Separation matrix and method for isolating target molecules - Patents.com

Abstract

The present disclosure is directed to a separation matrix comprising a plurality of chromatographic particles, each chromatographic particle comprising a core and a layer surrounding the core, the core having a first average pore diameter and the layer surrounding the core having a second average pore diameter, the second average pore diameter being at least 1.5 times the first average pore diameter, the first average pore diameter blocking diffusion of target molecules through the pores of the core and the second average pore diameter allowing at least some diffusion of target molecules through the pores of the layer surrounding the core. Also disclosed are methods for preparing such a separation matrix, the use of such a separation matrix, and methods for separating target molecules by using such a separation matrix, in particular methods for separating adeno-associated virus capsids in which genetic material is fully packaged from adeno-associated virus capsids in which genetic material is not fully packaged, and compositions obtained by said methods.

Description

[Technical field]

[0001] The present disclosure relates to the field of separation of target molecules and is directed to a separation matrix comprising a plurality of chromatographic particles, each chromatographic particle comprising a core and a layer surrounding the core, designed such that the target molecules are prevented from diffusing through the pores of the core, while the target molecules are allowed to diffuse at least in part through the pores of the layer surrounding the core.

[0002] Further disclosed are methods for preparing such separation matrices, uses of such separation matrices, and methods for separating target molecules by use of such separation matrices. In particular, methods for separating adeno-associated viral capsids in which genetic material is fully packaged from adeno-associated viral capsids in which the genetic material is not fully packaged, and compositions, including pharmaceutical compositions, obtainable by said methods, and uses of such compositions are disclosed. [Background technology]

[0003] Peak broadening in chromatography is influenced by different types of diffusion present in chromatography. Van Deemter described three different terms, A, B and C, that affect peak broadening in chromatography (see, for example, T. Edge or PJ Marriott). Term A is the so-called "eddy diffusion" and zone broadening is related to channeling due to non-ideal packing. Term B is longitudinal diffusion, and an example of longitudinal diffusion is the diffusion of analytes in the capillary of a chromatography system. Longitudinal diffusion applies well to low molecular weight molecules because they have a much higher diffusion rate in liquids than large proteins. It is important to have low dead volumes in chromatography systems to reduce longitudinal diffusion. Term C diffusion is caused by mass transfer resistance between the mobile phase and the solid support. Diffusion occurs as the analyte diffuses in and out of the pores, and the longer the distance (larger the particle size) and the more dilute the analyte, the more peak broadening appears. Increased flow rate also contributes to peak broadening since the rate of diffusion into the pores is constant and increased flow rate separates the analytes further.

[0004] To optimize the process related to the purification of a specific target molecule, unique operating conditions are required and the best separation matrix may vary. Specific processes need to be designed for the purification of peptides and proteins, nucleic acids, virus particles, etc. In the purification of antibodies, the type of antibody is decisive for the choice of separation matrix. The same applies to different serotypes of adeno-associated viruses.

[0005] Adeno-associated virus (AAV) is a non-enveloped virus that has a linear single-stranded DNA (ssDNA) genome and can be engineered to deliver DNA to target cells. Recombinant adeno-associated virus (rAAV) vectors have emerged as one of the most versatile and successful gene therapy delivery vehicles. There is an increasing demand to use viral vectors for gene therapy. AAV vectors are one of the most attractive gene transfer tools for developing novel gene therapies for muscle diseases and other disorders. Most of the early AAV gene transfer studies used AAV serotype 2 (AAV2). To further improve the efficiency and specificity of AAV-mediated gene transfer, numerous AAV serotypes and variants have been developed by viral genome engineering and / or capsid modification. The use of serotypes such as AAV8 and AAV9 has increased in recent years. The target organ determines the choice of serotype. In order to use AAV particles as vectors in therapy, it is necessary to purify the viral particles from cellular impurities such as DNA after transfection. Ultracentrifugation is efficient but not scalable. Typically, several filtration steps and several chromatography steps are used to separate AAV particles from cell cultures (eg, Weihong Qu et al.).

[0006] The therapeutic efficacy of AAV vectors depends on a high percentage of viral particles in which the genetic material of interest is fully packaged. Upstream expression systems produce a mixture of fully packaged AAV particles (containing the genetic material of interest), empty AAV particles, and AAV particles in which the genetic material of interest is partially packaged, along with impurities. Therefore, it is necessary to enrich fully packaged AAV particles in the purification process. However, there are several challenges associated with achieving efficient and scalable separation of fully packaged and empty adeno-associated virus capsids, including: - The large diversity of capsids (serotypes and variants) and cell culture differences in yield of intact capsids, which means that extensive optimization is required for the purification of each serotype or variant of adeno-associated virus. - Small differences between fully packaged and empty capsids related to some parameters relevant for purification, e.g., isoelectric point. - In addition to fully packaged and empty capsids, partially packaged capsid variants are also produced in infected host cells, and there are indications that such partially packaged, and therefore less therapeutically effective, capsids may in part be coeluted with fully packaged capsids.

[0007] Therefore, there remains a need in the art for alternative separation matrices to provide new chromatographic materials and purification strategies to speed up the purification process, improve resolution, and reduce costs, and to provide large-scale methods for downstream processing of a wide variety of target molecules. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] US6,602,990 [Patent Document 2] WO2006043896A1 [Patent Document 3] US6,428,707 [Non-patent literature]

[0009] [Non-Patent Document 1] "Styrene based polymer supports developed by suspension polymerization" (R. Arshady) Summary of the Invention [Problem to be solved by the invention]

[0010] The objective of the present disclosure is to provide a chromatographic material that is applicable to large-scale methods for the separation of target molecules and achieves improved resolution in industrial preparative chromatography. The main application area of ​​the chromatographic material disclosed here is the polishing step of separation methods, also called secondary or final purification. [Means for solving the problem]

[0011] More particularly, a first aspect of the present disclosure provides a separation matrix comprising a plurality of chromatography particles, each chromatography particle comprising a core and a layer surrounding the core, the core has a first average pore diameter and a layer surrounding the core has a second average pore diameter, the second average pore diameter being at least 1.5 times the first average pore diameter; The present invention is directed to a separation matrix in which a first average pore diameter prevents diffusion of target molecules through the pores of the core and a second average pore diameter allows at least some diffusion of target molecules through the pores of a layer surrounding the core.

[0012] A second aspect of the present disclosure relates to a method for preparing a separation matrix according to the first aspect, comprising the step of preparing a plurality of chromatographic particles.

[0013] A third aspect of the present disclosure is directed to the use of a separation matrix according to the first aspect for preparative applications, such as for separating target molecules from cell culture harvest or impurities.

[0014] A fourth aspect of the present disclosure relates to a method of preparative chromatography for separating one or more target molecules from one or more other compounds in a liquid sample, the method comprising contacting a mobile phase comprising the target molecules and the compounds with a separation matrix according to the first aspect of the present disclosure.

[0015] A fifth aspect of the present disclosure is directed to a particular method of preparative chromatography for separating one or more target molecules from one or more other compounds in a liquid sample. More specifically, the method is for separating adeno-associated viral capsids in which genetic material is fully packaged from adeno-associated viral capsids in which genetic material is not fully packaged, comprising: a) applying a liquid sample containing adeno-associated virus capsids to a weak cation exchange chromatography material, The liquid sample has a purity of at least 90% and 12 adeno-associated virus capsids at a concentration of adeno-associated virus capsids / ml, at least 10% of the adeno-associated virus capsids being fully packaged adeno-associated virus capsids in which the genetic material is; A weak cation exchange chromatography material, i. a ligand for binding to an adeno-associated virus capsid, or ii. a support comprising a separation matrix according to the first aspect of the present disclosure as described above, b) eluting the adeno-associated virus capsids in which the genetic material is fully packaged from the chromatographic material. wherein the adeno-associated virus capsids eluted in step (b) are eluted in an eluate fraction, the combined eluate fraction comprising at least 50% of the adeno-associated virus capsids of the liquid sample added in step (a), at least 60% of the adeno-associated virus capsids being fully packaged with genetic material.

[0016] The method disclosed above according to a fifth aspect comprises the steps of: separating the eluate fraction containing the adeno-associated virus capsids in which the genetic material is fully packaged, which is eluted in step (b), c1) concentrating the adeno-associated virus capsid to a pharma- ceutical suitable dose; c2) replacing the buffer applied in step (b) of claim 1 with a pharma- ceutically acceptable buffer, and / or c3) Sterilizing the eluate fraction containing the adeno-associated virus capsid

[0043] The method may further comprise subjecting the composition to one or more of: This results in a pharmaceutical composition comprising the adeno-associated virus capsid.

[0017] A sixth aspect of the present disclosure is directed to a composition comprising adeno-associated virus capsids obtained by carrying out the above method according to the fifth aspect, wherein the ratio of adeno-associated virus capsids in which the genetic material is fully packaged to adeno-associated virus capsids in which the genetic material is not fully packaged in the composition is at least 3:2.

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

[0019] [Figure 1] FIG. 1 shows a schematic diagram of a chromatography particle comprising a core and a layer surrounding the core, the chromatography particle being included in a separation matrix according to a first embodiment of the present disclosure. [Diagram 2] FIG. 1 shows a schematic diagram of a chromatography particle comprising a core, a first layer surrounding the core, and a second layer surrounding the first layer, the chromatography particle being included in a separation matrix according to a first embodiment of the present disclosure. [Figure 3A] 1 is a flow chart of a method for preparing a separation matrix comprising preparing a plurality of chromatographic particles according to a second embodiment of the present disclosure. [Figure 3B] 1 is a flow chart of a method for preparing a separation matrix comprising preparing a plurality of chromatographic particles according to a second embodiment of the present disclosure. [Figure 4] 1 is a flow chart of a method for isolating adeno-associated virus capsids according to a fifth embodiment of the present disclosure. [Diagram 5]5 is a flow chart of the method for isolating adeno-associated virus capsids according to FIG. 4, further comprising an additional step (a1) prior to step (a). [Figure 6] 5 is a flow chart of a method for isolating adeno-associated virus capsids according to FIG. 4, optionally including step (a1) before step (a) and further including one or more additional steps (c1), (c2) and / or (c3) after step (b). [Figure 7] FIG. 1 is a graph showing elution curves for fully packaged and non-fully packaged capsids following the separation method as described in Example 1 below. [Figure 8] 1 is a graph showing elution curves for monoclonal antibodies following the separation method as described in Example 2 below. [Figure 9] FIG. 1 is a graph showing pore size distribution curves for chromatography particles according to an embodiment of the invention obtained by loading polypeptides of various molecular weights into a chromatography column packed with the chromatography particles and plotting the log Mw of each polypeptide against the gel phase partition coefficient (Kav). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present disclosure solves or at least mitigates problems associated with peak broadening in chromatography associated with existing chromatography media by providing a separation matrix including a plurality of chromatography particles (10) according to a first aspect of the present disclosure. As shown generally by FIG. 1, each chromatography particle (10) includes a core (20) and a layer (30) surrounding the core (20), the core (20) having a first average pore diameter and the layer (30) surrounding the core (20) having a second average pore diameter, the second average pore diameter being at least 1.5 times the first average pore diameter, the first average pore diameter blocking diffusion of target molecules through the pores of the core (20) and the second average pore diameter at least partially allowing diffusion of target molecules through the pores of the layer (30) surrounding the core (20).

[0021] The core of the particle has an average pore diameter that does not allow diffusion of the target molecule into and within the core, so that diffusion of the target molecule is only within a layer surrounding the core (i.e., close to the surface of the particle). This results in shorter diffusion distances than would be the case if an equally sized particle with a uniform average pore diameter were used throughout its entire structure or volume.

[0022] The shorter diffusion distance leads to faster mass transfer of analytes in a chromatography column packed with the particles disclosed herein, which in turn leads to sharper and narrower peaks of eluted analytes in the resulting chromatogram, i.e., improved resolution of various analytes. The signal-to-noise ratio benefits from the sharper peaks. The resolution achieved by using the particles disclosed herein of a certain size is equal to the resolution achieved with smaller particles having a homogeneous average pore diameter. However, the back pressure generated in a chromatography column packed with particles disclosed herein of a certain size is equal to the back pressure generated in a chromatography column packed with particles of the same size having a homogeneous average pore diameter. Thus, the back pressure is lower for particles disclosed herein of a certain size than for smaller particles that may otherwise be selected to obtain the required resolution of target molecules. This is an advantage of the particles disclosed herein compared to smaller particles that cause higher back pressure and associated limitations / problems.

[0023] The chromatographic particle as disclosed herein can be said to include a porous core and a porous layer surrounding the porous core. As mentioned above, the core has a first average pore diameter, and the layer surrounding the core has a second average pore diameter, and the second average pore diameter is at least 1.5 times, for example, 1.5, 2, 2.5, 3, 3.5, 4, or 5 times, the first average pore diameter. For example, the second average pore diameter can be at least 10 times, for example at least 15 times, for example about 20 times, the first average pore diameter. The first average pore diameter prevents the diffusion of target molecules through the pores of the core, which means that the target molecules are prevented from diffusing into and through the porous structure of the core. Similarly, the second average pore diameter allows at least partial diffusion of the target molecules through the pores of the layer surrounding the core, which is intended to mean that the target molecules are allowed to diffuse completely or at least partially into and through the porous structure of the layer surrounding the core.

[0024] It should be understood that each chromatographic particle, core, or layer, defined as having a certain "average pore diameter" or "mean pore size", may nevertheless contain pores of different sizes or diameters, both pores large enough to readily allow the target molecule to diffuse into the particle and pores small enough to block the diffusion of the target molecule. This pore diameter or pore size diversity can be measured by the diffusion coefficient of molecules of well-defined molecular weight and hydrodynamic size. As a non-limiting example, dextran with a molecular weight of 140-225 KDa and a hydrodynamic diameter of 20-25 nm can be used to assess the degree of diffusion of adeno-associated virus capsids in the pores of the core and the pores of the layer surrounding the core, respectively.

