Methods and compositions for purifying adeno-associated virus particles

Abstract

The present invention relates to a method for purifying adeno-associated virus particles by cation exchange mixed-mode chromatography.

Description

[Technical field]

[0001] The present invention relates to a method for purifying adeno-associated virus particles by cation exchange mixed-mode chromatography. [Background technology]

[0002] Adeno-associated virus (AAV) has been characterized and developed as a powerful viral vector for gene delivery in cultured cells in vitro and also in vivo.Meanwhile, AAV is the main platform for in vivo delivery of gene therapy.AAV is a small non-enveloped virus that contains a single-stranded DNA genome of approximately 4.7 kb, consisting of one rep region that codes for four overlapping replication proteins Rep78, Rep68, Rep52, and Rep40, two inverted terminal repeats (ITRs) that can form a T-shaped secondary structure and act as the origin of genome replication, and one cap region that codes for three structural proteins VP1, VP2, and VP3 and assembly activation protein (AAP).

[0003] The naturally isolated serotypes 1 to 9 of AAV viruses share a common genome structure, but these serotypes may display different tissue tropism. Because AAVs appear to be nonpathogenic and exhibit efficient transduction and stable expression, they are considered to be one of the most promising gene delivery vehicles.

[0004] AAV vectors can be produced in various cell lines in adherent or suspension cell culture formats using transient transfection or superinfection methods. Depending on the specific serotype and production times, viral particles, including complete, partial, and empty seeds, can be secreted from outside the cell into the culture medium and contained inside the cell in various proportions.

[0005] Initially, stable AAV producer cells were generated by transfection and selection of human-derived cells, such as HeLa or HEK293 cells, with rAAV transfer vectors containing an ITR cassette and packaging constructs containing Rep and Cap. Then, production of recombinant AAV vectors (rAAV) was achieved by infection with auxiliary viruses, such as adenovirus (AdV), which provide helper functions. After identification of the AdV genes required for AAV vector packaging, a helper virus-free method was established using a double or triple transfection protocol consisting of two or three plasmids that contain constructed helper plasmids instead of auxiliary viruses. This system is widely used in research and drug discovery. In addition, the development of baculovirus expression vectors provides another method to produce rAAV virus in insect Sf9 cells. These various techniques have been shown to be capable of producing rAAV virus of sufficient quality for laboratory and clinical trials.

[0006] A cell lysis step is generally required to release viral particles into the supernatant upon harvesting. For this application, typical cell lysis reagents such as Triton X-100, Tween 20, and NaCl are commonly used.

[0007] After cell lysis, AAV needs to be purified.Typical AAV purification processes include clarification, concentration and diafiltration using tangential flow filtration, chromatographic purification by using affinity chromatography and ion exchange chromatography.In some processes, ultracentrifugation and gradient ultracentrifugation are used instead of or in addition to chromatography.The final step in AAV purification typically involves concentration and diafiltration into a suitable excipient buffer composition and sterile filtrate.

[0008] Although the demand for viral vectors as vehicles in gene therapy is increasing, current manufacturing processes suffer from purification methods that scale to production demands while also reducing the burden of impurities.Adeno-associated viruses (rAAV) are attractive vectors in the field due to their ability to induce diving and non-diving tissues, patient safety, and control for cell-specific application.However, producing these viruses with increased efficiency and lower manufacturing costs is a difficult task for downstream processing.

[0009] A major challenge in current AAV processes is the production of non-genome-containing particles. Although their mechanism of action is not fully understood, empty rAAV capsids are treated as a major process impurity that may interfere with the safety and efficacy of the final formulated drug. Regardless of being produced in mammalian or insect cell lines, the resulting rAAV feed stream typically contains 10-90% empty capsids that need to be removed further downstream. Typically downstream schemes consist of two steps: an initial affinity capture step that captures all rAAV particles from the feed stream and removes other process-related impurities, followed by a polishing step that separates complete from empty rAAV particles. It has been investigated that complete capsids have a higher charge density due to the negatively charged genome they harbor. And although this negatively charged genome only slightly changes the isoelectric point (pI) (empty: pI=5.9 vs. complete: pI=6.3), these attributes are sufficient that ion exchange chromatography, especially anion exchange chromatography (AEX), has been widely investigated for this type of application (Wang, Dan; Tai, Phillip WL; Gao, Guangping (2019): Adeno-associated viral vector as a platform for gene therapy delivery. In: Nature reviews. Drug discovery 18 (5), S. 358-378. DOI: 10.1038 / s41573-019-0012-9).

[0010] In WO04113494 a method is disclosed for separating empty and full particles by single or multiple anion and / or cation exchange steps.

[0011] Nonetheless, there remains a need for efficient and simple methods that provide purification of target rAAV from process-related impurities as well as separation of empty and complete capsids. Summary of the Invention

[0012] The inventors have surprisingly found that mixed-mode cation exchange chromatography is particularly effective in purifying rAAV from process-related impurities such as host cell proteins. Typically, empty capsids can also be partially or completely removed in the same chromatography step.

[0013] Thus, the present invention is directed to a method for purifying adeno-associated virus (AAV) particles by contacting a sample containing the AAV particles with a mixed-mode cation exchange chromatography matrix, the chromatography matrix comprising cation exchange groups and hydrophobic groups.

[0014] In a preferred embodiment, the method of the present invention comprises the following steps: a) contacting a sample containing AAV particles with a mixed-mode cation exchange chromatography matrix; b) optionally washing the chromatography matrix; c) eluting the AAV particles bound to the chromatography matrix in step a) with an elution buffer.

[0015] In a preferred embodiment, in step c) the elution buffer has a higher pH than the loading buffer. The elution buffer may be applied in a step or in a gradient. In another embodiment, the method includes collecting AAV particles that flow through the chromatography matrix without being bound to it.

[0016] In a preferred embodiment, the chromatography matrix is ​​a membrane or a monolith. In a highly preferred embodiment, the chromatography matrix is ​​a membrane, in particular a hydrogel membrane.

[0017] In another preferred embodiment, the sample contains empty and complete AAV capsids, and a mixed-mode cation exchange chromatography matrix is ​​contacted with the sample under conditions such that the majority of the AAV particles, both empty and complete, bind to the chromatography matrix, while the majority of process-related impurities flow through the matrix.

[0018] In another preferred embodiment, elution occurs under conditions such that the majority of empty AAV capsids emerge from the mixed-mode cation exchange chromatography matrix along with other impurities prior to the majority of intact AAV particles.

