30(4): 144-152, which is hereby incorporated by reference herein.

It is to be understood that the term "liquid sample" as used herein encompasses any type of sample obtainable from a cell culture, or from a fluid originating from a cell culture which fluid is at least partly purified, by any means of separation and purification.

The term "eluate" is used in its conventional meaning in this field, i.e., the part(s) of a liquid sample loaded onto a chromatography material, which bind to the chromatography material, and which are recovered by being eluted from the chromatography material.

In the method illustrated in Fig. 2, the flow rate used is dependent on the dimensions of the stationary phase (i.e., the chromatography material, or the chromatography device containing said chromatography material), and the residence time chosen. Feasible residence times of the adeno- associated virus capsids fully packaged with genetic material within the herein disclosed convectionbased chromatography material would be about 0.1 s - 2 min. Equivalent flow velocity in a 0.4 ml lab scale unit would be about 3000 - 2 cm/h. In a 2.4 litre unit a maximum feasible flow velocity over the stationary phase would be about 850 cm/h.

As appreciated by persons skilled in the art, since the purpose is to bind the adeno-associated virus capsids fully packaged with genetic material to the anion exchange ligands, step (a) of the method, i.e., adding the liquid sample to the chromatography material, is performed under conditions allowing said binding. In contrast, the adeno-associated virus capsids not fully packaged with genetic material do not bind to the anion exchange ligands or bind to the anion exchange ligands to a much lesser extent than the fully packaged capsids. Consequently, the not fully packaged capsids will exit the chromatography material before the fully packaged capsids, typically in the flow-through, i.e., without applying elution conditions.

Steps (a) and (b) of the above-disclosed method may comprise applying a buffer having a pH of from about 6.0 to about 10.5, such as from about 7.0 to about 10.0, such as from 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. According to non-limiting examples, as described in Example 1 below, a pH of about 9 may be applied for a chromatography material comprising a ligand defined by Formula I or Formula II.

Said buffer is suitably selected from buffers generally recommended for anion exchange chromatography and may for example comprise tris(hydroxymethyl)amino-methane (i.e., Tris), 1,3- bis(tris(hydroxymethyl)methylamino) propane (i.e., bis-Tris propane), triethanolamine, N- methyldiethanolamine, Diethanolamine, 1,3-diaminopropane, or ethanolamine. A person skilled in the art is able to choose a suitable concentration for any one of the above-listed buffers.

In the above-disclosed method, step (b) may comprise applying a buffer, optionally one of the buffers mentioned above, wherein the buffer comprises a compound which improves separation between capsids fully packaged with genetic material and capsids not fully packaged with genetic material. This compound may or may not be present in a buffer applied in step (a). Without being bound by theory, such a compound may for example improve separation by influencing interactions between capsid and ligand or interactions between capsid and capsid. Said compound which improves separation may for example be selected from a carbohydrate, a divalent metal ion, and a detergent.

Where said compound which improves separation is a carbohydrate, it may for example be selected from sucrose, sorbitol, and a polysaccharide.

Where said compound which improves separation is a divalent metal ion, it may for example be selected from Mg²⁺, Fe²⁺, and Mn²⁺. The metal ion may be present in the form of a salt, optionally in combination with for example chloride ions or sulphate ions. A non-limiting example of a suitable metal salt to include in the buffer of step (b) is MgCI₂. Non-limiting examples of suitable concentrations of MgCI₂ include from about 0.5 to about 30 mM of MgCI₂, such as from about 1 to about 20 mM, such as from about 2 to about 10 mM, or about 0.5, 1.0, 1.5, 2.0, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 mM, of MgCI₂.

Where said compound which improves separation is a detergent, it may for example be selected from poloxamer, such as poloxamer 188 or Pluronic™ F68, and polysorbate, such as Tween 20 or Tween 80.

In the above-described method, step (b) may comprise applying a buffer, optionally one of the buffers mentioned above, wherein the buffer comprises a compound which may help eluting capsids bound to the chromatography material. This compound is not present in a buffer applied in step (a). Non-limiting examples of such a compound is a salt, such as a salt of a monovalent metal ion. More particularly, the salt may be a kosmotropic salt. Salts in water solvent are defined as kosmotropic (order-making) if they contribute to the stability and structure of water-water interactions. In contrast, chaotropic (disorder-making) salts have the opposite effect, disrupting water structure, increasing the solubility of nonpolar solvent particles, and destabilizing solute aggregates.