[0025] The term "target molecule" has its conventional meaning in the field of bioprocessing, where the target molecule is produced (often recombinantly) by cells in cell culture and purified from the cell culture by any means of separation and purification. Alternatively, the target molecule is present in a biological solution that does not necessarily originate from a cell culture. Non-limiting examples of target molecules are biopolymers that are large biological polymers consisting of monomers linked together, such as enzymes, antibodies and antibody fragments, as well as peptides containing at least 30 amino acid residues, proteins (which may be natural or recombinant), including but not limited to oligonucleotides, nucleic acid sequences such as DNA and RNA (e.g., mRNA), and virus particles. The target molecule that is separated from other compounds by the use of the chromatographic particles disclosed herein is typically a virus particle, a protein or polypeptide, in particular a therapeutic protein or polypeptide such as an antibody, or a virus particle containing genetic material of interest (e.g., for therapeutic use), but may also be, for example, a nucleic acid. The target molecule may be, for example, a biological molecule, including but not limited to a biological polymer intended for use as a biologic, i.e., a pharmaceutical compound. More specifically, non-limiting examples of targeting molecules contemplated herein include virus particles, virus-like particles, monoclonal antibodies (including, but not limited to, monoclonal antibody variants such as bispecific, fusion, and chimeric monoclonal antibodies), and antibody fragments (including, but not limited to, antigen-binding fragments F(ab')2, Fab, Fab', and Fv). Non-limiting examples of virus particles are adeno-associated virus capsids, lentivirus capsids, adenovirus capsids, influenza virus capsids, and rotavirus capsids.Examples of adeno-associated virus capsids include capsids of various serotypes, such as adeno-associated virus serotype 9 (AAV9), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), and adeno-associated virus serotype 10 (AAV10), and variants thereof.

[0026] It should be understood that "target molecule" means a type of target molecule, and that the singular form of the term is intended to encompass multiple individual target molecules or representatives of the same type.

[0027] "Virus particle" is used herein to denote a complete infectious virus particle. It comprises a core containing the viral genome (i.e., the viral genome) in either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) form, which is surrounded by a morphologically defined shell. The shell is called the capsid. The capsid and the enclosed viral genome together constitute the so-called nucleocapsid. The nucleocapsid of some viruses is surrounded by a lipoprotein bilayer envelope. In the field of bioprocessing, with the aim of generating viral vectors for various applications, such as therapeutics, the genome of the viral particle is modified to contain a gene insert containing the genetic material of interest. The modified viral particle is allowed to infect host cells in cell culture, the viral particles grow in said host cells, and then the viral particles are purified from the cell culture by any means of separation and purification. As used herein, viral particles isolated from cell cultures by the methods disclosed herein may alternatively be referred to as "target molecules," "targets," "analytes of interest," or "analytes." "Viral particle" is intended to mean a type of viral particle, and it should be understood that the singular form of the term may encompass a large number of individual viral particles. As used herein, the term "viral particle" may be used interchangeably with the terms "vector" and "capsid," respectively, as further defined below.

[0028] The term "vector" is used herein to refer to a viral particle, usually a recombinant viral particle, intended to be used to achieve gene transfer to modify a particular cell type or tissue. Viral particles can be engineered to provide vectors that express, for example, therapeutic genes. Several types of viruses are currently being investigated for use in delivering genetic material (e.g., genes) to cells to provide transient or permanent transgene expression. These include adenoviruses, retroviruses (gamma-retroviruses and lentiviruses), poxviruses, adeno-associated viruses (AAV), baculoviruses, and herpes simplex viruses. In the present specification, the term "vector" can be used interchangeably with the terms "viral particle" and "capsid," respectively.

[0029] The term "capsid" refers to the shell of a viral particle. The capsid surrounds the core of the viral particle and should usually contain the viral genome. Modified (recombinant) capsids, as produced in the upstream process of manufacturing, are expected to contain the complete viral genome, which contains the genetic material of interest for one or more applications, for example, for various therapeutic applications. However, due to low packaging efficiency, the constructed capsids do not necessarily contain genetic material or only encapsidate truncated gene fragments, resulting in so-called empty capsids and partially filled capsids, respectively. These capsids have no therapeutic function, but still compete to bind to receptors during cell-mediated processes. This can reduce the overall therapeutic efficacy and cause undesirable immune responses. As a result, tracking these capsids throughout the production process is important to ensure consistent product quality and appropriate administration response (Xiaotong Fu et al.). In up to 20-30% of the population of artificially produced virus particles in cell culture, the capsid is only partially filled with genetic material. Moreover, in up to 98% of artificially produced virus particles, the capsid does not contain any part of the viral genome at all, i.e. is empty. However, typically between 80%-90% of artificially produced virus particles have empty capsids, and the best case currently achieves only 50% empty capsids.

[0030] As used herein, the term "capsid" may be used interchangeably with the terms "vector" and "viral particle," respectively. In the context of this disclosure, a capsid may or may not contain genetic material.

[0031] The term "genetic material of interest" is intended to mean genetic material that is deemed suitable and valuable in the field of bioprocessing to be produced by viral replication and purified so that it can be used in various applications, including but not limited to therapeutic applications. As a non-limiting example, genetic material of interest can include therapeutically relevant genetic material, such as therapeutically relevant nucleotide sequences.

[0032] The term "capsid in which the genetic material is fully packaged" refers to a capsid that is correctly produced (by a host cell), or in other words, The capsid containing the complete viral genome, or in other words, The capsid contains 100% of the viral genome, or in other words, Capsid containing a functional viral genome is used herein to represent

[0033] The viral genome comprises a genetic insert which contains the genetic material of interest as defined elsewhere herein.

[0034] A capsid that contains a complete viral genome may alternatively be referred to herein as a "complete capsid" or a "completely packaged capsid." The terms "complete capsid," "completely packaged capsid," and "capsid in which the genetic material is fully packaged" may be used interchangeably throughout this document.

[0035] The term "capsid in which the genetic material is not fully packaged" refers to a capsid that has not been properly produced (by the host cell), or in other words, Capsids that do not contain the complete viral genome, or in other words, Capsids that contain less than 100% of the viral genome is used herein to represent

[0036] Capsids in which the genetic material is not fully packaged are either partially filled with genetic material or are not filled with any genetic material at all.

[0037] The term "capsid in which genetic material is not completely packaged" encompasses the terms "partially filled capsid" and "empty capsid" as defined below.

[0038] A "partially filled capsid" refers to a capsid that contains a portion of the viral genome, such as a missing portion of the viral genome, or in other words, A capsid containing a partial viral genome, or in other words, Capsids containing incomplete viral genomes, or in other words, Capsids containing defective viral genomes, or in other words, A capsid that comprises more than 0% and less than 100% of the complete viral genome, for example, about 1% to about 99%, for example, about 5% to about 95%, for example, about 10% to about 90%, or for example, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% of the complete viral genome. As defined herein, partially filled capsids are incorrectly generated capsids, and therefore it is desirable to separate and remove as many partially filled capsids as possible from a population of capsids before using the population of capsids for its intended use, e.g., therapeutic use. As used herein, partially filled capsids may alternatively be referred to as "intermediate capsids."

[0039] An "empty capsid" is a capsid that does not contain any part of the viral genome, i.e., contains 0% of the viral genome, or in other words, Empty capsids are defined herein as capsids that are not filled with any genetic material. Thus, empty capsids do not contain any genetic material of interest. Therefore, it is desirable (e.g., may be required due to clinical regulations) to separate and remove as many empty capsids as possible from a population of capsids before using the population of capsids for its intended use, e.g., therapeutic use.

[0040] Before using a population of viral particles for its intended use, e.g., therapeutic use, it may be desirable (and may even be required, e.g., due to clinical regulations) to enrich for complete capsids, i.e., to increase the percentage of complete capsids at the expense of the percentage of partially filled and empty capsids.

[0041] The percentage of full and empty capsids in a population of capsids can be estimated or analyzed in several ways known in the art, some of which are briefly described below.

[0042] 1:A260:280 in the chromatogram will give an estimate of the percentage of complete capsids present in the peak (a ratio of 1-1.5 indicates enrichment in complete capsids, while a ratio of 0.5-0.7 contains mostly empty capsids).

[0043] 2. qPCR:ELISA ratio. qPCR quantifies viral genomes and ELISA quantifies total viral particles. The ratio of the two assays with variation will be less precise and uncertain. Orthogonal analysis is required for confirmation (see 3, 4 or 5 below).

[0044] 3. Analytical anion exchange to separate full and empty capsids (A260:280 ratio and peak area to calculate percentages). Precision dependent on peak definition.

[0045] 4. Analytical Ultracentrifugation (AUC): Detects and quantifies particles of different densities (corresponding to full, partially filled, and empty capsids). This is currently known in the art as the "gold standard". However, ultracentrifugation is not scalable and therefore not suitable for the analysis of large batches of capsids.

[0046] 5. Transmission Electron Microscopy (TEM). Image analysis to count particles (full, partially filled and empty capsids). Can introduce artifacts from sample preparation.

[0047] Some methods for estimating or analyzing the percentage of full and empty capsids in a population of capsids are described in more detail in Xiaotong Fu et al., which is hereby incorporated by reference.

[0048] The term "variant" in relation to an adeno-associated virus serotype is intended to mean an adeno-associated virus serotype in which the capsid structure has been modified, e.g., targeted to a specific target organ, to improve clinical performance. As a non-limiting example, an adeno-associated virus serotype 9 (AAV9) variant comprises the capsid portion of AAV9 and may further comprise the capsid portion of other AAV serotypes other than AAV9, such as AAV5. However, an AAV9 variant as referred to herein must retain significant structural similarity to the unmodified AAV9 capsid, e.g., retaining at least 50%, e.g., 60%, 70%, 80%, or 90% of the outer surface structure of the unmodified AAV9 capsid. In the context of purifying or isolating a variant of AAV9, a "variant" is defined herein as an adeno-associated virus that has a functionally equivalent binding ability to a ligand of a specified chromatographic material compared to the binding ability of the original AAV9 to the specified chromatographic material. A variant of an adeno-associated virus may be obtained, for example, by natural mutation or by engineered modification (i.e., by human interaction) of one or more nucleotides of the genome of the adeno-associated virus. This definition applies to all adeno-associated virus serotypes, such as adeno-associated virus serotype 9 (AAV9), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), and adeno-associated virus serotype 10 (AAV10).

[0049] In the present context of separation techniques within the field of bioprocessing, a target molecule may be defined by its molecular weight and / or by its hydrodynamic diameter. Non-limiting examples are target molecules with a molecular weight from about 50 kDa (e.g. Fab fragments, about 55 kDa), for example with a molecular weight of about 140 kDa (e.g. monoclonal antibodies, about 150 kDa), for example with a molecular weight of about 2000 kDa (e.g. adeno-associated virus capsid, about 3900 kDa). Further non-limiting examples are target molecules with a hydrodynamic diameter from about 7 nm (e.g. Fab fragments, about 7 nm), for example with a molecular weight of about 10 nm (e.g. monoclonal antibodies, about 10 nm), for example with a molecular weight of about 20 nm, for example with a molecular weight of about 25 nm (e.g. adeno-associated virus capsid, about 20-25 nm).

[0050] Similarly, the first average pore diameter of the core and the second average pore diameter of the surrounding layer of the chromatographic particles disclosed herein can be appropriately defined by the molecular weight and / or hydrodynamic diameter of the target molecule, the diffusion of which is intended to be prevented or allowed, respectively.

[0051] Thus, the first average pore diameter may block the diffusion of target molecules having a molecular weight of about 50 kDa, such as about 140 kDa, for example about 2000 kDa. Alternatively or in addition, the first average pore diameter may block the diffusion of target molecules having a hydrodynamic diameter of about 7 nm, such as about 10 nm, for example about 20 nm, for example about 25 nm.

[0052] To prevent the diffusion of the target molecule, the pores of the core have an average pore diameter that is substantially smaller than the diameter of the target molecule. In other words, the first average pore diameter is substantially smaller than the diameter of the target molecule. As used herein, the term "substantially smaller" is intended to mean at least 10% smaller, for example, but not limited to, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% smaller.

[0053] Non-limiting examples include: (i) the target molecule is an adeno-associated virus capsid and the first average pore diameter is <15 nm, including but not limited to 13, 12, 11, 10, 7.5, 5, 3, or 1.5 nm; (ii) the target molecule is a monoclonal antibody and the first average pore diameter is <10 nm, including but not limited to 9, 8, 7, 6, 5, 4, 3, 2, or 1 nm; (iii) the target molecule is an antigen-binding fragment of an antibody and the first average pore diameter is <5 nm, including but not limited to 4, 3, 2, 1, or 0.5 nm; (iv) the target molecule is a virus-like particle and the first average pore diameter is <40 nm, including but not limited to 35, 30, 25, 20, 15, 10, or 5 nm; (v) the target molecule is a lentiviral capsid and the first average pore diameter is <80 nm, including but not limited to 70, 60, 50, 40, 30, 20, or 10 nm; (vi) the target molecule is an adenovirus capsid and the first average pore diameter is <70 nm, including but not limited to 60, 50, 40, 30, 20, or 10 nm; or (vii) the target molecule is an influenza virus capsid and the first average pore diameter is <80 nm, including but not limited to 70, 60, 50, 40, 30, 20, or 10 nm.

[0054] Furthermore, the second average pore diameter can at least partially allow the diffusion of target molecules having a molecular weight of about 50 kDa, for example about 140 kDa, for example about 2000 kDa. Non-limiting examples of such target molecules are given above. Alternatively or in addition, the second average pore diameter can at least partially allow the diffusion of target molecules having a hydrodynamic diameter of about 7 nm, for example about 10 nm, for example about 20 nm, for example about 25 nm. Non-limiting examples of such target molecules are given above.

[0055] To allow the diffusion of target molecules, the pores of the layer surrounding the core have an average pore diameter that is substantially larger than the diameter of the target molecule. In other words, the second average pore diameter is substantially larger than the diameter of the target molecule. In this specification, the term "substantially larger" is intended to mean at least 10% larger, for example, but not limited to, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% larger, or 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times larger.