[0019] In another preferred embodiment, the AAV purified by the methods of the present invention has a ratio of full to empty AAV of more than 70% full AAV, preferably more than 90%, and most preferably more than 99% full AAV, meaning that empty particles have been reduced to less than 10%, more preferably less than 1%. In a preferred embodiment, the sample is a crude lysate.

[0020] In another preferred embodiment, the elution in step c) is performed with a linear pH gradient from a pH between 4 and 6.5 to a pH between 9 and 11, while keeping the conductivity at a constant level, preferably between 1 mS / cm and 50 mS / cm, most preferably between 13 and 20 mS / cm. Preferably, the target AAV elutes at a pH above 8.0, in particular at a pH between 8.5 and 10.5, while empty AAV as well as process related impurities such as HCPs elute ahead of the target AAV at a pH below 8.

[0021] In a preferred embodiment, in a fraction of the AAV eluted in step c), the HCPs are reduced by a log reduction of 2 logs or more compared to the sample applied in step a) and the percentage of intact AAV is greater than 90%. In a particularly preferred embodiment, this fraction results from elution with a linear pH gradient from a pH between 4 and 6.5 to a pH between 9 and 11, while keeping the conductivity at a constant level, and the fraction elutes at a pH above 8.0, in particular it is a fraction eluting between pH 8.5 and 10.5.

[0022] The present invention is further directed to a method for purifying AAV particles from a sample containing cells encapsulating viral particles by: a) lysing the cells; b) isolating and / or purifying the AAV particles by contacting them with a mixed-mode cation exchange chromatography matrix.

[0023] In a preferred embodiment, the lysis of the cells is achieved using a detergent selected from the group of alkyl dimethylamine oxides, and optionally sodium chloride.

[0024] In one embodiment, the method of the invention comprises one or more of the following steps: - Clarification - Filtration - Dialysis / Diafiltration - Tangential flow filtration - Treatment with nucleases, e.g. RNase and / or DNase - Treatment with chloroform - Ion exchange chromatography - Affinity Chromatography - Hydrophobic Interaction Chromatography - Centrifugation - PEG precipitation [Brief description of the drawings]

[0025] [Figure 1]Figure 1 shows a run on an Eshmuno® CMX column performed on an AEKTA™ system. (UV-280 nm blue; 260 nm red). No AAV was detectable during loading. A first peak occurred in the wash step, and a second peak could be eluted during the elution gradient. The overall recovery of all rAAV2 capsids was 78% and for intact rAAV2 capsids was 77%, however, no enrichment in the elution peaks was observed. Further details can be found in Example 1.

[0026] [Diagram 2] Figure 2 shows a chromatogram of AAV2 from a capture run using step gradient elution on a 1 mL volume mixed-mode membrane. The equilibration buffer consisted of a combination of salts and 120 mM NaCl and was at pH 5.3, whereas the elution buffer contained 500 mM NaCl and the same constituent salts and was at pH 8.5. Further details can be found in Example 2.

[0027] [Diagram 3] Figure 3 shows a chromatogram of AAV2 from a capture run using step gradient elution on a 1 mL volume mixed-mode membrane aimed at improving HCP clearance. Operating conditions: pH 6.5, 120 mM NaCl, where the elution buffer contains 500 mM NaCl and the same constituent salts, and is at pH 8.5. Further details can be found in Example 3.

[0028] [Figure 4] Figure 4 shows the chromatogram of AAV2 from a capture run using linear gradient elution on a 1 mL volume mixed-mode membrane aimed at improving HCP clearance and intact rAAV2 capsid on step 1. Binding conditions: pH 5.3, 150 mM NaCl, where the elution buffer contains 150 mM NaCl and the same constituent salts, and is at pH 10. Further details can be found in Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Before describing the invention in detail, it is to be understood that the invention is not limited to particular compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "ligand" includes a plurality of ligands, and reference to an "antibody" includes a plurality of antibodies, and so forth.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For purposes of the present invention as described herein, the following terms are defined.

[0031] Adeno-associated virus (AAV) is a member of the Parvoviridae family. The AAV genome is composed of a linear, single-stranded DNA molecule that contains approximately 4.7 kilobases (kb) and consists of two major open reading frames that code for the nonstructural Rep (replication) and structural Cap (capsid) proteins. Adjacent to the AAV coding region are two cis-acting inverted terminal repeat (ITR) sequences, approximately 145 nucleotides in length, with an interrupted palindrome that can fold into a hairpin structure that functions as a primer during the initiation of DNA replication. In addition to their role in DNA replication, ITR sequences have been shown to be necessary for viral assembly, rescue from the host genome, and encapsidation of viral nucleic acid into mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158:97-129).

[0032] Multiple serotypes of AAV exist and display different tissue tropisms. Known serotypes include, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11.

[0033] Vectors derived from AAV are particularly attractive for delivering genetic material because they can infect (transduce) a wide variety of non-dividing and dividing cell types, including muscle fibers and neurons, and because they lack viral structural genes, thereby eliminating natural host cell responses to viral infection, such as the interferon-mediated response. In addition, the wild-type virus has not been associated with any pathology in humans.

[0034] According to the present invention, scAAV is also within the group of AAV. Self-complementary adeno-associated vector (scAAV) is a viral vector modified from naturally occurring adeno-associated virus (AAV) for use in gene therapy. ScAAV is termed "self-complementary" because the coding region is designed to form an intramolecular double-stranded DNA template.

[0035] Thus, in some embodiments, "AAV" refers to vectors or viruses derived from adeno-associated virus serotypes, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 and AAV-11. AAV vectors can have one or more AAV wild-type genes deleted in whole or in part, e.g., the rep and / or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for rescue, replication and packaging of AAV virions. Thus, AAV vectors are defined herein to include at least those sequences (e.g., functional ITRs) that provide for viral replication and packaging. ITRs need not be wild-type nucleotide sequences and may be altered, e.g., by insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging. In one embodiment, the vector is an AAV-9 vector, which has ITRs from AAV-2. "AAV" also refers to a protein shell or capsid that provides an efficient vehicle for delivery of vector nucleic acid to the nucleus of a target cell.

[0036] The term "AAV" as used herein is also intended to cover recombinant AAV. Adeno-associated virus (AAV) is also referred to herein as a virus, viral particle or viral vector.

[0037] As used herein, the term "cell" or "cell line" refers to a single cell or a population of cells capable of continuous or sustained growth and division in vitro. In some embodiments, by way of example, the terms "HEK293 cells," "293 cells," or grammatical equivalents thereof are used interchangeably herein and refer to the host / packaging cell line used in the methods disclosed herein.