Kosmotropes cause water molecules to favorably interact, which in effect stabilizes intramolecular interactions in macromolecules such as proteins (Moelbert S et al). A scale can be established for example by referring to the Hofmeister series, or lyotropic series, which is a classification of ions in order of their ability to salt out or salt in proteins (Hyde A et al).

More particularly, the kosmotropic salt may comprise (i) an anion selected from a group consisting of CO3^2-, SO₄ ^2-, S2O3^2-, H2POT, HPO₄ ^2- , acetate-, citrate-, and Cl-, and (ii) a cation selected from a group consisting of NH₄ ⁺, K⁺, Na⁺, and Li⁺. In a currently preferred embodiment, the salt is sodium acetate (NaOAc). Non-limiting examples of suitable concentrations of NaOAc include from about 5 mM to about 500 mM, such as about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mM. However, it is to be understood that other salts consisting of a combination an anion as listed under (i) and a cation as listed under (ii) may alternatively be used to elute the capsids. Non-limiting examples are NaCI, LiCI, KCI, or other equivalent metal salt suitable to use for salt elution, as is well known in the art. Non-limiting examples of suitable concentrations of NaCI include from about 5 mM to about 2M of NaCI, such as about 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, or 2000 mM, of NaCI. Further, step (b) may comprise applying a gradient of such a compound to improve elution of the adeno-associated virus capsids fully packaged with genetic material from the chromatography material. Such a gradient may be a linear gradient or a step gradient, or a combination thereof.

A non-limiting example of a suitable buffer to be applied in step (b) may comprise 20 mM bis-Tris propane (BTP), pH 9, 2 mM MgCL and 250 mM sodium acetate.

In the herein disclosed method, the chromatography material referred to in steps (a) and (b) of the method may advantageously be a polishing chromatography material, meaning that the chromatography material is applied in a polishing step.

The term "polishing step" refers in the context of liquid chromatography to a final purification step, wherein trace impurities are removed to leave an active, safe product. Impurities removed during the polishing step are often conformers of the target molecule, i.e., forms of the target molecule having particular molecular conformations, or suspected leakage products. A polishing step may alternatively be called "secondary purification step".

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

The herein disclosed method may comprise a step (al), which comprises pre-purifying adeno- associated virus capsids by separating adeno-associated virus capsids from an adeno-associated virus capsid-containing cell culture harvest, thereby obtaining a pre-purified liquid sample comprising adeno-associated virus capsids, before adding said pre-purified liquid sample comprising adeno-associated virus capsids to the chromatography material according to step (a) of the method.

Such a pre-purifying step (al) may alternatively be called a "capture step" and refers in the context of liquid chromatography to the initial step(s) of a separation procedure. Most commonly, a capture step includes clarification (e.g., by filtration, centrifugation, or precipitation), and normally also concentration and/or stabilisation of the sample, and a significant purification from soluble impurities, for example by applying chromatography after the clarification, concentration, and stabilisation of sample. After the capture step, an intermediate purification may follow, which further reduces remaining amounts of impurities such as host cell proteins, DNA, viruses, endotoxins, nutrients, components of a cell culture medium, such as antifoam agents and antibiotics, and product-related impurities, such as aggregates, misfolded species, and aggregates.

Such a pre-purifying step may comprise subjecting the adeno-associated virus capsid-containing cell culture harvest to one or more of the following non-limiting examples of purification methods:

(i) affinity chromatography,

(ii) ion exchange chromatography,

(iii) precipitation or tangential flow filtration (TFF), followed by size-exclusion chromatography, such as by use of for example Capto™ Core 400 chromatography material (Cytiva, Sweden), which combines flow-through of the capsids with 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.

Non-limiting examples of chromatography materials suitable to apply in a pre-purifying step include affinity chromatography material, ion exchange chromatography material, and size-exclusion chromatography material, respectively. The chromatography material may be functionalised with a positively charged group, such as a quaternary amino, quaternary ammonium, or amine group, or a negatively charged group, such as a sulfonate or carboxylate group. The chromatography material may be functionalised with an ion exchanger group, an affinity peptide/protein-based ligand, a hydrophobic interaction ligand, an IMAC ligand, or a DNA based ligand such as Oligo dT.