[0056] Non-limiting examples include: (i) the target molecule is an adeno-associated virus capsid and the pores in the layer surrounding the core have an average pore diameter of >25 nm, including but not limited to 30, 50, 75, 100, 150, or 200 nm; (ii) the targeting molecule is a monoclonal antibody and the pores in the layer surrounding the core have an average pore diameter of >11 nm, including but not limited to 15, 30, 50, 75, 100, 150, or 200 nm; (iii) the targeting molecule is an antigen-binding fragment of an antibody and the pores in the layer surrounding the core have an average pore diameter of >7 nm, including, but not limited to, 10, 15, 30, 50, 75, 100, 150, or 200 nm; (iv) the target molecule is a virus-like particle and the pores in the layer surrounding the core have an average pore diameter of >60 nm, including but not limited to 75, 100, 150, or 200 nm; (v) the target molecule is a lentiviral capsid and the pores in the layer surrounding the core have an average pore diameter of >100 nm, including but not limited to 120, 150, or 200 nm; (vi) the target molecule is an adenovirus capsid and the pores in the layer surrounding the core have an average pore diameter of >90 nm, including but not limited to 110, 150, or 200 nm; or (vii) The target molecule is an influenza virus capsid, and the pores in the layer surrounding the core have an average pore diameter of >100 nm, including but not limited to 120, 150, or 200 nm.

[0057] Furthermore, it should be understood that the ability of target molecules to diffuse through the pores of a chromatographic particle is not only determined by the average pore diameter, but is also influenced by parameters such as flow rate and residence time in the chromatographic column, all potentially relevant parameters were considered in arriving at the above specified ranges and non-limiting examples of average pore diameters that are representative of suitable average pore diameters in relation to the separation of target molecule types as specified above.

[0058] The chromatography particles disclosed herein may have a diameter of about 20 μm to about 500 μm, such as about 30 μm to about 150 μm, for example about 40 μm to about 140 μm, for example about 60 μm to about 120 μm, or for example about 100 μm to about 450 μm, such as about 100 μm to about 400 μm, for example about 120 μm to about 380 μm, for example about 150 μm to about 350 μm, or about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 μm.

[0059] The core can have a diameter of about 60% to about 97% of the diameter of the chromatography particle, for example, about 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, or 97% of the diameter of the chromatography particle.

[0060] The layer surrounding the core can have a diameter of about 3% to about 40% of the diameter of the chromatography particle, for example, about 6% to about 10% of the diameter of the chromatography particle, or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, or 40%. It should be understood that the diameter of the layer surrounding the core is intended to mean the distance consisting of two radii of the layer surrounding the core, the two radii of the layer surrounding the core extending on opposite sides of the core, each radius extending from the inner end of the layer (i.e. adjacent the outer end of the core) to the outer end of the layer. In other words, the radius of the layer surrounding the core can be about 1.5% to about 20% of the diameter of the chromatography particle, for example, about 3% to about 5%, or about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20% of the diameter of the chromatography particle.

[0061] A non-limiting example of a chromatographic particle that is useful within the context of the present disclosure is a particle having a diameter of about 40 μm, comprising a core having a diameter of about 31 μm to about 37 μm, e.g., about 31, 32, 33, 34, 35, 36, or 37 μm, and a layer surrounding the core having a diameter of about 3 μm to about 9 μm, e.g., about 3, 4, 5, 6, 7, 8, or 9 μm.

[0062] Another non-limiting example of a chromatographic particle that is useful within the context of the present disclosure is a particle having a diameter of about 90 μm, comprising a core having a diameter of about 81 μm to about 87 μm, e.g., about 81, 82, 83, 84, 85, 86, or 87 μm, and a layer surrounding the core having a diameter of about 3 μm to about 9 μm, e.g., about 3, 4, 5, 6, 7, 8, or 9 μm.

[0063] Yet another non-limiting example of a chromatographic particle that is useful within the context of the present disclosure is a particle having a diameter of about 300 μm, comprising a core having a diameter of about 291 μm to about 297 μm, e.g., about 291, 292, 293, 294, 295, 296, or 297 μm, and a layer surrounding the core having a diameter of about 3 μm to about 9 μm, e.g., about 3, 4, 5, 6, 7, 8, or 9 μm.

[0064] As shown diagrammatically by Figure 2, the above-mentioned layer (30) surrounding the core (20) may be a first layer, and the chromatographic particle (10) may further comprise a second layer (40) surrounding the first layer (30), the second layer (40) having a third average pore diameter different from the first average pore diameter and the second average pore diameter. The third average pore diameter may allow a higher diffusion rate of the target molecules (i.e. through the pores of the second layer) than the diffusion rate allowed by the second average pore diameter (i.e. through the pores of the first layer).

[0065] The chromatography particles may be substantially spherical, elongated, or irregularly shaped. It should be understood that Figure 1 is a schematic diagram of one embodiment of a chromatography particle that is useful within the context of the present disclosure, and Figure 2 is a schematic diagram of another embodiment of a chromatography particle that is useful within the context of the present disclosure, and that chromatography particles having other morphologies than substantially spherical may alternatively be used.

[0066] The chromatography particles can be made of organic or inorganic materials. In one embodiment, the chromatography particles are prepared from natural polymers such as cross-linked carbohydrate materials, e.g., agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, pectin, starch, etc. Natural polymer chromatography particles are easily prepared and optionally cross-linked according to standard methods such as inverse suspension gelation (S. Hjerten). In a particularly advantageous embodiment, the chromatography particles are a type of relatively rigid but porous agarose, which is prepared by methods that enhance its flow properties, see for example US 6,602,990. In an alternative embodiment, the chromatography particles are prepared from synthetic polymers or copolymers, such as cross-linked synthetic polymers, e.g., styrene or styrene derivatives, divinylbenzene, acrylamide, acrylate esters, methacrylate esters, vinyl esters, vinyl amides, etc. Such synthetic polymers are easily prepared and optionally crosslinked according to standard methods, see for example "Styrene based polymer supports developed by suspension polymerization" (R. Arshady). Natural or synthetic porous polymeric chromatographic particles are also available from commercial sources such as Cytiva, Sweden. In yet an alternative embodiment, the chromatographic particles are prepared from inorganic polymers such as silica. Inorganic porous chromatographic particles are well known in the art and are easily prepared according to standard methods. The chromatographic particles may be of natural or synthetic origin, preferably a polysaccharide such as agarose. The chromatographic particles may be dried (e.g. dried agarose particles) and are immersed in a liquid during use in order to retain their original morphology.

[0067] The term "separation matrix" is used herein to describe a material comprising a support to which one or more ligands containing functional groups are coupled. The functional groups of the ligands bind to compounds, also referred to herein as analytes or target molecules, to be separated from the liquid sample and / or from other compounds present in the liquid sample. In the context of the present disclosure, the support of the separation matrix comprises or consists of the chromatographic particles disclosed herein, as described in detail herein. The separation matrix may further comprise a compound that couples the ligand to the support. The terms "linker", "extender", and "surface extender" may be used to describe such compounds, as further described below. The term "resin" is sometimes used for separation matrices in the art. The terms "chromatographic material" and "chromatographic matrix" are used herein to describe types of separation matrices.

[0068] The separation matrix can be contained in any type of separation device as further defined elsewhere herein. As a non-limiting example, the chromatographic material can be packed into a chromatographic column and then the liquid sample can be added to the chromatographic material contained in the chromatographic column.

[0069] Accordingly, chromatography columns comprising a plurality of the presently disclosed chromatography particles, or the presently disclosed separation matrices, as described in detail elsewhere herein, are also useful for the purposes of the present disclosure.

[0070] Optionally, the chromatography column can be a bioprocessing chromatography column, including but not limited to a fast protein liquid chromatography (FPLC) column.

[0071] Optionally, the chromatography column can be a disposable chromatography column.

[0072] In this context, a "ligand" is a molecule that has a known or unknown affinity for a given analyte and includes any functional group or capture agent immobilized on its surface, whereas an "analyte" includes any specific binding partner for a ligand. The term "ligand" may be used interchangeably herein with the terms "specific binding molecule," "specific binding partner," "capture molecule," and "capture agent." As used herein, a molecule in a liquid sample that is intended for separation from the liquid sample and that interacts with a ligand may be referred to as an "analyte of interest," "analyte," "target molecule," or "target."

[0073] The term "liquid sample" as used herein should be understood to encompass any type of sample obtained from a cell culture, or a fluid derived from a cell culture, which has been at least partially purified by any means of separation and purification. For example, the liquid sample may comprise a supernatant obtained from cell fermentation, or the liquid sample may comprise a crude feedstock.

[0074] The term "surface" as used herein means all external surfaces, and in the case of porous supports such as porous particles, includes the outer surfaces and pore surfaces.

[0075] The term "eluting agent" is used in its conventional sense in the art, ie a buffer of suitable pH and / or ionic strength to release one or more compounds from the separation matrix.

[0076] The term "eluate" is used in its conventional sense in the art, ie, the portion of a liquid sample that is eluted from a chromatography column after the liquid sample has been loaded onto the chromatography column.

[0077] The separation matrix may advantageously be a polishing chromatographic material, meaning that the chromatographic material is applied in a polishing step.

[0078] The term "polishing step", in the context of liquid chromatography, refers to a final purification step in which trace impurities are removed to leave an active and safe product. The impurities removed during the polishing step are often conformers of the target molecule, i.e., forms of the target molecule with a particular molecular conformation, or spurious breakthrough products. The polishing step may alternatively be referred to as a "secondary purification step".

[0079] As mentioned above, the separation matrix comprises a support in the form of a plurality of chromatographic particles (10) to which one or more ligands are coupled. The chromatographic particles (10) can therefore be said to comprise a ligand for binding to a target molecule. At least the layer (30) surrounding the core (20) of the chromatographic particles comprises a ligand. Optionally, the core (20) of the chromatographic particles (10) may also comprise a ligand. Whether the core comprises a ligand depends on which method was used to prepare the chromatographic particles, as described further below.

[0080] It should be understood that "a ligand" and "the ligand" refer to a type of ligand, and that the singular form of the term is intended to encompass multiple individual ligands or representatives of the same type.

[0081] The ligand may be a cationic ligand, an anionic ligand, a hydrophobic interaction ligand, a multimodal ligand, or a combination thereof. It should be understood that the type of ligand used depends on the type of target molecule to be separated from the liquid sample. Different types of target molecules have different binding characteristics, including but not limited to different surface charges, and therefore different types of ligands are required to separate different types of target molecules. For example, different serotypes of adeno-associated virus may require different types of ligands to achieve sufficient separation between fully packaged capsids and not fully packaged capsids.

[0082] Below are listed non-limiting examples of previously known chromatographic materials, including non-limiting examples of such different types of ligands, any one or combination of these different types of ligands can be coupled to the chromatographic particles disclosed herein in place of the support material of that type of known product.

[0083] Capto Q (www.cytivalifesciences.com) provided by Cytiva, Sweden is a non-limiting example of a strong anion exchange chromatographic material with about 100% quaternized amine groups. Capto DEAE (Cytiva, Sweden) is a non-limiting example of a strong anion exchange chromatographic material with a degree of quaternization of about 15% of the amine groups.

[0084] As used herein, the term "strong anion exchange chromatographic material" is intended to mean a chromatographic material that includes a ligand that includes a quaternized amine group. A quaternary amine group is a strong anion exchange group that is always positively charged regardless of the Ph to which it is exposed. For DEAE-based types of chromatographic materials, the degree of quaternization of the amine groups can vary among the amine groups contained in the chromatographic material. A degree of quaternization of about 12% to about 100% of the amine groups in the entire chromatographic material is generally considered to result in a chromatographic material that behaves like a strong anion exchange chromatographic material, since at least 12% of all these amine groups are always charged. In contrast to quaternized amine groups, almost all other ion exchange groups are weak, i.e., their charge varies from fully charged to uncharged within the reasonable range of Ph used (e.g., Ph 2-11), and have a neutral charge at Pi (equal amounts of + and - charges).

[0085] Chromatographic materials containing carboxymethyl groups (abbreviated as CM) as ligands are non-limiting examples of weak cation exchange chromatographic materials provided by Cytiva, Sweden.

[0086] A non-limiting example of a strong cation exchange material is Capto S ImpAct (Cytiva, Sweden), which contains sulfonate (S) as the strong cation exchange ligand.

[0087] Another non-limiting example of a strong cation exchange material is Capto SP ImpRes (Cytiva, Sweden), in which the ligand contains sulfopropyl (SP) groups.

[0088] A non-limiting example of a multimodal anion exchange chromatographic material containing hydrophobic groups is the chromatography resin called Capto adhere or Capto adhere ImpRes (Cytiva, Sweden). The multimodal anion exchange ligand is an N-benzyl-N-methylethanolamine ligand coupled to a support, said support being linked to the nitrogen atom of the ligand by a linker. This ligand and chromatographic materials comprising this ligand are further described, for example, in WO2006043896A1, which is incorporated herein by reference in its entirety.

[0089] Capto MMC or Capto MMC Impres are non-limiting examples of multimodal weak cation exchange chromatography materials (Cytiva, Sweden). The ligand contains a carboxyl group. Therefore, its characteristics are in part similar to weak cation exchangers. Hydrogen bonds and hydrophobic interactions are also included.

[0090] Further, a non-limiting example of a hydrophobic interaction chromatographic material is aromatic phenyl Sepharose 6 Fast Flow (Cytiva, Sweden), and another non-limiting example of a hydrophobic interaction chromatographic material is aliphatic butyl Sepharose 4 Fast Flow (Cytiva, Sweden).

[0091] Regardless of which type of ligand (i.e., cationic ligand, anionic ligand, hydrophobic interaction ligand, multimodal ligand, or a combination thereof) is selected, the ligand may be optionally linked to the surface of the chromatographic particles disclosed herein via a linker, i.e., the coupling of the ligand to the particle may be achieved by introducing a linker between the support and the ligand. The coupling may be carried out according to any conventional covalent coupling method, such as by using epichlorohydrin; epibromohydrin; allyl-glycidyl ether; bis-epoxides such as butanediol diglycidyl ether; halogen-substituted aliphatic substances such as di-chloro-propanol; and divinyl sulfone. Other non-limiting examples of suitable linkers are polyethylene glycols (PEG) having 2 to 6 carbon atoms, carbohydrates having 3 to 6 carbon atoms, and polyhydric alcohols having 3 to 6 carbon atoms. All of these methods are well known in the art and are easily carried out by the skilled artisan.