[0038] Suitable cells and cell lines are described for use in the production of AAV and AdV. The cells themselves may be selected from any biological organism, including prokaryotic (e.g., bacterial) cells and eukaryotic cells, including insect cells, yeast cells and mammalian cells. Particularly preferred host cells are selected from any mammalian species, including, but not limited to, A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, HEK 293 cells, Saos, C2C12, L cells, HT1080, and HepG2, as well as primary fibroblasts, hepatocytes and myoblasts from mammals, including humans, monkeys, mice, rats, rabbits and hamsters.

[0039] Generally, an expression cassette is composed, at a minimum, of a 5' AAV inverted terminal repeat (ITR), a nucleic acid sequence encoding a desired therapeutic immunogen or antigen operably linked to regulatory sequences that direct its expression, and a 3' AAV ITR. In one embodiment, the 5' and / or 3' ITR of AAV serotype 2 is used. However, 5' and / or 3' ITRs from other suitable sources may be selected. This expression cassette is packaged into a capsid protein to form an AAV virus or particle.

[0040] In addition to the expression cassette, the cell contains a sequence that drives the expression of AAV in the cell (cap sequence) and a rep sequence of the same source or cross-complementary source as the source of the AAV ITR found in the expression cassette. The AAV cap and rep sequences may be independently selected from different AAV parental sequences and introduced into the host cell in a suitable manner known to those skilled in the art. While the full-length rep gene may be utilized, smaller fragments thereof, namely rep78 / 68 and rep52 / 40, have been found to be sufficient to allow AAV replication and packaging.

[0041] The cell also requires helper functions to package the AAV of the present invention. Optionally, these helper functions may be provided by herpesvirus. In another embodiment, the necessary helper functions are provided by human or primate non-human animal adenovirus sources, such as those available from a variety of sources, including American Type Culture Collection (ATCC), Manassas, Va. (US), respectively.

[0042] During the production of AAV, some percentage of capsids may not incorporate any transgene, and are called empty capsids, empty AAVs or empty AAV particles.In addition, capsids containing fragments of transgenes are called partial capsids, partial AAVs or partial AAV particles.These undesirable product-related impurities are produced together with the complete capsids or complete AAVs that contain the full length of desired transgenes.

[0043] Purification means increasing the purity of a target molecule, in this case AAV, for example by removing one or more impurities. The term "impurity" or "contaminant" as used herein refers to any foreign or deleterious molecule or species, including DNA, RNA, one or more host cell proteins, nucleic acids, endotoxins, biological macromolecules such as lipids, impurities of synthetic origin such as detergents, partial and / or empty AAV or AdV, and one or more additives that may be present in a sample containing viral particles to be purified and thereby separated from one or more of the impurities.

[0044] As used herein, and unless otherwise stated, the term "sample" refers to any composition or mixture containing AAV. Samples may be derived from biological or other sources. Biological sources include eukaryotic and prokaryotic sources, such as plant and animal cells, tissues and organs. Samples may also include diluents, buffers, detergents, and contaminating species, debris, etc., found mixed with the target molecule. Samples may be "partially purified" (i.e., subjected to one or more purification steps, such as filtration steps) or may be obtained directly from host cells that produce AAV; for example, samples may include harvested cell culture fluid.

[0045] Alkyl dimethylamine oxides suitable for lysing virus-producing cells are amphiphilic, charged amine oxides coupled to saturated hydrocarbon chains of various lengths. Preferably, the length of the saturated hydrocarbon chain is between 8 and 18 carbon atoms. In a preferred embodiment, the alkyl dimethylamine oxide is selected from the group consisting of dimethyldecylamine oxide, dimethylundecylamine oxide, dimethyldodecylamine oxide (LDAO), dimethyltridecylamine oxide and dimethyltetradecylamine oxide (TDAO), with TDAO being particularly preferred.

[0046] These compounds are preferably used at concentrations above their critical micelle concentration. Critical micelle concentration (CMC) is defined as the concentration of a surfactant above which micelles are formed and any additional surfactant added to the system becomes micelles. The value of CMC for a given surfactant in a given medium depends on temperature, pressure, and (sometimes strongly) on the presence and concentration of other surfactants and electrolytes.

[0047] The terms "purifying," "separating," or "isolating," as used interchangeably herein, refer to increasing the purity of a target AAV from a composition or sample that contains the target AAV and one or more impurities.

[0048] The term "chromatography" refers to any type of technique for separating an analyte of interest (e.g., a target AAV) from other molecules present in a sample. Often, the target AAV is separated from other molecules as a result of differences in the rate at which individual molecules of a mixture bind to and / or migrate through a chromatographic matrix under the influence of a mobile phase.

[0049] The terms "matrix" or "chromatographic matrix" are used interchangeably herein and refer to a solid phase through which a sample passes during chromatographic separation. A matrix typically comprises a substrate and a ligand covalently bound to the substrate. Matrices of the present invention include or consist of particles, membranes or monoliths, for example, preferably the substrate is a membrane or monolith, most preferably a membrane.

[0050] A "ligand" is a functional group that is part of a chromatography matrix, typically attached to the matrix substrate, and that determines the binding and interaction properties of the matrix. Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic interaction groups, metal affinity groups, affinity groups, bioaffinity groups, and mixed mode groups (combinations of the foregoing). It is also possible for one ligand to have more than one binding / interaction property.

[0051] The matrix of the present invention comprises at least cation exchange groups and hydrophobic interaction groups. It is a mixed mode cation exchange chromatography matrix. The cation exchange groups may be strong cation exchange groups, such as, for example, sulfonic acid groups. They may also be weak cation exchange groups, such as carboxymethyl or carboxylic acid. Examples of hydrophobic interaction groups are hydrophobic groups, such as phenyl, butyl, propyl, hexyl. The matrix may comprise two or more different cation exchange groups, for example, weak and strong cation exchange groups. It may also comprise two or more different hydrophobic interaction groups. It may additionally comprise further other types of ligands. The groups may be part of the substrate or they may also be part of the ligand. One ligand may comprise one or more different cation exchange groups and / or hydrophobic groups.

[0052] The ligand can be attached to the substrate of the matrix by any type of covalent attachment. Covalent attachment can be performed by directly binding a functional group to a suitable residue on the substrate, such as, for example, OH, NH2, carboxyl, phenol, anhydride, aldehyde, epoxide, or thiol. It is also possible to attach the ligand via a suitable linker. It is also possible to produce the matrix by polymerizing a monomer that contains the ligand and a polymerizable moiety. Examples of matrices produced by polymerization of suitable monomers are polystyrene, polymethacrylamide or polyacrylamide-based matrices produced by polymerizing suitable styrene or acryloyl monomers.