Herein, the term "cell culture" refers to a culture of cells or a group of cells being cultivated, wherein the cells may be any type of cells, such as bacterial cells, viral cells, fungal cells, insect cells, or mammalian cells. A cell culture may be unclarified, i.e., comprising cells, or may be cell-depleted, i.e., a culture comprising no or few cells but comprising biomolecules released from the cells before removing the cells. Further, an unclarified cell culture may comprise intact cells, disrupted cells, a cell homogenate, and/or a cell lysate.

The term "cell culture harvest" is used herein to denote a cell culture which has been harvested and removed from the vessel or equipment, in which the cells have been cultivated.

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

The herein disclosed method may comprise separating adeno-associated virus capsids of adeno- associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 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.

The term "variant" in relation to an adeno-associated virus (AAV) serotype 1, 2, 3, 4, 5, 6, 7, 8, or 10, as listed above, is intended to mean a modified or engineered AAV, in which the capsid structure has been modified to improve clinical performance, for example towards a specific target organ. As a non-limiting example, an AAV8 variant comprises capsid parts of AAV8 and may additionally comprise capsid parts of other AAV serotypes than AAV8, such as AAV5. However, an AAV8 variant as referred to herein must retain a significant structural similarity to a non-modified AAV8 capsid, such as retaining at least 50%, such as 60%, 70%, 80%, or 90%, of the external surface structure of a non-modified AAV8 capsid. This applies equally to a variant of AAV serotype 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, or 13, as compared to a non-modified AAV serotype 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, or 13, respectively. Further, as a non-limiting example, in the context of purification or separation of a variant of AAV8, a "variant" is herein defined as an adeno-associated virus which has a functionally equivalent binding capacity to the ligand of a specified chromatography material, compared to the binding capacity of the original AAV8 to said specified chromatography material. This applies equally to a variant of AAV serotype 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, or 13, as compared to the original AAV serotype 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, or 13, respectively. The specified chromatography material may, for example, be a strong, or partially strong, anion exchange chromatography material as disclosed in more detail elsewhere herein. A variant of an adeno-associated virus may for example be obtained by spontaneous mutation, or by engineered modification (i.e., obtained by human interaction), of one or more nucleotides of the genome of the adeno-associated virus.

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

A non-limiting example of the presently disclosed method comprises adding the liquid sample in step (a) to a chromatography material comprising a ligand defined by Formula I, wherein each of Rl, R2, and R3 is CH3, and a linker comprising vinyl sulfone, and optionally further comprising glycidol, and eluting the capsids in step (b) by use of an elution buffer comprising magnesium chloride and/or sodium acetate. Optionally, this non-limiting example of the method is for separating capsids of serotype AAV2, AAV5, AAV8 or a variant thereof.

Another non-limiting example of the presently disclosed method comprises adding the liquid sample in step (a) to a chromatography material comprising a ligand comprising N,N-diethylethylenediamine and a linker comprising vinyl sulfone, and eluting the capsids in step (b) by use of an elution buffer comprising magnesium chloride and/or sodium acetate. Optionally, this non-limiting example of the method is for separating capsids of serotype AAV2, AAV5, AAV8 or a variant thereof.

The present disclosure further provides use of the chromatography material as disclosed elsewhere herein, or use of the chromatography device as disclosed elsewhere herein, for separating adeno- associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material.

According to a non-limiting example, the present disclosure provides use of a chromatography material comprising a ligand defined by Formula I, wherein each of Rl, R2, and R3 is CH3, and a linker comprising vinyl sulfone, and optionally further comprising glycidol. Said use may be directed to separating 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 a variant thereof. Optionally, this non-limiting example of the use is for separating capsids of serotype AAV2, AAV5, AAV8 or a variant thereof. Optionally, said use may comprise applying an elution buffer comprising magnesium chloride and/or sodium acetate. According to another non-limiting example, the present disclosure provides use of a chromatography material comprising a ligand comprising N,N-diethylethylenediamine and a linker comprising vinyl sulfone. Said use may be directed to separating 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 a variant thereof. Optionally, this nonlimiting example of the use is for separating capsids of serotype AAV2, AAV5, AAV8 or a variant thereof. Optionally, said use may comprise applying an elution buffer comprising magnesium chloride and/or sodium acetate.