[0092] The ligand may optionally be coupled to the support via a longer linker molecule, also known as a "surface extender" or simply an "extender". Extenders are well known in the art and are commonly used to sterically increase the distance between the ligand and the support. Extenders may be represented as tentacles or flexible arms. The function of linkers, especially extenders, is to increase the surface area of ​​the support and to allow its functionalization. For a more detailed description of possible chemical structures, see, for example, US 6,428,707, which is incorporated herein by reference in its entirety. Briefly, the extender may be in the form of a polymer, such as a homopolymer or copolymer. The hydrophilic polymeric extender may be of synthetic origin, i.e. with a synthetic backbone, or of biological origin, i.e. with a biopolymer with a naturally occurring backbone. Typical synthetic polymers are polyvinyl alcohol, polyacrylamide and polymethacrylamide, polyvinyl ethers, etc. Typical biopolymers are polysaccharides, such as starch, cellulose, dextran, agarose, etc.

[0093] According to a second aspect, the present disclosure provides a method for preparing a separation matrix according to the first aspect, comprising preparing a plurality of chromatographic particles (10), either according to steps (a) and (b) as illustrated by the flow chart in FIG. 3a, or according to steps (a′) and (b′) as illustrated by the flow chart in FIG. 3b: (a) providing as a starting material a chromatographic particle having a uniform average pore diameter, the uniform average pore diameter at least in part allowing target molecules to diffuse through the pores of the chromatographic particle throughout the entire structure or volume of the chromatographic particle; (b) adding a polymeric compound to the interior (i.e., inner or central portion) of the core of the chromatography particle provided in step (a), thereby reducing the average pore diameter of the core compared to the homogeneous average pore diameter of the chromatography particle provided in (a), thereby obtaining a chromatography particle comprising a core and a layer surrounding the core, the core having a first average pore diameter and the layer surrounding the core having a second average pore diameter, the second average pore diameter corresponding to the homogeneous average core diameter of the chromatography particle provided in (a); Contains, or (a') providing a chromatography particle having a first average pore diameter that inhibits diffusion of a target molecule through the pores of the chromatography particle throughout the entire structure or volume of the chromatography particle; (b') embedding the chromatography particles provided in step (a') in a surrounding layer having a second average pore diameter; Including, the first average pore diameter and the second average pore diameter are different from each other, and the second average pore diameter is at least 1.5 times the first average pore diameter; The method provides a method in which the first average pore diameter prevents diffusion of the target molecules through the pores of the core and the second average pore diameter at least partially allows diffusion of the target molecules through the pores of a layer surrounding the core.

[0094] In this specification, the term "homogeneous average pore diameter" is intended to mean that a particle with a homogeneous average pore diameter has a homogeneous (or essentially the same) average pore diameter throughout its entire structure or volume. The chromatography particle with a homogeneous average pore diameter provided as starting material in step (a) of the method according to the second embodiment has an average pore diameter that allows the target molecules to diffuse completely or at least partially through the pores of the particle throughout its entire structure or volume.

[0095] Further, step (b) of the above method according to the second aspect may further comprise the steps of (i) or (ii): (i) the coupling of polymer molecules to the core of the particle, or (ii) polymer grafting, which involves adding a monomer to the core of the particle and polymerizing the monomer inside the core, thereby creating polymer molecules inside the core; may include either This reduces the average pore diameter of the core.

[0096] To reduce the average pore diameter of the core of a particle having a uniform average pore diameter, polymer molecules can be coupled to the inside of the core of the particle. For example, the average pore diameter can be reduced by a factor of 2 or less, for example, a factor of 3, a factor of 4, a factor of 5, a factor of 6, a factor of 7, a factor of 8, a factor of 9, or a factor of 10. For example, polymer natural molecules such as dextran (e.g., dextran T5 (molecular weight 5 kDa) or dextran T3.5 (molecular weight 3.5 kDa)), or polymer synthetic molecules such as polyvinyl alcohol or polyvinyl pyrrolidone, or combinations thereof, can be coupled to the core to reduce the average pore diameter. Alternatively, polymer molecules can be produced directly in the core of the particle through polymer grafting, i.e., by adding a monomer to the core and then polymerizing this monomer inside the core of the particle.

[0097] Thus, the core of the chromatographic particle may include a polymeric compound that reduces the average pore diameter of the core compared to the average pore diameter of the layer surrounding the core. Optionally, such a polymeric compound may be dextran or a synthetic polymer.

[0098] The chromatographic material used in Example 1 herein further comprises a support in the form of a substantially spherical chromatographic particle or bead, more precisely called ImpRes (Cytiva, Sweden), which, as mentioned above, has a diameter of about 40 μm and which, for the purposes of the present disclosure, has been modified to obtain a smaller average pore diameter in the core of the particle than in the layer surrounding the core. The unmodified ImpRes particle is a non-limiting example of a particle with a homogeneous average pore diameter throughout its entire structure or volume, allowing at least in part the diffusion of target molecules of approximately the same size as the adeno-associated virus capsid. As mentioned above, dextran with a molecular weight of 140-225 kDa and a hydrodynamic diameter of 20-25 nm (i.e. a diameter of the same size as the adeno-associated virus capsid) can be used to evaluate the degree of diffusion of the adeno-associated virus capsid in the pores of the particle. Unmodified ImpRes particles have a Kd for dextran between 0.3 and 0.61, indicating that capsids have the potential to diffuse into approximately 30-60% of the pores.

[0099] ImpRes particles may be modified by coupling or grafting polymer molecules to the interior of their core to create particles with smaller average pore diameters in their core than in the layers surrounding the core, resulting in chromatographic particles that are useful for the purposes of the present disclosure.

[0100] The chromatographic material used in Example 2 herein comprises a support in the form of substantially spherical chromatographic particles or beads, more precisely called Sepharose FF (Cytiva, Sweden), having a diameter of about 90-100 μm and modified for the purposes of this disclosure to obtain a smaller average pore diameter in the core of the particle than in the layer surrounding it. Unmodified Sepharose FF particles are a non-limiting example of particles with a homogeneous average pore diameter throughout their entire structure or volume that allows at least in part the diffusion of target molecules of approximately the same size as monoclonal antibodies. Unmodified Sepharose FF particles have a Kd for Dextran 50k (i.e., dextran with a molecular weight of about 50000 Da and a diameter of approximately the same size as an IgG antibody) between 0.5 and 0.6, indicating that monoclonal antibodies have the possibility of diffusing in approximately 50-60% of the pores.

[0101] Sepharose FF particles may be modified by coupling or grafting polymer molecules to the interior of their core to create particles with an average pore diameter in their core that is smaller than in the layers surrounding the core, thereby resulting in chromatographic particles that are useful for the purposes of the present disclosure.

[0102] Chromatographic particles comprising a core and a layer surrounding the core, where the layer surrounding the core at least partially allows diffusion of a target molecule and the core blocks diffusion of the same type of target molecule, may be alternatively referred to herein as "semi-solid beads," "semi-solid particles," "semi-solid resins," "semi-solid matrices," or the like.

[0103] Semi-solid particles for use in the separation of target molecules may for example be produced by preparing semi-solid particles according to any one of the methods according to the second aspect as described above and by coupling a ligand having affinity for the target molecule to the particle. The coupling of the ligand having affinity for the target molecule may be carried out before or after preparing the semi-solid particles according to the method according to the second aspect.

[0104] Since the core of the particle is occupied by polymer molecules, the ligands accessible to the target molecule are only in the layer surrounding the core. Furthermore, since the core of the particle has an average pore diameter that does not allow the target molecule to diffuse into the core, the diffusion of the target molecule is only in the layer surrounding the core (i.e., close to the surface of the particle). This results in a shorter diffusion distance than when using an equal-sized particle with a homogeneous average pore diameter throughout its entire structure or volume. As further explained in more detail above, this means that the use of semi-solid particles according to the present disclosure can result in advantageous effects such as improved resolution and faster separation compared to an equal-sized particle with a homogeneous average pore diameter. When the core of the particle is filled with polymer molecules, the total surface area of ​​the chromatographic material is reduced, and therefore the binding capacity of the chromatographic material is reduced. However, in some chromatographic applications, the resolution between chromatographic peaks is more important than the binding capacity of the target molecule.

[0105] The results shown in Example 1 below show that the use of semi-solid beads improves the separation of fully packaged and not fully packaged adeno-associated virus capsids when the chromatographic material comprises a weak cation exchange chromatographic material. More specifically, the results show that the resolution of peaks in chromatograms is improved when semi-solid beads are used compared to the use of beads with a uniform average pore diameter throughout their entire structure or volume. Thus, it is demonstrated that the use of semi-solid beads improves the separation of full capsids on the one hand and empty and partially filled capsids on the other hand.

[0106] When the target molecule is a monoclonal antibody (mAb), as in this Example 2, the layer surrounding the core of the particle allows at least some diffusion of the monoclonal antibody. The size of a monoclonal antibody is approximately 150 kD, which is approximately 10-15 nm in diameter. The results shown in Example 2 below show that the use of semi-solid beads can result in reduced peak widths when a linear elution gradient is used to separate various proteins, including monoclonal antibodies. The results also showed that when the flow rate was increased, the peak width and resolution of the resolved peaks were not affected for commercial chromatographic beads SP Sepharose FF (Cytiva, Sweden), which have a homogenous average pore diameter equal to that of the layer surrounding the core of the semi-solid beads tested in Example 2. A drawback of using semi-solid beads was that the dynamic protein binding capacity was reduced. However, as mentioned above, the resolution between chromatographic peaks may be more important than the binding capacity of the target molecule.

[0107] For the purposes of the present disclosure, a method for manufacturing a chromatography column is also contemplated, the method comprising the steps of preparing a separation matrix (according to the first aspect of the present disclosure), optionally preparing a plurality of chromatography particles by carrying out the method according to the second aspect (as described above), followed by adding the separation matrix comprising a plurality of the thus prepared chromatography particles to a chromatography column, and sterilizing the chromatography particles or the chromatography column comprising the separation matrix.

[0108] A third aspect of the present disclosure is directed to the use of a separation matrix (according to the first aspect as described in detail elsewhere herein) for preparative applications, such as for separating target molecules from cell culture harvest or impurities.

[0109] As used herein, the term "cell culture" refers to a culture of cells or a group of cultured cells, where the cells can be any type of cell, such as bacterial cells, viral cells, fungal cells, insect cells, or mammalian cells. The cell culture may be unclarified, i.e., contain cells, or may be cell-depleted, i.e., cultures that contain no or few cells, but contain biomolecules released from the cells prior to cell removal. Additionally, unclarified cell cultures may contain intact cells, disrupted cells, cell homogenates, and / or cell lysates.

[0110] The term "cell culture harvest" is used herein to refer to a cell culture that has been harvested and removed from the vessel or device in which the cells were cultured.

[0111] As described in more detail elsewhere herein, the target molecule to be separated from the cell culture harvest or impurities by use of the chromatography particles disclosed herein can be, for example, an adeno-associated virus capsid, a monoclonal antibody, an antigen-binding fragment of an antibody, a virus-like particle, a lentivirus capsid, an adenovirus capsid, or an influenza virus capsid.

[0112] A fourth aspect of the present disclosure relates to a method of preparative chromatography for separating one or more target molecules from one or more other compounds in a liquid sample, comprising contacting a liquid sample containing the target molecules and compounds with a separation matrix (according to the first aspect) or with a chromatography column containing such a separation matrix, and exposing the liquid sample to a differential pressure (or "delta pressure") of <5 bar, such as <3 bar, or such as 4.5, 4, 3.5, 3, 2.5, or 2 bar. The term "preparative chromatography" is used herein in conjunction with the main application area of ​​the chromatographic particles disclosed herein, as opposed to analytical applications and high pressure liquid chromatography (HPLC), for which the chromatographic particles disclosed herein are not suitable. Preparative chromatography may be defined herein by the application of a delta column pressure not exceeding 5 bar, preferably not exceeding 3 bar.

[0113] When the method according to the fourth aspect comprises the step of contacting a liquid sample comprising the target molecule with a chromatography column containing a separation matrix, the mobile phase containing the liquid sample may be passed through said chromatography column by gravity and / or by pumping, (a) the target molecule may be recovered in the flow-through of a chromatography column, in which case the compounds adsorbed to the separation matrix are host cell proteins and / or other impurities, or (b) The target molecule may be recovered in the eluate fraction from a chromatography column, where host cell proteins and other impurities are found in the column flow-through.

[0114] The method according to the fourth aspect may further comprise a step of clarification (e.g. by filtration, centrifugation or precipitation), and optionally concentration and / or stabilization, and / or chromatography, prior to the above-mentioned step of contacting the mobile phase comprising the target molecule and the compound with a separation matrix or with a chromatography column. Said step of clarification and / or chromatography may comprise a step of pre-purifying the target molecule, for example by isolating it from the cell culture harvest, thereby obtaining a pre-purified liquid sample comprising the target molecule. Said pre-purified liquid sample may then be contacted with a separation matrix or with a chromatography column.

[0115] Such a pre-purification step may alternatively be referred to as a "capture step" and, in the context of liquid chromatography, refers to an initial step in a separation procedure. Such a pre-purification step may include subjecting the target molecule-containing cell culture harvest to one or more of the following non-limiting examples of purification methods: (i) affinity chromatography; (ii) ion exchange chromatography; (iii) precipitation or tangential flow filtration (TFF) followed by size exclusion chromatography, for example by using Capto Core 400 chromatographic material (Cytiva, Sweden), which combines flow-through of the target molecule and binding of impurities to the chromatographic material; (iv) TFF followed by ion exchange chromatography; and (v) TFF followed by ion exchange chromatography and Capto Core.

[0116] Non-limiting examples of chromatographic materials suitable for application in the pre-purification step of the above method include affinity chromatographic materials, ion exchange chromatographic materials, and size exclusion chromatographic materials, respectively. The chromatographic materials may be functionalized with positively charged groups such as quaternary amino, quaternary ammonium, or amine groups, or negatively charged groups such as sulfonate or carboxylate groups. The chromatographic materials may be functionalized with ion exchange groups, affinity peptide / protein-based ligands, hydrophobic interaction ligands, IMAC ligands, or DNA-based ligands such as Oligo dT.