[0053] In another embodiment, the chromatography matrix can be produced by grafting ligands onto or from the substrate. Grafting is suitable from processes involving controlled free radical polymerization, such as the method of atom transfer free radical polymerization (ATRP). A highly preferred one-step grafting, for example from the polymerization reaction of acrylamides, methacrylates, acrylates, methacrylates, etc. functionalized with ionic, hydrophilic or hydrophobic groups, can be initiated by cerium (IV) on hydroxyl-containing supports without the need to activate the support.

[0054] When a chromatography matrix is ​​used in a chromatographic separation, it is typically used within a separation device, also called a housing, as a means for holding the matrix.

[0055] In one embodiment, the device comprises a housing with an inlet and an outlet and a fluid path between the inlet and the outlet. In one embodiment, the device is a chromatography column. Chromatography columns are known to those skilled in the art. They usually comprise a cylindrical tube or cartridge packed with a stationary phase, and a filter and / or a means for fixing the stationary phase in the tube or cartridge, and optionally a connection for solvent delivery to and from the tube or cartridge. The size of the chromatography column varies depending on the application, for example analytical or preparative. In one embodiment, the column, or generally the separation device, is a single-use device.

[0056] In an embodiment, the housing comprises: A housing unit, wherein the housing unit comprises: (a) Entrances and exits; (b) a fluid flow path between the inlet and the outlet; and (c) Chromatography matrix within the housing unit.

[0057] In one embodiment, the chromatography matrix comprises: a support member including a plurality of pores extending therethrough; and a non-free-standing macroporous crosslinked gel comprising macropores having an average size of 10 nm to 3000 nm, the macroporous gel being positioned within the pores of a support member; wherein the macropores of the macroporous crosslinked gel are smaller than the pores of the support member; Here, the pores of the support member are substantially perpendicular to the fluid flow path.

[0058] In one aspect, the present invention relates to a fluid treatment device comprising: A plurality of housings, in particular housing units, where each housing unit comprises: (a) Entrances and exits; (b) a fluid flow path between the inlet and the outlet; and (c) Chromatography matrix within the housing unit.

[0059] In certain embodiments, particularly when the chromatography matrix is ​​a membrane, the chromatography matrix is ​​arranged within the housing in a substantially coplanar stack of substantially coextensive sheets, in a substantially tubular configuration, or in a substantially spirally wound configuration.

[0060] A "buffer" is a solution that resists changes in pH by the action of its acid-base conjugate components. For example, various buffers that can be employed depending on the desired pH of the buffer are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975). Non-limiting examples of buffers include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, and combinations thereof.

[0061] According to the present invention, the term "buffer" or "solvent" is used for any liquid composition used to load, wash, elute, re-equilibrate, strip and / or sanitize a chromatography matrix.

[0062] When "loading" a chromatography column in binding and elution mode, a sample or composition containing a target molecule and one or more impurities is loaded onto the chromatography column. In preparative chromatography, the sample is preferably loaded directly without the addition of a loading buffer. If a loading buffer is used, the buffer has a composition, conductivity, and / or pH such that the target AAV binds to the stationary phase, while ideally all impurities such as host cell proteins or empty AAV flow through the column without being bound. Typically, if used, the loading buffer has the same or similar composition as the equilibration buffer used to prepare the column for loading. The final composition of the sample loaded onto the column is called the feed. The feed may include the sample and the loading buffer, but preferably it is only the sample.

[0063] "Washing" or "washing" a chromatography matrix means passing a suitable liquid, e.g., a buffer, through or over the matrix. Typically, washing is used to remove weakly bound impurities from the matrix in bind / elute mode before eluting the target molecule. In addition, washing steps can be used to reduce residual detergent levels, enhance virus clearance, and / or modify conductivity carryover during elution.

[0064] "Eluting" a molecule (e.g., a target AAV) from a matrix means that the molecule is removed from it. Elution can occur by changing solution conditions such that a buffer different from the loading buffer and / or wash buffer competes with the molecule of interest for ligand sites on the matrix, or by altering the equilibrium of the target molecule between the stationary and mobile phases such that the target molecule is preferentially present in the elution buffer.

[0065] A non-limiting example is to elute molecules from a mixed mode cation exchange resin by changing the ionic strength of the buffer surrounding the mixed mode cation exchange material such that the buffer competes with the molecules for charged sites on the ion exchange material. Alternatively, a pH change is also suitable.

[0066] The terms "flow-through process," "flow-through mode," and "flow-through operation," as used interchangeably herein, refer to a chromatographic process in which at least one target molecule (e.g., AAV) contained in a sample along with one or more impurities is intended to flow through a chromatographic matrix that generally binds the one or more impurities, where the target molecule generally does not bind (i.e., flows through) and elutes from the chromatographic matrix along with the loading buffer.

[0067] The terms "binding and elution mode" and "binding and elution process" as used herein refer to a separation technique in which at least one target molecule (e.g., AAV) contained in a sample is bound to a suitable chromatographic matrix and then eluted with a buffer different from the loading buffer.

[0068] The term "ion density" as used herein refers to the number of ions per unit volume or mass of a given separation material, more specifically, the number of ions of a given type (e.g., positive or negative ions) per unit volume or mass of the separation material. Most often, the number of ions is estimated by titrating a given separation material. Moreover, the amount of ions is given in equivalents (eq) per unit of mass or volume for the separation material.

[0069] The term "conductivity" as used herein refers to an intrinsic property of most materials that quantifies how strongly it resists or conducts electric current. In an aqueous solution, such as a buffer solution, electric current is carried by charged ions. Conductivity is determined by the number of charged ions, the amount of charge they carry, and how fast they move. Thus, in most aqueous solutions, the higher the concentration of dissolved salts, the higher the conductivity. Increasing the temperature allows the ions to move faster, thus increasing the conductivity. Typically, unless otherwise specified, conductivity is defined at room temperature. The base unit of conductance is the Siemens (S). It is defined as the reciprocal of the resistance in Ohms measured between the opposing faces of a 1 cm cube of liquid. Thus, values ​​are estimated in S / cm.

[0070] Log reduction is a measure of how thoroughly a purification process reduces the concentration of a contaminant. It is defined as the common logarithm of the ratio of the level of contamination before and after the process, with an increase of 1 corresponding to a reduction in concentration by a factor of 10. In general, a log reduction of n means that the remaining concentration of the contaminant is only 10-n times lower than the original. Thus, for example, a log reduction of 0 is no reduction at all, while a log reduction of 1 corresponds to a 90 percent reduction from the original concentration, and a log reduction of 2 corresponds to a 99 percent reduction from the original concentration.