Adeno-associated virus capsids fully packaged with genetic material, obtainable by the herein disclosed method, as well as by the herein disclosed use of a chromatography material or chromatography device, may be subjected to subsequent steps of concentrating to a pharmaceutically relevant dose, replacing of elution buffer with a pharmaceutically acceptable buffer, and/or sterilising, thereby obtaining a pharmaceutical composition comprising adeno- associated virus capsids. Such a pharmaceutical composition may be for use in therapy, optionally for use in gene therapy. The pharmaceutical composition may be administered to a subject in a method for preventing or treating a disease or disorder related to an organ or tissue in the subject.

A person skilled in the art understands that the pharmaceutically relevant dose will depend on various factors such as, but not limited to, the disease or disorder to be treated as well as the weight and condition of the subject to be treated with a pharmaceutical composition. Pharmaceutically acceptable buffers are well known in the art and can easily be chosen by the skilled person.

It is to be understood that the present disclosure is not restricted to the below-described exemplifying embodiments thereof and that several conceivable modifications of the present disclosure are possible within the scope of the following claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated. The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. EXPERIMENTAL SECTION

Example 1:

Prototypes of chromatography material were prepared by functionalising a support material comprising a non-woven web of cellulose acetate nanofibres with an anion ligand comprising N,N- diethylethylenediamine group (herein alternatively referred to as "DAX") or a quaternary amine group (herein alternatively referred to as "Q"), by use of an activation linker either comprising glycidol ("G") or not comprising glycidol (herein alternatively referred to as "non-glycidol" or "NG") (Table 1).

Table 1. HiTrap Fibro™ 0.4 mL prototypes

Preparation of Fibro-NG-DAX (a):

Fibro-VS (NG) preparation: Fibro cellulose acetate (CA) sheets were inserted into a reactor with gauzes in between. The sheets were washed with MQ-water (3 x 5 L, 15 min per wash). The reactor was drained, the temperature increased to 30 °C and KOH reaction solution (309 g in 5500 mL 33% EtOH) added. The solution was circulated for 2 h. The reactor was drained and the sheets washed with MQ-water (4 x 5 L) and acetone (2 x 5 L). The reactor was drained and the sheets left to dry overnight. The reactor was then filled with 3 L sodium carbonate buffer (306.6 g NajCOs in 2960 mL MQ-water) and acetonitrile (1224 mL). The solution was cooled to 15 °C and then pumped to the reaction chamber. Divinyl sulfone (DVS) (945 mL) was added directly to the reaction chamber. The circulation was turned back on and the reaction left to proceed at room temperature for 6 h. The reaction mixture was drained and the sheets washed with 1:1 acetone/MQ-water (4 x 5 L) followed by MQ-water (4 x 5 L).

DAX coupling: The sheets were placed in food boxes and washed with MQ-water (4 x 100 mL, 20 min) on shaking table (84 rpm). To each box 25 mL MQ-water and 2 mL DAX were added. The boxes were placed on heated shaking tables (45 °C, 75 rpm) for 23 h. The reaction solution was decanted and the sheets were washed with MQ-water (6 x 100 mL, 20 min). Deactivation: Thioglycerol (15.2 mL) was added to Tris buffer (600 mL, Tris 0.1 M, EDTA 0.001 M, pH 10) and the pH adjusted to 8.6. This solution (153 mL) was added to the food boxes and the reactions sealed and put on the shaking table (84 rpm) in room temperature for 16 h. The reaction solution was decanted, and the sheets were washed with 20% EtOH (3 x 100 mL, 20 min) and MQ- water (3 x 100 mL, 20 min). Titration gave an ionic capacity of 162 pmol/mL of membrane.

Preparation of Fibro-NG-DAX (b):

Fibro-NG-DAX (b) was prepared in similar fashion as Fibro-NG-DAX (a) except that the coupling was performed with 2.25 mL per membrane sheet yielding a material with an ionic capacity of 203 pmol/mL of membrane.