[0117] The term "separation device" has its conventional meaning in the art of bioprocessing and should be understood to encompass any type of separation device capable of and suitable for separating and purifying a compound from a fluid containing a by-product from the production of the compound. A separation device may include a separation matrix as further defined elsewhere herein.

[0118] Non-limiting examples of separation devices suitable for use in the capture step or pre-purification step as described herein are filtration devices, chromatography columns and membrane devices. Chromatography columns suitable for use in the capture step can be packed with, for example, affinity chromatography material, ion exchange chromatography material, mixed mode chromatography material or hydrophobic interaction chromatography material.

[0119] A fifth aspect of the present disclosure is directed to a particular method of preparative chromatography for separating one or more target molecules from one or more other compounds in a liquid sample. More specifically, the present disclosure provides a method for separating adeno-associated viral capsids in which genetic material is fully packaged from adeno-associated viral capsids in which genetic material is not fully packaged, as shown in FIG. 4, comprising: a) applying a liquid sample containing adeno-associated virus capsids to a weak cation exchange chromatography material, The liquid sample has a purity of at least 90% and 12 adeno-associated virus capsids at a concentration of adeno-associated virus capsids / ml, at least 10% of the adeno-associated virus capsids being fully packaged adeno-associated virus capsids with genetic material therein; A weak cation exchange chromatography material, i. a ligand for binding to an adeno-associated virus capsid, or ii. A support comprising a separation matrix according to the first aspect of the present disclosure as described above. A process comprising: b) eluting the adeno-associated virus capsids in which the genetic material is fully packaged from the chromatographic material. wherein the adeno-associated virus capsids eluted in step (b) are eluted in an eluate fraction, which combined eluate fraction comprises at least 50% of the adeno-associated virus capsids of the liquid sample added in step (a), at least 60% of the adeno-associated virus capsids having fully packaged genetic material, thereby solving or at least alleviating problems associated with existing methods for separating fully packaged from non-fully packaged adeno-associated virus capsids.

[0120] Notable advantages of the method according to the fifth aspect of the present disclosure include that it is suitable for large-scale separation of fully packaged capsids and not fully packaged capsids, and provides an improved ratio of fully packaged capsids to not fully packaged capsids compared to prior art methods. By carrying out the method, it is possible to obtain a composition having a ratio of fully packaged capsids to not fully packaged capsids of at least 3:2. More specifically, the method disclosed herein provides an improved ratio of fully packaged capsids to empty capsids, and an improved ratio of fully packaged capsids to partially packaged capsids.

[0121] Definitions of the following terms can be found further above in this document: "genetic material of interest," "capsid in which the genetic material is fully packaged," "capsid in which the genetic material is not fully packaged," "partially filled capsid," and "empty capsid."

[0122] Before using a population of viral particles for its intended use, e.g., therapeutic use, it may be desirable (and may even be required, e.g., due to clinical regulations) to enrich for complete capsids, i.e., to increase the percentage of complete capsids at the expense of the percentage of partially filled and empty capsids. The percentage of complete and empty capsids in a population of capsids can be estimated or analyzed in several ways known in the art. Some of these methods have been further briefly described above.

[0123] As mentioned above, in the method for separating fully packaged capsids from not fully packaged capsids, the liquid sample applied to the chromatographic material in step (a) has a purity of at least 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and a purity of at least 10 12 , e.g., 10 13 , 10 14 , or 10 15adeno-associated virus capsids at a concentration of adeno-associated virus capsids / ml, of which at least 10%, e.g., 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% are adeno-associated virus capsids in which the genetic material is fully packaged. With respect to the purity of adeno-associated virus capsids in a liquid sample, a purity of at least 90%, e.g., up to 99%, is intended to mean that at least 90%, e.g., up to 99%, of the biological material in the liquid sample is represented by adeno-associated virus capsids (including full, empty, and partially filled capsids), while the remainder, up to 10%, e.g., 1%, is represented by host cell proteins and DNA.

[0124] The objective of step (b) of the method disclosed above is to obtain fully packaged capsids of the highest possible purity. Those skilled in the art will readily understand that this can be achieved by applying a variety of different separation conditions. A non-limiting example of a separation condition for obtaining fully packaged capsids of the highest possible purity is one that allows the binding of incompletely packaged capsids to a chromatographic material, but does not allow the binding of incompletely packaged capsids to a chromatographic material. (i) allowing fully packaged capsids to substantially flow through the chromatographic material (i.e., fully packaged capsids that do not substantially bind to the chromatographic material); or (ii) separation conditions that allow fully packaged capsids to bind to the chromatographic material and subsequently elute them from the chromatographic material. It should be understood that in the bind-elute process described in item (ii), fully packaged capsids may be eluted from the chromatographic material before or after non-fully packaged capsids, depending on which separation conditions are applied.

[0125] As mentioned above, there are slight differences between fully packaged and not fully packaged capsids in relation to some parameters relevant for purification, such as their isoelectric points. This often leads to (at least partial) co-elution of fully packaged and not fully packaged capsids. Thus, in reality, the adeno-associated virus capsids eluted in step (b) of the method disclosed above will not be completely separated into full, empty, and partially filled capsids. However, there will be an eluate fraction that contains a substantially higher percentage of full capsids than in the liquid sample applied to the chromatographic material in step (a). More specifically, as disclosed above, the adeno-associated virus capsids eluted in step (b), i.e., adeno-associated virus capsids with fully packaged genetic material, are eluted in an eluate fraction, which combined eluate fraction comprises at least 50%, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, of the adeno-associated virus capsids of the liquid sample added in step (a), of which at least 60%, e.g., 65%, 70%, 75%, 80%, 85%, or 90% of the adeno-associated virus capsids are fully packaged with genetic material. A non-limiting example of the recovery and purification of intact capsids achieved by the methods disclosed herein is a recovery of at least 50% of the capsids of the liquid sample added in step (a), of which at least 60% are intact capsids, e.g., a recovery of at least 70% of the capsids of the liquid sample added in step (a), of which at least 80% are intact capsids. Further in Example 1 described below, results show a recovery of 60-70% of the capsids from the harvest, of which at least 60% are intact capsids, further demonstrating improved resolution of partially filled capsids by the use of chromatography particles according to the present disclosure, compared to the use of chromatography particles having a uniform average pore diameter throughout their entire volume. Currently, there are no publicly available large-scale methods that provide recovery and purification as high as the methods disclosed herein.

[0126] As mentioned above, the chromatographic material applied in the methods disclosed herein comprises a weak cation exchange chromatographic material comprising a support comprising a ligand for binding to an adeno-associated virus capsid and a separation matrix according to the first aspect of the present disclosure.

[0127] The ligand of the weak cation exchange chromatography material has the following formula I:

[0128] [ka]

[0129] (In the formula, X1 is selected from O, S, and NHCO; n is an integer from 1 to 4, i.e., 1, 2, 3, or 4; Each R1 and R2 is independently selected from hydrogen, C1-C3 alkyl, and OH. It can be defined by:

[0130] As a non-limiting example, X1 is O, n is 1, and each R1 and R2 is hydrogen.

[0131] There are currently available chromatography materials provided by Cytiva, Sweden (www.cytivalifesciences.com) that contain a ligand defined by formula I, where X1 is O, n is 1, and each R1 and R2 is hydrogen, i.e., the ligand is a carboxymethyl group (abbreviated as CM).

[0132] According to another non-limiting example, X1 is S, n is 2, and each R1 and R2 is hydrogen.

[0133] By yet another non-limiting example, X1 is NHCO, n is 2 or 3, and each R1 and R2 is hydrogen.

[0134] By yet another non-limiting example, X1 is O, n is 1, R1 is CH3, and R2 is hydrogen.

[0135] The density of the ligand can be about 30 to about 110 μmol, such as about 40 to about 90 μmol, for example about 50 to about 80 μmol, or about 30, 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 70, 75, 80, 85, 90, 95, 100, 105 or 110 μmol of ligand per ml of weak cation exchange chromatography material.

[0136] Steps (a) and (b) of the method disclosed above according to the fifth aspect of the present disclosure may comprise applying a buffer having a pH of about 2.0 to about 12.0, such as about 3.0 to about 9.0, such as about 4.0 to about 6.0, such as about 4.5, or about 2.0, 2.5, 3.0, 3.5, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, or 12.0.

[0137] The buffer is suitably selected from buffers commonly recommended for cation exchange chromatography and may include, for example, citric acid, lactic acid, acetic acid, phosphoric acid, MES (2-(N-morpholino)ethanesulfonic acid), ADA (N-(2-acetamido)-2-iminodiacetic acid), MOPS (3-(N-morpholino)propanesulfonic acid), HEPES (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), tris(hydroxymethyl)amino-methane (i.e., Tris), or 1,3-bis(tris(hydroxymethyl)methylamino)propane (i.e., Bis-Trispropane).

[0138] A person skilled in the art can select a suitable concentration for any one of the buffers listed above. Non-limiting examples of buffers applied in steps (a) and (b) of the method according to the fifth embodiment, in which the chromatographic material is a weak cation exchange chromatographic material, may include about 10 to about 100 mM acetate, for example, about 20 to about 60 mM acetate, or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mM acetate. Such a buffer may have, for example, a pH of about 2.0 to about 12.0, such as about 3.0 to about 9.0, such as about 4.0 to about 6.0, such as about 4.5, or about 2.0, 2.5, 3.0, 3.5, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, or 12.0.

[0139] Step (b) of the method according to the fifth aspect may comprise the step of applying a buffer, optionally one of the buffers mentioned above, which comprises a compound that improves the separation of capsids in which the genetic material is fully packaged and capsids in which the genetic material is not fully packaged. This compound may or may not be present in the buffer applied in step (a) of the method according to the fifth aspect. Without being bound by theory, such a compound may improve the separation, for example, by affecting capsid-ligand or capsid-capsid interactions. The compound that improves the separation may be selected, for example, from carbohydrates, divalent metal ions, and detergents.

[0140] When the compound that improves separation is a carbohydrate, it may be selected, for example, from sucrose, sorbitol, and polysaccharides.

[0141] When the compound that improves separation is a divalent metal ion, it can be, for example, Mg 2+ , Fe 2+, and Mn 2+ The metal ion may be selected from the group consisting of: 1) a cation, 2) a phosphate buffer, 3) a phosphate buffer, 4) a phosphate buffer, 5) a phosphate buffer, 6) a phosphate buffer, 7) a phosphate buffer, 8) a phosphate buffer, 9) a phosphate buffer, 10) a phosphate buffer, 11) a phosphate buffer, 12) a phosphate buffer, 13) a phosphate buffer, 14) a phosphate buffer, 15) a phosphate buffer, 16) a phosphate buffer, 17) a phosphate buffer, 18) a phosphate buffer, 19) a phosphate buffer, 20) a phosphate buffer, 25) a phosphate buffer, 26) a phosphate buffer, 27) a phosphate buffer, 28) a phosphate buffer, 29) a phosphate buffer, 30) a phosphate buffer, 31) a phosphate buffer, 32) a phosphate buffer, 33) a phosphate buffer, 34) a phosphate buffer, 35) a phosphate buffer, 36) a phosphate buffer, 37) a phosphate buffer, 38) a phosphate buffer, 39) a phosphate buffer, 40) a phosphate buffer, 41) a phosphate buffer, 42) a phosphate buffer, 43) a phosphate buffer, 44) a phosphate buffer, 45) a phosphate buffer, 46) a phosphate buffer, 47) a phosphate buffer, 48) a phosphate buffer, 49) a phosphate buffer, 50) a phosphate buffer, 51) a phosphate buffer, 52) a phosphate buffer, 53) a phosphate buffer, 54) a phosphate buffer, 55) a phosphate buffer, 56) a phosphate buffer, 57) a phosphate buffer, 58) a phosphate buffer, 59) a phosphate buffer, 58) a phosphate buffer, 59)

[0142] When the compound improving separation is a surfactant, it may be selected, for example, from poloxamers, such as Poloxamer 188 or Pluronic™ F68, and polysorbates, such as Tween 20 or Tween 80.

[0143] In the above method according to the fifth aspect, step (b) may comprise applying a buffer, optionally one of the buffers mentioned above, which comprises a compound that may aid in eluting the capsids bound to the chromatographic material. This compound is not present in the buffer applied in step (a) of the method according to the fifth aspect. Non-limiting examples of such compounds are salts, such as salts of monovalent metal ions, e.g., NaCl, LiCl, KCl, or other equivalent metal salts suitable for use in salt elution, as known in the art. Non-limiting examples of suitable concentrations of NaCl include about 50 mM to about 2 M NaCl, e.g., about 50, 100, 150, 200, 250, 500, 600, 700, 800, 900, 1000, 1500, or 2000 mM NaCl. Furthermore, step (b) may comprise applying a gradient of such compounds to improve the elution of the adeno-associated virus capsids, whose genetic material is fully packaged, from the chromatographic material.

[0144] Non-limiting examples of suitable buffers to be applied in step (b), where the chromatographic material is a weak cation exchange chromatographic material, may include: (i) 25 mM acetate, 10 mM MgCl2, 500 mM NaCl, pH 4.5; (ii) 25 mM acetate, 2 mM MgCl2, 800 mM NaCl, pH 4.5; (iii) 25 mM acetate, 500 mM NaCl, pH 4.5; (iv) 25 mM acetate, 500 mM NaCl, pH 4.5; or (v) 25 mM acetate, 500 mM NaCl, 10 mM MgCl2, pH 4.5; (vi) 50 mM acetate, 500 mM NaCl, pH 4.5.

[0145] The liquid sample added in step (a) of the method disclosed herein according to the fifth aspect may advantageously be a pre-purified liquid sample.

[0146] The present disclosure further provides a method for separating fully packaged adeno-associated virus capsids from non-fully packaged adeno-associated virus capsids, comprising carrying out the method as shown in FIG. 4 and described in detail above, further comprising step (a1) comprising pre-purifying the adeno-associated virus capsids by separating the adeno-associated virus capsids from an adeno-associated virus capsid-containing cell culture harvest as illustrated in FIG. 5, thereby obtaining a pre-purified liquid sample comprising adeno-associated virus capsids, and then adding said pre-purified liquid sample comprising adeno-associated virus capsids to a chromatographic material according to step (a) of the method as shown in FIG. 4 and FIG. 5, respectively.