[0071] Membranes as chromatographic matrices can be distinguished from particle-based chromatography by the fact that the interaction between solutes, for example target AAV or impurities, and the matrix does not occur in the dead-end pores of the particle, but mainly occurs in the through-pores of the membrane. Exemplary membrane types are flat sheet systems, stacks of membranes, microporous polymer sheets with integrated cellulose, polystyrene or silica-based membranes, as well as radial flow cartridges, hollow fiber modules and hydrogel membranes. Preferred are hydrogel membranes. Such membranes include a membrane support and a hydrogel formed in the pores of the support. The membrane support provides mechanical strength to the hydrogel. The hydrogel determines the properties of the final product, such as pore size and binding chemistry.

[0072] The membrane support can consist of any porous membrane, such as a polymeric membrane, a ceramic-based membrane, a woven or non-woven fibrous material, etc. Suitable polymeric materials for the membrane support are cellulose or cellulose derivatives, as well as other, preferably inert, polymers, such as polyethylene, polypropylene, polybutylene terephthalate or polyvinylidene difluoride.

[0073] Hydrogels can be formed through the in situ reaction of one or more polymerizable monomers with one or more crosslinkers and / or one or more crosslinkable polymers to form crosslinked gels, preferably with macropores. Suitable polymerizable monomers include monomers containing vinyl or acrylic groups. Preferred are monomers containing additional functional groups that either directly form the ligands of the matrix or are suitable for attaching ligands. Suitable crosslinkers are compounds that contain at least two vinyl or acrylic groups.

[0074] Further details on suitable membrane supports, monomers, crosslinkers, etc., as well as suitable production conditions can be found in WO04073843 and WO2010 / 027955. Particularly preferred are membranes made of inert flexible fibrous reticular supports, such as Natrix® type membranes, Merck KGaA, Germany, which contain an assembly of porous polyacrylamide hydrogels with cation exchange groups and hydrophobic groups inside and around the fibrous reticular support. Further details on suitable hydrogel mixed-mode membranes can be found in WO2014018635.

[0075] Examples of membranes that are suitable for use in the method of the present invention are: - Mustang® type membranes, such as Pall, which have a polyethersulfone (PES) based support and a cross-linked polymer coating functionalized with suitable ligands. - Sartobind® type membranes, such as Sartorius, made of stabilized reinforced cellulose functionalized with suitable ligands. - Membranes made of stabilized reinforced cellulose containing a hydrogel with suitable ligands, such as Sartobind® Jumbo Membranes, Sartorius, made of stabilized reinforced cellulose. - 3M™ Emphaze™ Hybrid Purifier type membranes, such as 3M, made of a fine fiber nonwoven scaffold containing a hydrogel with suitable ligands. - Membranes made of inert flexible fibrous reticulated supports, such as Natrix® type chromatography membranes, Merck KGaA, Germany, which contain a porous polyacrylamide hydrogel with suitable cation exchange and hydrophobic ligands within and around the fibrous reticulated support.

[0076] Monoliths or monolithic adsorbents, like membranes, have through-holes, such as interconnected channels, that allow liquid to flow from one side of the monolith through the monolith to the other side of the monolith. As the mobile phase flows through these through-holes, the molecules to be separated are transported by convection rather than by diffusion. Due to their structure, monolithic adsorbents exhibit flow-rate independent separation efficiency and dynamic capacity.

[0077] The monolith is typically formed in situ from the reactant solution and can have any shape or constrained geometry, typically a frit-free structure, which ensures convenience of operation. Preferably, the monolithic material has a dual porous structure, mesopores and macropores. The micron-sized macropores are through-holes and ensure fast dynamic transport and low back pressure in applications, while the mesopores contribute to sufficient surface area and thus high loading capacity.

[0078] The monoliths can be made of organic, inorganic or organic / inorganic hybrid materials, with organic polymer-based monoliths being preferred. The synthesis of organic polymer monoliths is typically carried out by one-step polymerization, which provides tunable porous structures with tailored functional groups. Generally, a prepolymerization mixture consisting of monomers, crosslinkers, porogenic solvents, and initiators in appropriate ratios is polymerized in a suitable container, also called a mold, which determines the form of the monolith. Polymerization is typically initiated by the use of heat, UV radiation, microwaves, or gamma radiation in the presence of an initiator. After reacting at the appropriate temperature for a specified time, the resulting material is typically washed with a solvent to remove unreacted components and the porogenic solvent.

[0079] Suitable organic polymers are polymethacrylates, polyacrylamides, polystyrenes, polyurethanes, and the like, such as poly(methacrylic acid-ethylene dimethacrylate), poly(glycidyl methacrylate-ethylene dimethacrylate) or poly(acrylamide-vinylpyridine-N,N'-methylenebisacrylamide).

[0080] Inorganic monoliths can be made of silica or other inorganic oxides. Preferably, they are made of silica. Silica monoliths are usually prepared via a sol-gel process with phase separation. This mainly involves hydrolysis, condensation, and polycondensation of silica precursors. Typically, tetraethoxysilane (TEOS) or tetramethyl orthosilicate (TMOS) is dispersed in a suitable solvent in the presence of a porogen (e.g., poly(ethylene glycol) (PEG)), followed by the addition of a catalyst, acid or base, or a dual catalyst, acid and base, in sequence. After reacting for a given time, the resulting gel-like product is washed with a solvent to remove unreacted precursor, porogen, and catalyst, followed by a suitable post-treatment, typically a thermal treatment.

[0081] The monolith can be modified with suitable functional groups, in this case cation exchange groups and hydrophobic interaction groups, to allow targeted interaction with samples containing target molecules and thus targeted separation. Typically, the monolith is contained within a housing, such as a column. Membranes and monoliths can also be produced by 3D printing processes.

[0082] The particulate substrate can be prepared, for example, from organic polymers. This type of organic polymer can be polysaccharides, such as agarose, dextran, starch, cellulose, etc., or synthetic polymers, such as poly(acrylamide), poly(methacrylamide), poly(acrylate), poly(methacrylate), hydrophilically substituted poly(alkylaryl ether), hydrophilically substituted poly(alkylvinyl ether), poly(vinyl alcohol), poly(styrene), and copolymers of the corresponding monomers. These organic polymers can also be preferably employed in the form of crosslinked hydrophilic networks. This also includes polymers made of styrene and divinylbenzene, which, like other hydrophobic polymers, can preferably be employed in hydrophilized form.

[0083] Alternatively, inorganic materials such as silica, zirconium oxide, titanium dioxide, aluminum oxide, etc. can be employed as particulate substrates. It is also possible to employ composite materials, i.e. particles that can themselves be magnetized, for example by copolymerization of magnetic particles or magnetic cores. It is also possible to use shells, i.e. core-shell materials, where at least the surface or coating bears OH groups.