Preparation of Fibro-G-Q:

Fibro-Glycidol-AI lyl-VS preparation: Fibro-CA laser cut discs (32 mm, 12 pieces) were washed with MQ-water (4 x 100 mL, 10 min) in a food box on a shaking table. The discs were placed between two chemically inert nets and rolled on a chemically inert plastic roll. The two rolls were placed in a beaker (600 mL) and a magnetic stirrer added in the middle. A glass stopper was used to stop the rolls from spinning during stirring. KOH (0.5 M, 150 mL) was added to the beaker and the solution stirred for 10 min. Glycidol (40 mL) was added to the beaker and the reaction stirred for 3 h. The reaction solution was decanted, and the rolls washed with MQ-water (4 x 200 mL, 10 min). KOH (1 M, 172 mL) was added, and the reaction stirred for 10 min. To the reaction, allyl glycidyl ether (AGE) (28 mL) was added and the reaction was stirred overnight.

The reaction solution was then decanted. The rolls were washed with acetone/MQ-water (1:1, 2 x 300 mL, 10 min) followed by wash with MQ-water (3 x 300 mL, 10 min). A solution of NajCOs (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 reactions stirred for 16 h. The reaction solution was decanted. The rolls were washed with Acetone/MQ-water (1:1, 2 x 300 mL, 10 min) followed by wash with MQ- water (3 x 300 mL, 10 min).

Coupling of triethylamine ammonium chloride (TMAC): In the beaker containing the functionalized membrane from above, N-bromosuccinimide (NBS) (7 g) was added in HzO:MeCN (250 mL, 75% v/v) and the reaction was left under stirring for 4 hours. The reaction solution was decanted, and the membrane washed with MQ-water (6 x 300 mL). Phosphate buffer (pH 12.01, 250 mL) was added, and the beaker was put into a water bath (30 °C). TMAC (40 mL) was added, and the pH adjusted to 11.3 with 50% KOH. The reaction was left stirring overnight. The reaction solution was then decanted, and the discs washed with water (6 x 300 mL, 10 min). Titration gave an ionic capacity of 118 pmol/mL of membrane.

Preparation of Fibro-NG-Q:

Fibro-Allyl-VS preparation: Fibro-CA laser cut discs (32 mm, 12 pieces) were washed with MQ-water (4 x 100 mL, 10 min) in a food box on a shaking table. The discs were placed between two chemically inert nets and rolled on a chemically inert plastic roll. The two rolls were placed in a beaker (600 mL) and a magnetic stirrer added in the middle. A glass stopper was used to stop the rolls from spinning during stirring. KOH (1 M, 172 mL) was added, and the reaction stirred for 10 min. AGE (28 mL) was added and the reaction was stirred overnight.

The reaction solution was decanted. The rolls were washed with Acetone/MQ-water (1:1, 2 x 300 mL, 10 min) followed by wash with MQ-water (3 x 300 mL, 10 min). A solution of NajCOs (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 reactions stirred for 16 h.

The reaction solution was then decanted. The rolls were washed with Acetone/MQ-water (1:1, 2 x 300 mL, 10 min) followed by wash with MQ-water (3 x 300 mL, 10 min). Add NBS (7 g) in H₂O:MeCN (250 mL, 75% v/v) to the beaker and leave stirring for 4 hours. Decant the reaction solution and wash with MQ-water (6 x 300 mL).

Coupling of triethylamine ammonium chloride (TMAC): Phosphate buffer (pH 12.01, 250 mL) was added to the functionalized membrane from above, and the beaker was put into a water bath (30 °C). TMAC (40 mL) was added, and the pH adjusted to 11.3 with 50% KOH. The reaction was left stirring overnight.

The reaction solution was then decanted, and the discs washed with water (6 x 300 mL, 10 min). Titration gave an ionic capacity of 100 pmol/mL of membrane.

Preparation of chromatography device:

Each of the Fibro chromatography material prototypes was assembled into a chromatography device of the type HiTrap Fibro™ (Cytiva, Sweden) 0.4 mL, herein alternatively called a Fibro unit.

Separation of AAV full and empty capsids:

The prototypes were tested using an AKTA pure P25 system with a flow rate of 10 mL/minute and peaks were detected using UV 280 and 260 nm. The Fibro unit was equilibrated in 30 membrane units (MV) of 20 mM BTP pH 9, 2 mM MgCL (buffer A). Affinity purified samples of AAV5 and AAV8 containing both full and empty capsids (approx. 30-40 % full capsids) were neutralized and diluted to approx. 1 x 10¹² viral capsids/mL in equilibration buffer (buffer A) and loaded onto the Fibro unit. After loading the unit was washed for 20 MV, followed by step elution with 20 mM BTP pH 9, 2 mM MgCLand 250 mM sodium acetate (buffer B).