[0147] As further described above, such a pre-purification step (a1) may alternatively be called a "capture step". Most commonly, the capture step involves clarification (e.g. by filtration, centrifugation, or precipitation) and usually also concentration and / or stabilization of the sample, and significant purification from soluble impurities, e.g. by applying chromatography, after sample clarification, concentration, and stabilization. The capture step may be followed by intermediate purification, which further reduces the residual amounts of impurities such as host cell proteins, DNA, viruses, endotoxins, nutrients, components of cell culture medium such as antifoaming agents and antibiotics, and product-related impurities such as aggregates, misfolded species, and aggregates.

[0148] Such a pre-purification step may include subjecting the adeno-associated virus capsid-containing cell culture harvest to one or more of the following non-limiting examples of purification methods: (i) affinity chromatography; (ii) ion exchange chromatography; (iii) precipitation or tangential flow filtration (TFF) followed by size exclusion chromatography, for example by using Capto Core 400 chromatographic material (Cytiva, Sweden), which combines capsid flow-through and binding of impurities to the chromatographic material; (iv) TFF followed by ion exchange chromatography; and (v) TFF followed by ion exchange chromatography and Capto Core.

[0149] As described above, the adeno-associated virus has a diameter of about 20 to 25 nm. The capsid is the shell of the virus particle, and the adeno-associated virus does not have a lipoprotein bilayer envelope surrounding the capsid, so the size of the adeno-associated virus capsid is about 20 to 25 nm in diameter.

[0150] Thus, the layer surrounding the core of the plurality of chromatographic particles contained in the separation matrix applied in the method according to the fifth aspect of the present disclosure can suitably comprise an average pore diameter of >25 nm, i.e., larger than the diameter of the adeno-associated virus capsid to be separated, thereby allowing diffusion of the capsid within the layer surrounding the core. It should be understood that for the specific purpose of separating adeno-associated virus capsids, an average pore diameter of >25 nm can be any size >25 nm, including, but not limited to, 30, 50, 75, 100, 150, or 200 nm.

[0151] In contrast, the average pore diameter of the core does not allow the diffusion of the adeno-associated virus capsid. Based on the size of the adeno-associated virus, the core can suitably comprise an average pore diameter of <20 nm, i.e., smaller than the diameter of the adeno-associated virus capsid to be separated, thereby not allowing or preventing the diffusion of the capsid within the core. Preferably, the core can comprise an average pore diameter of <15 nm, substantially smaller than the size of the adeno-associated virus, such as a diameter of 10 nm or 5 nm.

[0152] Chromatographic particles having a smaller average pore diameter in their core than in the layers surrounding the core can be prepared, for example, by carrying out the method according to the second aspect of the present disclosure.

[0153] The method disclosed herein for separating fully packaged adeno-associated virus capsids from non-fully packaged adeno-associated virus capsids comprises: separating the eluate fraction containing the adeno-associated virus capsids in which the genetic material is fully packaged, which is eluted in step (b) of the method as described above, from the c1) concentrating the adeno-associated virus capsid to a pharma- ceutical suitable dose; c2) replacing the buffer applied in step (b) of the process with a pharma- ceutically acceptable buffer, and / or c3) Sterilizing the eluate fraction containing the adeno-associated virus capsid to obtain a pharmaceutical composition comprising the adeno-associated virus capsid.

[0154] Those skilled in the art will appreciate that the pharma- ceutically appropriate dose will depend on a variety of factors, including, but not limited to, the disease or disorder being treated and the weight and condition of the subject being treated with the pharmaceutical composition.

[0155] Pharmaceutically acceptable buffers are well known in the art and can be readily selected by one of ordinary skill in the art.

[0156] In order for the resulting composition to meet all regulatory requirements for pharmaceutical compositions, usually all three steps c1 to c3 listed above must be carried out.

[0157] In the above disclosed method according to the fifth aspect of the present disclosure, the adeno-associated virus capsid may advantageously be a capsid of adeno-associated virus serotype 9 (AAV9) or a variant thereof.

[0158] The term "variant" in relation to adeno-associated virus serotype 9 (AAV9) is intended to mean an AAV9 whose capsid structure has been modified, altered or engineered to improve clinical performance, e.g., target specific target organs. AAV9 variants include the capsid portion of AAV9 and may further include the capsid portion of other AAV serotypes other than AAV9, such as AAV5. However, AAV9 variants as referred to herein must retain significant structural similarity to the unmodified AAV9 capsid, e.g., retaining at least 50%, e.g., 60%, 70%, 80%, or 90% of the outer surface structure of the unmodified AAV9 capsid. In the context of purifying or isolating variants of AAV9, a "variant" is defined herein as an adeno-associated virus that has a functionally equivalent binding ability to a ligand of a specified chromatographic material compared to the binding ability of the original AAV9 to said specified chromatographic material. The specified chromatographic material may be, for example, a weak cation exchange chromatographic material, as disclosed in more detail elsewhere herein. Variants of adeno-associated viruses may be obtained, for example, by natural mutation or by engineered modification (i.e., obtained by human interaction) of one or more nucleotides of the genome of the adeno-associated virus.

[0159] The present disclosure is further useful for carrying out a method for preventing or treating an organ or tissue-related disease or disorder in a subject, comprising administering to the subject a pharmaceutical composition comprising adeno-associated virus capsids obtained by carrying out the above-disclosed isolation method comprising one or more of steps c1 to c3 (as described in detail above), wherein in the pharmaceutical composition, the ratio of adeno-associated virus capsids in which the genetic material is fully packaged to adeno-associated virus capsids in which the genetic material is not fully packaged is at least 3:2, i.e., the number of full capsids is at least 1.5 times the total number of empty and partially filled capsids. Preferably, the ratio of adeno-associated virus capsids in which the genetic material is fully packaged to adeno-associated virus capsids in which the genetic material is not fully packaged is at least 2:1, for example, 3:1, 4:1, or 5:1. That is, preferably the number of complete capsids is at least 2-fold, such as 3, 4, or 5-fold, the total number of empty and partially filled capsids.

[0160] Said methods for preventing or treating an organ or tissue associated disease or disorder may suitably include gene therapy, It should be understood that the therapeutically relevant gene or genetic material is present in the fully packaged adeno-associated virus capsid, which genetic material is contained in the pharmaceutical composition administered to the subject.

[0161] In the above described methods for preventing or treating an organ or tissue associated disease or disorder, the adeno-associated virus capsid may advantageously be a capsid of adeno-associated virus serotype 9 (AAV9), or a variant thereof.

[0162] In the above methods for preventing or treating a disease or disorder associated with an organ or tissue, the organ or tissue may optionally be selected from the central nervous system, heart, liver, lung, and skeletal muscle, particularly when the adeno-associated virus capsid is a capsid of adeno-associated virus serotype 9 (AAV9) or a variant thereof.

[0163] It should be understood that the tissue types mentioned above are non-limiting examples of organ and tissue types that may be amenable to treatment by administration of a pharmaceutical composition comprising an adeno-associated virus capsid, in particular, the capsid of adeno-associated virus serotype 9 (AAV9), or a variant thereof.

[0164] A non-limiting example of an organ- or tissue-related disease or disorder is spinal muscular atrophy, where a method of treatment or prevention can be suitably carried out by administration of an adeno-associated virus, in particular AAV9.

[0165] Another non-limiting example of an organ- or tissue-related disease or disorder is an inherited retinal dystrophy, the treatment or prevention of which can be suitably carried out by administration of an adeno-associated virus, in particular AAV2.

[0166] Those skilled in the art will understand that to achieve the desired medical effect, the pharmaceutical composition must be administered to the subject in a pharma- ceutical effective amount or dose. The pharma- ceutical effective amount and dose depend on various factors, including, but not limited to, the disease or disorder being treated and the weight and condition of the subject being treated.

[0167] The present disclosure further provides, according to a sixth aspect, a composition comprising adeno-associated virus capsids obtained by carrying out a method for separating fully packaged adeno-associated virus capsids from those that are not fully packaged, as described in detail above (i.e., the method according to the fifth aspect), in which the ratio of adeno-associated virus capsids in which genetic material is fully packaged to those in which genetic material is not fully packaged is at least 3:2, i.e., the number of full capsids is at least 1.5 times the total number of empty and partially filled capsids. Preferably, the ratio of adeno-associated virus capsids in which genetic material is fully packaged to those in which genetic material is not fully packaged is at least 2:1, e.g., 3:1, 4:1, or 5:1. That is, preferably the number of complete capsids is at least 2-fold, such as 3, 4, or 5-fold, the total number of empty and partially filled capsids.

[0168] The present disclosure is further useful for providing a pharmaceutical composition comprising adeno-associated virus capsids obtained by carrying out the above-disclosed separation method comprising one or more of steps c1 to c3 (as described in detail above), in which the ratio of adeno-associated virus capsids in which genetic material is fully packaged to adeno-associated virus capsids in which genetic material is not fully packaged is at least 3:2, i.e., the number of full capsids is at least 1.5 times the total number of empty capsids and partially filled capsids. Preferably, the ratio of adeno-associated virus capsids in which genetic material is fully packaged to adeno-associated virus capsids in which genetic material is not fully packaged is at least 2:1, e.g., 3:1, 4:1, or 5:1. That is, preferably, the number of full capsids is at least 2 times, e.g., 3, 4, or 5 times the total number of empty capsids and partially filled capsids.

[0169] The pharmaceutical compositions described above may be for use in therapy, optionally for use in gene therapy.

[0170] In the above pharmaceutical compositions for use in a treatment, such as a gene therapy, the adeno-associated virus capsid may advantageously be the capsid of adeno-associated virus serotype 9 (AAV9), or a variant thereof.

[0171] Furthermore, the pharmaceutical composition may advantageously be for use in the prevention or treatment of a disease or disorder associated with an organ or tissue selected from the central nervous system, the heart, the liver, the lungs, and skeletal muscles.

[0172] A device or composition that "comprises" one or more recited components may also include other components not specifically recited. The term "comprising" includes as a subset "consisting essentially of," which means that the device or composition has the recited components, with no other features or components present. Similarly, a method that "comprises" one or more recited steps may also include other steps not specifically recited.

[0173] The singular forms "a" and "an" are intended to be construed as including the plurals as well. EXAMPLES

[0174] Separation of AAV9 capsids on weak cation-exchange chromatographic materials containing semi-solid particles Preparation of chromatographic materials a) Carboxymethyl (CM) ligand prototypes on beads with homogeneous average pore diameter (resins 1, 2 and 3) General steps: Highly cross-linked agarose base matrix (40 mL beads with homogenous average pore diameter) was washed 5 times with water (2 gel volumes (GV)), drained and transferred to a 100 mL 3-neck RB flask, then 2.5 mL of water was added. The gel mixture was kept in a water bath at room temperature (RT). NaOH solution (50% in water, 7.8 mL) was added and the mixture was kept stirring with a mechanical stirrer. After 30 min, sodium chloroacetate was added in one portion (13.2 g for resin 1, 6.6 g for resin 2 and 19.88 g for resin 3, respectively). The reaction is exothermic and the temperature should be tightly maintained at 20 °C. After dissolution of sodium chloroacetate, the water bath temperature was increased to 33 °C. The reaction was kept stirring at 33 °C for 16 h. Water (10 mL) was then added and the solution was cooled to RT. Acetic acid (60%) was added to adjust the pH of the solution to 6.8-7.2. The resulting gel was washed three times with water (1 GV).

[0175] Titration of the resulting resins indicated an ionic capacity (ie, ligand density) of 45 μmol / mL for resin 1, 30 μmol / mL for resin 2, and 56 μmol / mL for resin 3.

[0176] b) Carboxymethyl (CM) prototype on semi-solid beads (i.e., an example of a chromatographic particle as comprised in a separation matrix according to the first embodiment of the present disclosure) b.1) Allylation of cross-linked agarose-based matrices Highly cross-linked agarose base matrix (300 mL of beads with homogenous average pore diameter) was washed with 10×GV of distilled water. The gel was then suction dried and transferred to a 2000 mL round bottom flask. 300 mL of 50% NaOH was added, mechanical propeller agitation (250 rpm) was applied, and the flask was placed in a 50°C water bath. After 30 minutes, 93.2 mL of allyl glycidyl ether (AGE) was added. The reaction was allowed to proceed for 18 hours. The gel was washed with 1×GV of distilled water, 5×GV of ethanol, then 8×GV of distilled water. Titration indicated an allyl level of 266 μmol / mL of resin.

[0177] b.2) Partial activation (outer part) The allylated resin from above (70.1 mL) was transferred to a 1000 mL round bottom flask. 350 mL of distilled water was added and mechanically stirred. A solution of 529 μL of bromine in 83.3 mL of water was prepared. The bromine solution was added slowly with stirring for approximately 2-3 minutes. After 20 minutes at room temperature, the gel was washed with 10×GV distilled water. Titration of remaining allylic groups indicated a 3 μm shell (i.e., the outer layer; the layer surrounding the core of the bead).

[0178] b.3) Coupling of 3-mercaptopropionic acid The partially activated gel from b.2) (30.03 mL) was transferred to a 100 mL round bottom flask. 10.48 mL of distilled water was added and mechanical stirring was applied. NaOH (5.42 mL of a 50% solution) was added followed by 3-mercaptopropionic acid (120.22 μL). The flask was left at 50 °C for 18 h. The gel was washed with 15 × GV of distilled water.

[0179] b.4) Coupling of allyl-dextran (allyl-Dx) to an allyl core, shell containing 3-mercaptopropionic acid-carboxymethyl ligand: Solubilization of Allyl-Dx: 19.96 g of Allyl-Dx was added to a 250 mL round bottom flask with 20 mL of water at room temperature overnight (23 h) and then at 30° C. with stirring (75 rpm) for 110 min.

[0180] b.4.a) Bromination of the allylic core 20 g of resin from b.3), 20.05 mL of water, and 0.8 g of NaOAc*3H2O were mixed in a 100 mL E-flask. Bromine water was added until a persistent yellow color was obtained. The reaction was stirred for 5 min before the excess bromine was quenched by adding sodium formate. The gel was then washed with 5×GV of water.