[0084] However, since the materials according to the invention should preferably withstand extended periods of use, e.g. alkaline cleaning or regeneration at basic pH, the use of hydrophilic substrates that are hydrolytically stable or difficult to hydrolyze is preferred.

[0085] The base matrix may consist of irregularly shaped or spherical particles, the particle size of which may be between 2 and 1000 μm. An average particle size between 3 and 300 μm is preferred, and in the most preferred embodiment the average particle size is between 20 and 63 μm.

[0086] The particulate substrate may be in the form of, inter alia, non-porous or preferably porous particles. The average pore size may be between 2 and 300 nm. Pore sizes between 5 and 200 nm are preferred, and the most preferred average pore size is between 40 and 110 nm.

[0087] In a highly preferred embodiment, the particulate substrate is formed by copolymerization of a hydrophilic substituted alkyl vinyl ether selected from the group of 1,4-butanediol monovinyl ether, 1,5-pentanediol monovinyl ether, diethylene glycol monovinyl ether or cyclohexanedimethanol monovinyl ether and divinylethyleneurea (1,3-divinylimidazolin-2-one) as a crosslinker. An example of a suitable commercially available vinyl ether-based substrate is Eshmuno®, Merck KGaA, Germany.

[0088] Preferred particle matrices are those with weak cation exchange, strong cation exchange and hydrophobic groups, e.g. matrices with sulfonic acid, carboxylic acid and phenyl groups, such as Eshmuno® HCX, Merck KGaA, Germany.

[0089] Also preferred is a particulate chromatography matrix comprising a hydroxyl-containing substrate, preferably a vinyl ether-based substrate, having polymer chains grafted to its surface by covalent bonds, characterized in that: a) the polymer chains are covalently attached to the base matrix via hydroxyl groups; b) the polymer chain comprises an end group -N(Y)-R3; Y are each independently H or CH3, preferably H; R3 is -CHCOOMR4, R4 is C1-C4 alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, preferably isopropyl and isobutyl, very preferably isobutyl, or C1-C4 perfluoroalkyl; and M is, independently of each other, H, Na, K, or NH4 + It is.

[0090] One example is Eshmuno® CMX, Merck KGaA. The substrate may also be in the form of a fiber, hollow fiber, or coating.

[0091] The present invention provides a method for separating or purifying AAV. This means that one or more AAVs can be separated from one or more other AAVs, from empty or partial AAVs, and / or from other impurities in a sample. Preferably, at least one AAV is separated from at least one impurity. This is done by chromatographic separation on a mixed-mode cation-exchange hydrophobic chromatography matrix that contains at least one type of cation-exchange group and one type of hydrophobic group.

[0092] The method of the present invention can separate, concentrate and / or purify AAV, and can provide efficient separation.In a particular aspect of the present invention, the target AAV can be separated from impurities and empty AAV in one chromatographic step.High resolution separation can be achieved, and different AAV species can be isolated.

[0093] The production of cells containing AAV is known to those skilled in the art. Typically, selected cells are expanded in a suitable culture medium in a bioreactor under suitable conditions. The cells may be grown as adherent or suspension cultures. For example, in suspension cultures of HEK293 cells, the suitable seeding number before transfection is 0.5-1.1 e6 viable cells per ml.

[0094] Suitable methods for transduction are known in the art. In one embodiment, cells can be transduced in vitro by combining the rAAV with the cells, e.g., in an appropriate medium, and screening for those cells that harbor the DNA of interest using conventional techniques such as Southern blot and / or PCR or by using a selectable marker.

[0095] Transfection can be carried out using any of the techniques known in the art, including but not limited to electroporation, lipofection, for example, using lipofectamine, cationic polymers, and cationic lipids. Any suitable transfection medium can be used. In one embodiment of the transfection process, adherent or suspension human lung kidney (HEK293) cells are transfected using triple DNA plasmid polyethylenimine (PEI) co-precipitation.

[0096] In one aspect, the disclosure provides a method for producing an AAV-based viral vector comprising the steps of: (i) culturing cells in a bioreactor or flask; (ii) transfecting the cells with a plasmid to enable production of AAV particles; (iii) lysing the mixture of cells and viral particles to release the viral particles from the cells; and iv) isolating and / or purifying the viral particles, where step iv) comprises purification on a mixed-mode cation exchange chromatography matrix.

[0097] After a suitable virus production period after transfection or infection, the cells are lysed and the virus particles are harvested. In some embodiments, the cells are dissociated from the bioreactor before the cell lysis process is initiated. In some embodiments, the cells are lysed in situ.

[0098] Preferably, for lysis, the cells are contacted with a composition comprising an alkyldimethylamine oxide and optionally sodium chloride. Preferably, the lysis solution is added to a bioreactor containing a suspension of cells such that a mixture of the cell suspension and the composition is produced.

[0099] Incubation of the mixture comprising the cells and the composition comprising alkyldimethylamine oxide and optionally sodium chloride is typically for an incubation time of 30 to 180 minutes, preferably between 60 and 90 minutes. Shorter and longer times may also be appropriate.

[0100] The pH of the mixture during incubation can vary in a wide range: it can be, for example, between pH 4 and pH 10, typically it is between pH 6 and pH 9. The temperature of the mixture during incubation can also vary within a wide range: it can be, for example, between 20°C and 37°C.

[0101] The concentration of the alkyldimethylamine oxide in the composition is such that it effectively induces cell lysis, meaning that after incubation with the composition under suitable conditions as described above, at least 80%, preferably 100%, of the cells are lysed. For this reason, the concentration of the alkyldimethylamine oxide in the final mixture with the cells is preferably above its CMC. For TDAO, N,N-dimethyltridecylamine N-oxide, the concentration in the mixture with the cells is typically between 0.1% and 5% (w / w), preferably between 1% (w / w) and 4% (w / w). Typically, the volume of the composition added is smaller than the volume of the cell culture.

[0102] As a result, the preferred concentration of alkyldimethylamine oxide, especially TDAO, in the composition is between 10 and 30% (w / w).The preferred concentration of sodium chloride in the composition to be added to the cell suspension is between 1 and 6 mol / l, typically around 3 to 5 mol / l.

[0103] After incubation with the lysis composition, the released AAV can then be isolated and / or purified, including by a single chromatographic purification on a cation exchange mixed-mode matrix.

[0104] Optionally, after dissolution, the resulting mixture is first filtered or centrifuged. In one embodiment, the mixture is filtered through a filter that removes large molecular contaminants and cellular debris, but allows the AAV to pass through.