For separation of AAV8 full and empty capsids, elution using 5% incremental steps of buffer B (25 MV each) resulted in empty capsids coming in the flow through (FT) and the full capsids eluting in 5% buffer B for all Fibro prototype units tested (Figure 3).

More particularly, non-glycidol Q. with IC 100 pmol/mL (NG-Q, Fig. 3A), Glycidol Q. with IC 118 pmol/mL (G-Q, Fig. 3B), non-glycidol DAX (a) with IC 162 pmol/mL (NG-DAX, Fig. 3C) and non- glycidol DAX (b) with IC 203 pmol/mL (NG-DAX, Fig. 3D) showed similar separation results.

The UV 260:280 ratios in the flowthrough peak (empty capsids, peak 1, ratio approx. 0.5 - 0.7), and in the bound and eluted peak (full capsids, peak 2, ratio approx. 1.1-1.3) were calculated and plotted for the different Fibro anion exchange ligand prototypes run in duplicate. The results were similar for Q. and DAX anion exchange ligands with different ligand density (Figure 4).

For separation of AAV5 full and empty capsids, using a Fibro non-glycidol DAX prototype with IC 162 pmol/mL, NG-DAX (a), a prescreening with 1% incremental elution steps with buffer B was used to determine the optimal elution conditions, i.e., to identify % of buffer B required for eluting the empty capsids before the full capsids start to elute (before breaking point when UV 260:280 is 1) (Figure 5A). The UV 260:280 ratio of 1 (breaking point) is indicated by an arrow at 3% buffer B. For this AAV serotype, also the empty capsids could bind to the Fibro anion exchange prototype. The conductivity during load and wash can be adjusted (increased, but still binding the full capsids) in order to have the empty capsids coming in flow-through without binding.

Based on the pre-screening results, 2.5% buffer B was used in a step 1 eluting the empty capsids, followed by a step 2 using 15% buffer B to elute the full capsids (Figure 5B). The UV260:280 ratios obtained for peak 1 and 2 correspond to typical empty and full capsid values. The calculated UV 260:280 ratios in peak 1 (empty capsids) and peak 2 (full capsids) are indicated in Fig. 5B.

Example 2: Separation of AAV8 capsids under variable conditions

Experimental designs for separation of fully packaged AAV8 capsids from empty AAV8 capsids are performed with equipment and samples as in Example 1 above, and further by use of anion exchange chromatography material as in Example 1, with the following variations:

In terms of ligand chemistry:

1) Fibro Q. analogues wherein the ligand is defined by Formula I: a. Rl, R2 is ethyl; R3 is methyl; b. Rl, R2 is methyl; R3 is CH2CHOHCH3;

2) Fibro DAX analogues, wherein the ligand is defined by Formula II: a. wherein X, for each occurrence independently, is selected from H, OH and a C1-3 group, and Ri, R?, R3 and R₄ are independently selected from H and a C1-3 group, b. wherein a C3 group is straight or branched, c. wherein a C1-3 group comprises groups independently selected from OH, O-C1-2, S-Ci-

2, NH, NHR, and NR₂, d. wherein R is selected from H and a C1-3 group.

In terms of buffers and elution conditions:

1) Different concentrations of MgCL between 1-20 mM;

2) Different NaCI linear gradient 0.1 -IM, with or without MgCL as in 1);

3) Different pH linear gradient pH 4 -10, with or without MgCL as in 1);

4) Different buffers: a. Tris b. N-Methyldiethanolamine

5) Step elutions with pH, NaCI, MgCL as in 1), 2) and 3);

6) Conditions suitable for binding and eluting both empty and full capsids;

7) All of the above with or without additives like sucrose (0.1-5%) and poloxamer 188 detergent (0.01- 1%).

Example 3: Separation of capsids of different adeno-associated virus serotypes under variable conditions

Experimental designs for separation of full capsids from empty capsids of adeno-associated virus serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV10, AAV11, AAV12, and AAV13 are performed according to the variable conditions of Example 1 and Example 2 above.