[0181] b.4.b) Allyl-Dx coupling The brominated gel from b.4.a) was added to the Allyl-Dx solution and the temperature was raised to 50°C. An additional 5 mL of water was added. After 90 min, 2.6 mL of 50% NaOH and 106 mg of NaBH4 were added. The reaction was allowed to proceed for 18 h with stirring. The mixture was then diluted with water and the gel was washed with 10x GV of water. The dry mass of the gel was measured.

[0182] b.5) ​​Polymerization of Allyl-Dx core with Vinylpyrrolidone / ADBA: The resin from b.4.b) (18.8 mL) was added to a 250 mL flask along with water (122.33 g). 0.94 g of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) initiator and 46.18 g of vinylpyrrolidone were added to the flask. After bubbling with nitrogen (0.1 NL / min) for 5 min, the flask was left at 55°C for 19 h. The very viscous solution was then diluted and washed on a glass filter with 5 x GV of water, at least 5 x 2 GV of EtOH, then 10 x 5 GV of water.

[0183] Titration indicated an ionic capacity (i.e., ligand density) of 45 μmol / mL of semi-solid resin.

[0184] Thus, the ligand density of the layer surrounding the core of the semi-solid bead in (b) above is equivalent to the ligand density of Resin 1 in (a) above.

[0185] Equipment and Samples All resins were packed in Tricorn 5 columns (2 mL), 10 cm bead height, and run using an Agilent Bio-Inert 1260 system or an AKTA pure P25 system at a flow rate of 1 mL / min, using linear gradients and different mobile phase systems according to the results below. For all different resins, the applied samples were pre-purified by affinity chromatography and contained approximately 1 x 10 6 ribosomal fractions of AAV9, containing both full and empty capsids (>15% full capsid). 13 The viral capsid was 100 μg / ml. Detection was performed by fluorescence (excitation at 280 nm, emission at 348 nm) or UV (280 and 260 nm).

[0186] result For comparison, currently available (i.e. prior art) cationic and cationic / multimodal resins (SP Sepharose XL, Capto SP ImpRes, Capto SP ImPact and Capto MMC ImpRes, CM Sepharose Fast Flow, and CM Sepharose High performance, all provided by Cytiva, Sweden) were evaluated in 50 mM acetate pH 4.5 and 5.5 with a salt gradient up to 1 M NaCl. When applying AAV9 pre-purified by affinity chromatography, the results showed no separation, only a single peak (results not shown).

[0187] Furthermore, CM prototype resin 2 as mentioned in (a) above, with a ligand density of 30 μmol / mL and homogenous porosity, was run at 25 mM acetate, pH 4.5, 500 mM NaCl, 10 mM MgCl2, and results showed separation of AAV9 full and empty capsids (results not shown).

[0188] Using CM prototype resin 3 as mentioned in (a) above, with an increased ligand density of 56 μmol / mL resin and homogeneous porosity, under the same chromatographic conditions as above, the separation performance was improved (Figure 7). The two peaks marked F and E on the right of Figure 7 indicate full (F) capsids and empty (E) capsids, respectively, separated on CM prototype resin 3 with homogeneous porosity. It can be further inferred from Figure 7 that separation on CM prototype resin 3 did not result in a clear intermediate peak.

[0189] Even better resolution was achieved when a semi-solid CM prototype resin with a correspondingly higher ligand density (referred to as CM prototype on semi-solid beads (b) above) was evaluated (Figure 7). The three peaks marked F, I, and E on the left of Figure 7 contain the majority of full (F), partially filled (I; intermediate), and empty (E) capsids, respectively, that were separated on the semi-solid CM prototype resin.

[0190] Preparative runs using the high ligand density semi-solid CM prototype resin were performed on an AKTA system. Pooled peak 1 and pooled peak 3 fractions from the run were each analyzed using TEM to confirm that peak 1 contained full capsids and peak 3 contained empty capsids (Table 1). qPCR and ELISA ratios also indicated enrichment of full capsids in peak 1 (data not shown).

[0191] [Table 1]

[0192] conclusion In the above experiments, it was found that the CM prototype with the highest ligand density (i.e., resin 3) exhibited better resolution than the CM prototype with a lower ligand density (i.e., resin 2). The semi-solid CM prototype (i.e., an example of a chromatographic particle included in a separation matrix according to the first aspect of the present disclosure) exhibited even higher resolution than the CM prototype resin 3. Furthermore, MgCl2 added to the elution buffer significantly increased the resolution when using the semi-solid CM column, but not when using either of the CM prototypes (i.e., resins 2 and 3). EXAMPLES

[0193] Separation of monoclonal antibody variants on strong cation-exchange chromatography materials containing semi-solid particles Preparation of chromatographic materials a) Sulfonate (S) prototypes on semi-solid beads (i.e., examples of chromatographic particles included in a separation matrix according to the first embodiment of the present disclosure) a.1) Allylation of cross-linked agarose-based matrices The cross-linked agarose base matrix (300 mL beads with homogenous porosity) was washed with 10×GV of distilled water. The gel was then suction dried and transferred to a 2000 mL round bottom flask. 250 mL of 50% NaOH and 0.3 g of sodium borohydride were added, mechanical propeller agitation (250 rpm) was applied, and the flask was placed in a 50° C. water bath. After 30 min, 92 mL of AGE was added. The reaction was allowed to proceed for 18 h. The gel was washed with 1×GV of distilled water, 5×GV of ethanol, and then 8×GV of distilled water. Titration indicated an allyl level of 288 μmol / mL of resin.

[0194] a.2) Partial activation (outer part of the bead) 600 g (mL) of allylated cross-linked agarose base matrix was transferred and discharged into a 4 L round bottom flask together with 3 L of distilled water. Mechanical stirring was applied (250 rpm). A solution of 3.375 mL of bromine in 400 mL of water was prepared. The bromine solution (equivalent to the ally in the 7 μm shell of the 95.8 μm beads) was added by dropping funnel at a stirring speed of 300 rpm for approximately 15 min. After 20 min, the gel was washed with 10 × 1 GV of distilled water. Titration of the remaining allylic groups indicated a 4.5 μm shell (i.e., the outer part of the bead; the layer surrounding the core of the bead).

[0195] a.3) S-shell coupling 619 g (mL) of shell-activated gel from the previous step was transferred to a 2 L round bottom flask. 124 mL of distilled water and 148.5 g of sodium sulfite were added. Mechanical propeller agitation was applied and the solution was left at 250 rpm for 30 minutes. The pH was adjusted to 12.5 by adding 50% NaOH and the flask was placed in a 50°C water bath. The reaction proceeded for 21 hours. The gel was washed with 10×GV of distilled water. Titration of the sulfonic acid groups indicated an ionic capacity (i.e., ligand density) of 73 μmol / mL.

[0196] a.4) Vinylpyrrolidone core polymerization 25.4 g of S-functionalized partially allylated material from the previous step was transferred to a glass filter. The resin was washed with 7 x 2 GV of distilled water, followed by 3 x 1.5 GV of 1 M Na2SO4 and suction dried. The dried gel was transferred to a 100 mL Duran flask and 1 M Na2SO4 solution was added to a total mass of 36.6 g, followed by 4.3 mL of vinylpyrrolidone and 0.089 g of ADBA. Nitrogen gas was bubbled through the reaction solution with a Pasteur pipette for 20 min. The Duran flask was placed in a glycerol bath with a temperature set at 45 °C and a stirring speed of 250 rpm. After 17 h, 32.5 g of distilled water was added and the suspension was stirred for 30 min at the reaction temperature. The gel was transferred to a glass filter and washed again with 10 GV of distilled water, then 5 x 1 GV of ethanol and finally 10 x 1 GV of water. Dry mass analysis showed an increase in mass of 126 mg / mL of resin (corresponding to the amount of polyvinylpyrrolidone (PVP) added to the core). The ionic capacity of the material was measured again and was 73 μmol / mL.

[0197] Equipment and Samples Sulfonate semi-solid resin prototypes were packed into a Tricorn 10 / 150 column (Cytiva, Sweden) using water. The resin was packed at 0.8 mL / min until the resin stabilized. After the resin stabilized, the resin was packed at 3 mL / min for >10 min. The top adapter was adjusted to the top of the resin bed plus 3 / 4 turn of the top adapter clockwise. The bed height of the column was 14.6 cm and the asymmetry of the column was tested using water and an AKTA Avant 25 system (Cytiva, Sweden) by injecting 100 μL of acetone (20 mg / mL) and a flow rate of 1.0 mL / min. The asymmetry was calculated to be 1.27 (spec 0.8-1.8). The reference resin, SP Sepharose Fast Flow (SP FF), with the same particle size distribution as the sulfonate semi-solid resin prototypes, was also packed in the same manner into a Tricorn 10 / 150 column with a bed height of 14.3 cm similar to the sulfonate semi-solid resin prototypes. The asymmetry of the reference column (SP FF) was 1.48 (specification 0.8-1.8).

[0198] The columns were installed in an AKTA Pure 25 system (Cytiva, Sweden). Each column was equilibrated with 3 column volumes (CV) of A buffer, 50 mM Na acetate pH 5.0 at 2 mL / min. 200 μL of protein mixture containing monoclonal antibody (mAb) 17 mg / mL, cytochrome C (CytC) 4 mg / mL and lysozyme (Lys) 4 mg / mL at a flow rate of 1 mL / min. The columns were washed with 2 CV of A buffer. Proteins were eluted using 25 mM Na phosphate + 0.4 M NaCl pH 7.0 as B buffer at 1 mL / min using a linear gradient between 0-100% B for 15 CV. Finally the columns were washed with 2 CV of B buffer and 2 CV of A buffer at 2 mL / min. Detection was at 280 nm.

[0199] result The peak widths of the eluted proteins in FIG. 8 were determined and the resolution Rs between the mAb5-CytC and CytC-Lys peaks was assessed and summarized in Table 2 below.

[0200] [Table 2]

[0201] conclusion Even though the particle size distribution was equivalent between the prototypes, the sulfonate semi-solid resins showed narrower and more resolved chromatographic peaks than the reference SP Sepharose FF using a linear gradient. EXAMPLES

[0202] Isolation of AAV9 capsids under variable conditions The experimental design for separation of fully packaged AAV9 capsids from empty and partially packaged AAV9 capsids is carried out using equipment and samples as in Example 1 above, with the use of weak cation exchange chromatography material on semi-solid beads as prepared in Example 1, items (a)+(b), with the following variations.

[0203] Regarding semi-solid bead preparation: 1) Reversing the order of the method steps compared to items (a)+(b) of Example 1, i.e., this variation includes a method comprising a first step of preparing semi-solid beads and a second step of functionalizing the semi-solid beads by coupling a ligand to the semi-solid beads, the ligand being for binding to a target molecule. More specifically, the variation includes a method for preparing chromatographic particles according to the present disclosure, comprising a first step of preparing chromatographic particles comprising a core and a layer surrounding the core, the core having a first average pore diameter and the layer surrounding the core having a second average pore diameter, the second average pore diameter being at least 1.5 times the first average pore diameter, and a subsequent step of coupling a ligand for binding to the semi-solid beads for binding to the target molecule.

[0204] Regarding ligand density on the semi-solid beads: 1) CM prototype on semi-solid beads of Example 1, item (b): lower and higher density of the same ligand.

[0205] Regarding the chemistry of the ligands on the semi-solid beads: 1) Weak cation exchange chromatography materials: a. Dextran CM, ie, a carboxymethyl (CM) ligand coupled to dextran coupled to a chromatographic material support (as in Example 1). b. CM linked to the chromatography material via an amide bond. c. PolyCM or conjugated CM attached to a short linker or scaffold coupled to a chromatographic material support. 2) Strong cation exchange chromatography materials: a. Sulfonate (S) Ligand (as in Example 2) EXAMPLES

[0206] Isolation of target molecules under variable conditions Experimental designs for the separation of various target molecules are carried out using the equipment and samples as in Example 1 or Example 2 above, by using chromatographic materials on semi-solid beads as prepared in Example 1, item (a)+(b) or Example 2, item (a), with the following variations.

[0207] Regarding the chemistry of the ligands on the semi-solid beads: 1) Strong anion exchange chromatography materials: a. Ligand for the chromatography material Capto Q (Cytiva, Sweden) with approximately 100% quaternized amine groups. b. Dextran Capto Q ligand, ie, Capto Q ligand coupled to dextran which is coupled to a chromatographic material support. c. Ligand of the chromatographic material Capto DEAE (Cytiva, Sweden) with a degree of quaternization of the amine groups of about 15%. d. Dextran Capto DEAE Ligand, ie, Capto DEAE Ligand coupled to dextran which is coupled to a chromatographic material support. 2) Strong cation exchange material: a. Ligand of the chromatographic material Capto S ImpAct (Cytiva, Sweden), which contains a sulfonate (S) group as a strong cation exchanger ligand. b. Ligand of the chromatography material Capto SP ImpRes (Cytiva, Sweden), which contains a sulfopropyl (SP) group. 3) Multimodal anion exchange chromatography materials containing hydrophobic groups: a. The ligand of the chromatography material Capto adhere ImpRes (Cytiva, Sweden), which comprises an N-benzyl-N-methylethanolamine ligand coupled to a support, said support being linked to the nitrogen atom of the ligand by a linker. 4) Multimodal weak cation exchange chromatography materials: a. Ligand of the chromatographic material Capto MMC (Cytiva, Sweden), which contains a carboxyl group and involves hydrogen bonding and hydrophobic interactions. 5) Hydrophobic interaction chromatography materials: a. Ligand for chromatographic material Phenyl Sepharose 6 Fast Flow (Cytiva, Sweden). b. Chromatography Materials Butyl Sepharose 4 Fast Flow (Cytiva, Sweden) Ligand.