[0105] In one embodiment, the released virus particles can be separated and purified from cell culture medium using clarification.Clarification can be a microfiltration process in which relatively larger components such as lysed cells and / or impurities are removed from the solution.Clarification filters include depth filtration, charged depth filtration, and similar microfiltration techniques.

[0106] Tangential flow filtration can be used to concentrate the mixture of purified virus particles and to remove salts and proteins.Tangential flow filtration (TFF) refers to a generally rapid and efficient method for filtration or purification of solutions containing target products and / or impurities, during which the solution or liquid stream flows parallel to the filtration membrane.

[0107] Centrifugation can be a low speed centrifugation to remove larger particles such as cell debris. This can be done, for example, at 10000-12000 g for 10-30 minutes. The released viral particles can be found in the supernatant.

[0108] Isolation and / or purification of AAV typically involves one or more of the following process steps: - Clarification - Filtration - Dialysis / Diafiltration - Tangential flow filtration - Treatment with nucleases, e.g. RNase and / or DNase - Treatment with chloroform - Ion exchange chromatography - Affinity Chromatography - Hydrophobic Interaction Chromatography - Centrifugation - PEG precipitation

[0109] In some embodiments, a nuclease, typically an endonuclease, is added, for example to reduce the amount of host cell DNA. It can be added directly to the mixture in the bioreactor before, during, or after lysis. The nuclease may be one that degrades both DNA and RNA. In one embodiment, the endonuclease is a genetically modified endonuclease from Serratia marcescens sold under the name Benzonase® (EMD Millipore, US).

[0110] In the cation exchange mixed chromatography step, the target AAV is separated from at least one impurity in the sample by contacting the sample containing the AAV with a chromatography matrix. The contact time usually ranges from 6 seconds to 24 hours. It is advantageous to work according to the principles of liquid chromatography by passing a liquid through a chromatography column or other type of housing containing the mixed mode cation exchange chromatography matrix. The liquid can simply pass through the column or housing by gravity or be pumped by means of a pump.

[0111] An alternative method is batch chromatography, in which the separation material is mixed with the liquid by stirring or shaking for as long as necessary to allow the AAV to bind to the separation material. It is also possible to function according to the principle of a chromatographic fluidized bed by introducing the liquid to be separated into a suspension containing, for example, a chromatographic matrix, where the chromatographic matrix is ​​selected such that it is suitable for the desired separation due to its high density and / or magnetic core.

[0112] When the chromatographic process is carried out in binding and elution mode, the target AAV binds to the chromatographic matrix. The chromatographic matrix can then be washed with one or more washing buffers, which preferably have the same ionic strength and the same pH as the liquid in which the target molecule contacts the chromatographic matrix. The washing buffer removes substances that do not bind to the chromatographic matrix. Further washing steps with other suitable buffers may follow this without desorbing the target AAV.

[0113] The desorption of the bound AAV is carried out by changing the ionic strength in the eluent, and / or by changing the pH of the eluent, and / or by changing the solvent. Thus, the target AAV can be obtained in a purified and concentrated form in the eluent. The target AAV usually has a purity of more than 70%, for example 70% to 99%, preferably more than 80%, for example 85% to 99%, and particularly preferably more than 90%, for example 90% to 99%, after desorption.

[0114] However, if the chromatographic process is carried out in flow-through mode, the target AAV remains in the liquid, but other accompanying substances bind to the separation material. The target AAV is then directly obtained by collecting the column eluate in the flow-through. The skilled person knows how the conditions, especially the pH and / or conductivity, must be adapted to bind certain biopolymers to the separation material, or whether it is advantageous for the purification operation not to bind the target AAV.

[0115] Cation exchange mixed mode materials can be used for the purification of AAV, allowing for the efficient removal of impurities such as host cell proteins. Moreover, preferably, the same chromatography step additionally provides for the removal of empty particles. Unexpectedly, it has been found that empty particles can be eluted together with host cell proteins from the cation exchange hydrophobic chromatography matrix under the preferred elution conditions of pH 4.5 to pH 7.8 and 13 to 20 mS / cm, and separated by peak fractionation, while the target AAV is still bound to the chromatography matrix. In a preferred embodiment, the chromatography matrix is ​​a membrane, in particular a hydrogel membrane.

[0116] The nature of the chromatography matrix used (i.e., strong and / or weak cation exchanger, type of hydrophobic group), as well as the salt concentration, buffer used, and pH conditions will vary depending on the AAV capsid variant (i.e., AAV capsid serotype or pseudotype). While all known AAV capsid variants share features such as size and shape, they differ in the details of molecular topology and surface charge distribution. Thus, while it is expected that all capsid variants are suitable for purification by mixed-mode cation exchange chromatography, the optimal method can be systematically determined using screening experiments of chromatography resins and buffers, and different conditions will be required for each AAV capsid variant to achieve efficient AAV particle purification. The determination of such conditions is readily apparent to those skilled in the art.

[0117] Preferably, chromatographic purification is performed by using a pH change from 4-6.5 to a pH above 9, preferably between 9-11, most preferably around pH 10, in gradient or step mode, while keeping the conductivity at a constant level in the range of 13-20 mS / cm. When using a pH gradient, the sample applied to the chromatographic matrix is ​​adjusted to the pH at which the pH gradient begins.

[0118] Generally, any and all buffer substances known to those skilled in the art could be used to produce a pH and conductivity stable aqueous solution. Examples of suitable buffers to use are listed for equilibration in Table 1 and for elution in Table 2. Buffer concentrations may vary if pH and conductivity need to be adjusted.

[0119] [Table 1]

[0120] [Table 2]

[0121] It is also possible to carry out elution on a chromatographic matrix by using a linear or step mode salt gradient. The salt may be selected from the group consisting of NaCl, KCl, sulfate, formate and acetate, preferably NaCl. Typically, the gradient starts with a low salt concentration, for example between 10-150 mM salt, which is then increased until the target AAV is eluted, for example to 150-1000 mM salt. Alternatively, a pH and salt gradient can be combined.

[0122] The term "column volume" refers to the volume inside a packed column that is not occupied by the chromatography matrix. This volume includes both the interstitial volume (the volume outside the matrix) and the porosity (pore volume) within the matrix itself.

[0123] In a preferred embodiment, the applied linear gradient lasts for approximately 30-50 column volumes (CV), plus an additional hold step with a buffer of the target elution buffer of at least 20 CV. In another preferred embodiment, the target AAV product recovery is greater than 30%, preferably greater than 60%.

[0124] In addition, this application is not limited to binding and elution applications, but can also be used in flow-through mode, which is particularly suitable for the separation of different types of AAV, with the target AAV remaining in the flow-through and the other types of AAV binding to the chromatographic matrix.