REFERENCES

WO2023285011 Al

W02015052460 Al

WO2015052465 Al 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: modelling preferential exclusion, binding and aggregate stability, Biophysical Chemistry, 2004 Dec, 112(1): 45-57

Hyde Adam M et al, General Principles and Strategies for Salting-Out Informed by the Hofmeister Series, Organic Process Research & Development, 2017, 21 (9): 1355-1370.

Claims

1. A chromatography material (1) comprising a support material (2) in the form of a convectionbased membranous structure comprising nanofibres, wherein the support material is functionalised with an anion exchange ligand (3) at a ligand density of <300 pmol/mL, wherein the chromatography material comprises a linker (4) connecting the ligand to the support material, the linker comprising a linear backbone having a length of 2-16 atoms.

2. The chromatography material according to claim 1, wherein the support material has a mean flow pore size of 0.1-2.0 pm.

3. The chromatography material according to claim 1 or 2, wherein the anion exchange ligand is defined by Formula I: wherein Ri is selected from H and C1-C3 alkyl, and Rj and R3 are independently selected from H, C1-C3 alkyl, CH2OH, and CH2CHOHCH3, preferably wherein each of Ri, R2, and R3 is CH3.

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

5. The chromatography material according to claim 1 or 2, wherein the anion exchange ligand is defined by Formula II: wherein X, for each occurrence independently, is selected from H, OH and a C1-3 group, and

Ri, R2, R3 and R₄ are independently selected from H and a C1-3 group, wherein a C3 group is straight or branched, wherein a C1-3 group comprises groups independently selected from OH, O-C1.2, S-C1.2, NH, NHR, and NR2, wherein R is selected from H and a C1-3 group.

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-methylethylenediarnine, 1,3-diaminopropane, l,3-diamino-2- hydroxypropane, 2-methyl-l,3-propanediamine and N,N-diethylethylenediamine, and preferably is N,N-diethylethylenediamine.

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

8. The chromatography material according to any one of the preceding claims, wherein the convection-based membranous structure comprises a non-woven web of polymer nanofibres, optionally wherein the polymer is a cellulosic polymer, such as cellulose acetate, or a synthetic polymer, or a combination thereof.

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

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

11. A method (100) for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material, the method comprising the following steps: a. adding (110) a liquid sample comprising adeno-associated virus capsids (6) to the chromatography material according to any one of claims 1-9, wherein the liquid sample comprises adeno-associated virus capsids of a purity of at least 90% and of a concentration of at least 10¹² adeno-associated virus capsids/ml, of which at least 10% of the adeno-associated virus capsids are adeno-associated virus capsids fully packaged with genetic material; b. eluting (120) the adeno-associated virus capsids fully packaged with genetic material from the chromatography material; wherein the adeno-associated virus capsids eluted in step (b) are eluted into at least one eluate fraction, which eluate fraction comprises at least 50% of the fully packaged adeno-associated virus capsids present in the liquid sample added in step (a), and wherein at least 60% of the adeno-associated virus capsids eluted in step (b) are fully packaged with genetic material.

12. The method according to claim 11, wherein the residence time of the adeno-associated virus capsids fully packaged with genetic material within the chromatography material is from about 0.1 seconds to about 2 minutes.

13. The method according to claim 11 or 12, wherein steps (a) and (b) comprise applying a buffer having a pH of from about 6.0 to about 10.5, such as from about 7.5 to about 9.5, optionally wherein said buffer comprises tris(hydroxymethyl)amino-methane (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 11-13, wherein step (b) comprises applying a buffer comprising a compound which improves separation between capsids fully packaged with genetic material and capsids not fully packaged with genetic material, optionally wherein said compound is selected from a carbohydrate, a divalent metal ion, a detergent, and/or a salt of a monovalent metal ion, such as a kosmotropic salt.

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

16. The method according to any one of claims 11-15, wherein the adeno-associated virus capsids are capsids of adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno- associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 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, optionally wherein the adeno-associated virus capsids are capsids of AAV2, AAV5, or AAV8.

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

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

19. The use according to the preceding claim, wherein the chromatography material is as defined in claim 4 or 7 , and wherein the adeno-associated virus capsids are capsids of adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 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, optionally wherein the adeno- associated virus capsids are capsids of AAV2, AAV5, AAV8 or a variant thereof.