[0208] Regarding the target molecules to be separated on the semi-solid beads: 1) Adeno-associated virus capsid of serotype 8 (AAV8), 5 (AAV5), 1 (AAV1), 2 (AAV2), 4 (AAV4), 6 (AAV6), 7 (AAV7), or 10 (AAV10); separated on semi-solid beads containing an anion exchange ligand as exemplified in Example 2 or Example 4 herein. 2) Monoclonal antibody variants, such as bispecific, fusion, or chimeric monoclonal antibodies; separated on semi-solid beads containing a ligand as exemplified in Examples 2 and 4 herein. 3) Antibody fragments (including but not limited to antigen-binding fragments F(ab')2, Fab, Fab', and Fv; are separated on semi-solid beads containing a ligand as exemplified in Examples 2 and 4 herein. 4) Oligonucleotides; separated on semi-solid beads containing anion exchange ligands, multimodal anion exchange ligands or hydrophobic interaction ligands as exemplified in Example 4 herein. 5) RNA; separated on semi-solid beads containing anion exchange ligands, multimodal anion exchange ligands or hydrophobic interaction ligands as exemplified in Example 4 herein. 6) DNA; separated on semi-solid beads containing anion exchange ligands, multimodal anion exchange ligands or hydrophobic interaction ligands as exemplified in Example 4 herein. EXAMPLES

[0209] Determination of pore size distribution and relative mean pore diameter This example describes how to verify that the chromatographic particles described herein have two different pore size distributions, a core pore size distribution and an outer layer pore size distribution, respectively. In summary, proteins and peptides of various sizes were injected and run through a column packed with chromatographic beads produced as described herein. The retention volume of each polypeptide was determined and the partition coefficient was calculated for each polypeptide. The partition coefficient was plotted against the log molecular weight of each polypeptide (Figure 9).

[0210] Equipment and Materials Chromatography resin (semi-solid beads) prepared essentially as described in Example 2a above was used. The particle size (d50v) of the agarose-based matrix was 96.5 μm. Titration revealed an allyl level (C) of 296.8 μmol / mL after activation. アリル1 ) and an allele level of 224.4 μmol / mL after partial inactivation (C アリル2 ) (see steps 2a.1 and 2a.2 above). The thickness of the outer layer (shell) t s is the formula

[0211]

number

[0212] (where r0 is d50v / 2, which corresponds to the radius of the bead, and C アリル1 and C アリル2 represents the allele levels before and after partial inactivation, respectively, as shown above), was calculated to be 4.3 μm.

[0213] Additionally, polypeptides were used as outlined in Table 3.

[0214] [Table 3]

[0215] Mobile phase buffer: PBS (2 tablets; Medicago, product number 09-9400-00) and 58.4 g NaCl were dissolved in 2000 ml mQ water. The buffer contained 0.64 M NaCl, 0.0027 M KCl, and 0.01 M phosphate, pH 7.4.

[0216] procedure The resin was packed into a Tricorn 10 / 300 column (Cytiva, Sweden) at a flow rate of 7 mL / min using 0.2 M NaCl (Merck) as the packing eluent. The bed height was 29.3 cm with a total bed volume of 23.0 mL. Asymmetry was determined using an AKTA Pure 25 chromatography system (Cytiva, Sweden) by injecting 100 μL of 2% (w / v) acetone into the column, using a flow rate of 0.75 mL / min and UV detection at 280 nm. The asymmetry was found to be 0.9, which is acceptable. Generally, column asymmetry values ​​between 0.8 and 1.8 are considered acceptable.

[0217] After the asymmetry test, the column was run and connected to a UHPLC system (Agilent 1260 Infinity). The proteins / peptides listed in Table 3 were weighed and individually diluted to a final concentration of 3 mg / mL each in 200 μL of mobile phase buffer. 50 μL was individually injected and the molecules were run through the column at a flow rate of 0.8 mL / min. Detection was performed using UV at 280 and 220 nm.

[0218] After chromatography, the individual peaks in the chromatogram were integrated and retention times and volumes were determined.

[0219] Calculations and Results Table 4 shows calculations performed on columns packed with the resin of the present invention. The void volume was determined using thyroglobulin, which has a molecular weight of 660 kDa.

[0220] The column volume was calculated by multiplying the bed height (29.3 cm) by the square of the column radius (0.5 cm) and then by π.

[0221] The bead volume (Vb) was calculated by subtracting the void volume from the column volume.

[0222] Full particle volume (Vfp) is calculated by the formula: Vfp = 4 / 3 x π x (particle diameter / 2) 3It was calculated by:

[0223] The layer volume (Vl) is calculated by the formula: Vl = Vfp-4 / 3 x π x (particle diameter - (layer thickness x 2) / 2) 3 It was calculated by:

[0224] [Table 4]

[0225] Table 5 shows the retention volume and gel phase partition coefficient (K av ) is shown. av was calculated by subtracting the void volume Vo from the retained volume (Vr) and dividing by the bead volume (Vb; 8.36 mL).

[0226] K av The shell was calculated for the largest polypeptide by subtracting the void volume Vo from the retained volume (Vr) and dividing by the bed volume in the column (3.58 mL).

[0227] [Table 5]

[0228] Figure 9 shows the K av Figure 1 shows the log Mw of polypeptides plotted against the MW. Trend lines were generated in Microsoft Excel using the linear equation with R>0.98 for both trend lines. It can be clearly seen that the slope of the pore size distribution curves is different between the larger biomolecules (29-660 kDa) and the smaller biomolecules (0.6-14 kDa). From the difference in slope, it can be concluded that the resin beads have two pore size distributions. Since the volume of the porous outer layer was small compared to the total bead volume, the larger polypeptides could only migrate into the outer layer and not into the core, thus eluting earlier and producing a steeper pore size distribution curve.

[0229] For the dense core of the beads, based on the linear function in Figure 9, the exclusion limit (where the linear curve passes through 0 on the y-axis) is 18 kDa, followed by the fractionation range (K av 1–0) is 0.7–18 kDa. For the outer layer, the slope of the pore size distribution curve indicates that it exceeds the exclusion limit of 18 kDa for the dense core and impedes larger biomolecules with a linear relationship with log molecular weight up to 440 kDa.

[0230] conclusion The average molecular weight for the linear fractional range of the core (0.7-18 kDa) is 9 kDa, and for the fractional range of the outer layer (18-440 kDa) the average molecular weight is approximately 200 KDa, which means that the average pore diameter of the outer layer is approximately 20 times that of the core.

[0231] It should be understood that the present disclosure is not limited to its above-described exemplary embodiments, and that several possible modifications of the present disclosure are possible within the scope of the following claims. [Explanation of symbols]

[0232] 10 Chromatography particles 20 cores 30 layers, 1st layer 40 Second Layer (References) TIFF2025503396000009.tif141159

Claims

1. A separation matrix comprising a plurality of chromatography particles (10), each chromatography particle (10) comprising a core (20) and a layer (30) surrounding the core (20), the core (20) having a first average pore diameter and the layer (30) surrounding the core (20) having a second average pore diameter, the second average pore diameter being at least 1.5 times the first average pore diameter; A separation matrix, wherein a first average pore diameter prevents diffusion of target molecules through the pores of the core (20) and a second average pore diameter allows at least some diffusion of target molecules through the pores of a layer (30) surrounding the core (20).

2. 2. The separation matrix according to claim 1, wherein the first average pore diameter blocks the diffusion of target molecules having a molecular weight of from about 50 kDa, such as from about 140 kDa, such as from about 2000 kDa.

3. 3. A separation matrix according to claim 1 or 2, wherein the first average pore diameter blocks the diffusion of target molecules having a hydrodynamic diameter of from about 7 nm, such as from about 10 nm, such as from about 20 nm, such as from about 25 nm.

4. The separation matrix according to any one of claims 1 to 3, wherein the chromatography particles (10) have a diameter of from about 20 μm to about 500 μm, such as from about 60 μm to about 120 μm, or such as from about 100 μm to about 400 μm.

5. The separation matrix according to any one of claims 1 to 4, wherein the core (20) has a diameter that is from about 60% to about 97% of the diameter of the chromatography particle (10).

6. 6. The separation matrix according to claim 1, wherein the layer (30) surrounding the core (20) has a diameter of about 3% to about 40%, such as about 6% to about 10%, of the diameter of the chromatographic particle (10).

7. 7. The separation matrix according to claim 1, wherein the chromatographic particles (10) comprise ligands for binding to target molecules, the ligands being located in a layer (30) surrounding the core (20), and optionally the ligands being further located in the core (20).

8. 8. The separation matrix according to claim 7, wherein the ligands are cationic ligands, anionic ligands, hydrophobic interaction ligands, multimodal ligands, or combinations thereof.

9. 9. The separation matrix according to claim 7 or 8, wherein the ligands are linked to the surface of the chromatography particles (10) via linkers or extenders.

10. 10. The separation matrix according to claim 1, wherein the chromatographic particles (10) are substantially spherical.

11. 11. A separation matrix according to any one of claims 1 to 10, wherein the chromatographic particles (10) are of natural or synthetic origin, preferably polysaccharides such as agarose.

12. the layer (30) surrounding the core (20) is a first layer, and the chromatography particle (10) further comprises a second layer (40) surrounding the first layer (30); 12. A separation matrix according to any one of the preceding claims, wherein the second layer (40) has a third average pore diameter different from the first average pore diameter and the second average pore diameter.

13. 13. A separation matrix according to any one of claims 1 to 12, wherein the core (20) comprises a polymeric compound which reduces the average pore diameter of the core (20) compared to the average pore diameter of the layers (30, 40) surrounding the core (20), and optionally the polymeric compound is a natural polymer such as dextran or a synthetic polymer.

14. A method for preparing a separation matrix according to any one of claims 1 to 13, comprising the step of preparing a plurality of chromatographic particles (10), said step comprising: (a) providing as a starting material a chromatographic particle having a uniform average pore diameter, the uniform average pore diameter at least in part allowing target molecules to diffuse through the pores of the chromatographic particle throughout the entire structure or volume of the chromatographic particle; (b) adding a polymeric compound to the interior of the core of the chromatography particle provided in step (a), thereby reducing the average pore diameter of the core compared to the homogeneous average pore diameter of the chromatography particle provided in (a), thereby obtaining a chromatography particle comprising a core and a layer surrounding the core, the core having a first average pore diameter and the layer surrounding the core having a second average pore diameter, the second average pore diameter corresponding to the homogeneous average core diameter of the chromatography particle provided in (a); Contains or (a') providing a chromatography particle having a first average pore diameter that inhibits diffusion of a target molecule through the pores of the chromatography particle throughout the entire structure or volume of the chromatography particle; (b') embedding the chromatography particles provided in step (a') in a surrounding layer having a second average pore diameter; Including, the first average pore diameter and the second average pore diameter are different from each other, and the second average pore diameter is at least 1.5 times the first average pore diameter; A method wherein the first average pore diameter prevents diffusion of the target molecules through the pores of the core and the second average pore diameter allows at least some diffusion of the target molecules through the pores of a layer surrounding the core.

15. The addition of step (b) (i) the coupling of polymer molecules to the core of the particle, or (ii) polymer grafting, which involves adding a monomer to the core of the particle and polymerizing the monomer inside the core, thereby creating polymer molecules inside the core; 15. The method of claim 14, further comprising:

16. 14. Use of a separation matrix according to any one of claims 1 to 13 for preparative applications, such as for separating target molecules from cell culture harvest or impurities.

17. 17. The use according to claim 16, wherein the target molecule is an adeno-associated virus capsid, a monoclonal antibody, an antigen-binding fragment of an antibody, a virus-like particle, a lentivirus capsid, an adenovirus capsid, or an influenza virus capsid.

18. 14. A method of preparative chromatography for separating one or more target molecules from one or more other compounds in a liquid sample, comprising contacting a liquid sample comprising the target molecules and the compounds with a separation matrix according to any one of claims 1 to 13, and exposing the liquid sample to a pressure of <5 bar, for example <3 bar.

19. 20. The method of claim 18, further comprising a clarification and / or chromatography step prior to the step of contacting the liquid sample containing the target molecules and compounds with the separation matrix.

20. 1. A method for separating adeno-associated virus capsids in which genetic material is fully packaged from adeno-associated virus capsids in which genetic material is not fully packaged, comprising: a) applying a liquid sample containing adeno-associated virus capsids to a weak cation exchange chromatography material, The liquid sample has a purity of at least 90% and 12 adeno-associated virus capsids at a concentration of adeno-associated virus capsids / ml, at least 10% of the adeno-associated virus capsids being fully packaged adeno-associated virus capsids in which the genetic material is; A weak cation exchange chromatography material, i. a ligand for binding to an adeno-associated virus capsid, and ii. A support comprising a separation matrix according to any one of claims 1 to 13. A process comprising: b) eluting the adeno-associated virus capsids in which the genetic material is fully packaged from the chromatographic material. wherein the adeno-associated virus capsids eluted in step (b) are eluted in an eluate fraction, the combined eluate fraction comprising at least 50% of the adeno-associated virus capsids of the liquid sample added in step (a), at least 60% of the adeno-associated virus capsids being fully packaged with genetic material; Optionally, the method, wherein the adeno-associated virus capsid is a capsid of adeno-associated virus serotype 9 (AAV9) or a variant thereof.

21. The ligand of the weak cation exchange chromatography material has the following formula I: 【Chemistry 1】 (In the formula, X 1 is selected from O, S, and NHCO; n is an integer from 1 to 4, Each R 1 and R 2 are independently selected from hydrogen, C1-C3 alkyl, and OH.

21. The method of claim 20, wherein said first and second inputs are defined by:

22. X 1 is O and n is 1, or X 1 is S, n is 2, and each R 1 and R 2 22. The method of claim 21 , wherein is hydrogen.

23. 23. The method according to claim 21 or 22, wherein the density of the ligand is from about 30 to about 110 μmol, such as from about 40 to about 90 μmol, such as from about 50 to about 80 μmol of ligand per ml of weak cation exchange chromatography material.

24. The eluate fraction containing the adeno-associated virus capsids in which the genetic material is fully packaged, which is eluted in step (b) as defined in claim 20, is c1) concentrating the adeno-associated virus capsid to a pharma- ceutical suitable dose; c2) replacing the buffer applied in step (b) as defined in claim 20 with a pharma- ceutically acceptable buffer, and / or c3) Sterilizing the eluate fraction containing the adeno-associated virus capsid 24. The method of claim 20, further comprising subjecting the adeno-associated virus capsid to one or more of the following steps to obtain a pharmaceutical composition comprising the adeno-associated virus capsid:

25. 25. A composition comprising adeno-associated virus capsids obtained by carrying out the method of any one of claims 20 to 24, wherein the ratio of adeno-associated virus capsids in which genetic material is fully packaged to adeno-associated virus capsids in which the genetic material is not fully packaged is at least 3:2, preferably at least 4:1.

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