[0125] Furthermore, the present invention provides a chromatography-based AAV purification step that is regenerative and applicable over a wide operating window, e.g., pH 3-10, conductivity 1 mS / cm-50 mS / cm. In a preferred embodiment, the methods of the present invention are used to purify samples containing a wide range of viral particle concentrations, for example ranging from samples with 1e11 AAV viral particles per ml (1e11 vp / mL) to samples with 2.5e13 vp / mL.

[0126] The pH window span is between pH 4.5 and pH 11, and in a more preferred embodiment, the conductivity range is between pH 5.3 and pH 10. The window span of the conductivity is between 1 mS / cm and 50 mS / cm, and in a more preferred embodiment, the conductivity range is between 2 and 30 mS / cm.

[0127] The methods of the invention are applicable to all rAAV and wt serotypes without the need for prior process steps.Preferably, the crude lysate (treated and clarified with nucleases) can be applied directly to the chromatography matrix.

[0128] Production of a crude lysate of AAV typically involves one or more of the following process steps, which are performed after cell lysis: - Clarification - Filtration - Dialysis / Diafiltration - Tangential flow filtration - Treatment with nucleases, e.g. RNase and / or DNase

[0129] The crude lysate was not treated with any other chromatography and / or ultracentrifugation steps. In a highly preferred embodiment, the sample, for example in the form of a crude lysate treated with a nuclease such as Benzonase® (Merck GaA, Germany) and clarified, is applied to a membrane with cation exchange and hydrophobic groups at a pH between 4.5 and 6.5 and a conductivity of approximately 14 to 19 mS / cm. Optionally, the loaded membrane is washed with an equilibration buffer with a pH of 4.5 to 6.5 and a conductivity of approximately 14 to 19 mS / cm.

[0130] Elution is performed by applying a linear pH gradient from a pH between 4.5 and 6.5, depending on the sample pH, to a pH between 9.5 and 10.5, at a constant conductivity of less than 20 mS / cm.

[0131] Elution preferably results in two fractions depending on the pH reached during this step: Fraction 1 elutes from the matrix in the pH range between 5.5 and 7.8 and contains the majority of the co-eluting HCPs and primarily empty rAAV particles. Fraction 2 elutes from the matrix in the pH range between 8.7 and 10.1 and contains a portion highly enriched in intact rAAV and a 3 log reduction in HCPs.

[0132] Preferred buffer compositions are those with a broad pH range of 4-11, examples of which include, but are not limited to, the Good buffer system containing Tris, phosphate and acetate, as well as amino acids.

[0133] The method of the present invention can efficiently purify AAV. Cation exchange mixed-mode chromatography can be used as an orthogonal method to capture rAAV from crude lysate with extremely high recovery in the eluate and regardless of serotype and expression host. Typically, contaminants can be reduced by more than 2 log logarithmic reduction. Preferably, this method achieves a host cell protein reduction of more than 2 log. Further reduction or removal of empty particles provides an overall reduction in process steps, since no separate additional process steps are required for this. Process and product-related impurities can be removed in one single step.

[0134] The enrichment of intact AAV capsids can be calculated as the quotient of the amount of genome or DNA-containing particles divided by the amount of total virus particles.In the enriched elution fraction produced by the method of the present invention, this quotient reaches 1, which means that all AAV present contains DNA and is therefore considered as 100% intact particles.In unenriched fractions, this quotient is typically less than 0.5.In crude lysate, this quotient is typically less than 0.2.This indicates that the purity of crude lysate is increased by at least 80% through a single purification step using cationic and hydrophobic interaction matrix capture and pH elution.

[0135] In particular, the use of membrane-based matrix provides effective and fast purification.It is known to those skilled in the art that the increase in purity depends heavily on the quality of crude lysate.However, even if crude lysate is used as sample, the method of the present invention provides target AAV product recovery rate of more than 30%, preferably more than 60%.

[0136] The invention is further illustrated by the following figures and examples, without, however, being limited thereto. The entire disclosures of all applications, patents, and publications cited above and below, as well as the corresponding EP application EP 22162928.0, filed March 18, 2022, are hereby incorporated by reference.

[0137] example The following examples represent practical applications of the present invention. Example 1 A 1 mL column of Eshmuno® CMX was loaded with 7 mL of affinity purified rAAV2. The column was eluted with a pH gradient from pH 4.5 to 9.0. The conductivity was kept constant at 400 mM NaCl.

[0138] [Table 3-1] [Table 3-2]

[0139] Figure 1 shows a run on an Eshmuno® CMX column performed on an AEKTA™ system. No AAV was detectable during loading. A first peak occurred in the wash step, and a second peak could be eluted during the elution gradient. Overall recovery of all rAAV2 capsids was 78% and for intact rAAV2 capsids was 77%, however, no enrichment in the elution peaks was observed. The overall recovery of all rAAV2 capsids was 78% and for intact rAAV2 capsids was 77%, however, no enrichment in the elution peaks was observed.

[0140] Example 2 In this case study, the critical experimental parameters considered are pH ranging from 4.5 to 6.5 and salt concentration ranging from 20 mM NaCl to 120 mM NaCl, as this is more reliable than conductivity, which is strongly temperature dependent. The focus was on binding conditions, while elution conditions were fixed. The required output values ​​are the dynamic binding capacity of the device, calculated as vp / mL, and the elution yield, calculated as the ratio of the concentration in the load volume to the concentration in the elution volume.

[0141] [Table 4-1] [Table 4-2]

[0142] A second step is required to achieve the desired pH values ​​of 4.5 (1), 5.5 (2), and 6.5 (3). For each pH value, we prepared a salt-free buffer (a) and a 1 M NaCl buffer (b). The membrane was first disinfected by rinsing it with 1 M NaOH solution and allowing the device to statically soak for 30 min. It was then rinsed with equilibration buffer until the desired conditions were reached.

[0143] Prior to loading, the pH and conductivity of the samples had to be adjusted: for this purpose, the feed was diluted 1:10 with equilibration buffer for each condition investigated. Equilibration (25 mM sodium acetate, 25 mM phosphate, 120 mM NaCl, pH 5.3, 5 MV) Load (AAV2 crude lysate, pH 5.3, 17 mS / cm). Total loaded volume is approximately 640 mL, roughly 2E+13 vp / mL device. Washing (25 mM sodium acetate, 25 mM phosphate, 120 mM NaCl, pH 5.3, 20 MV) Elution (25 mM sodium acetate, 25 mM phosphate, 500 mM NaCl, pH 8.5 20 MV). A step gradient elution was applied. ·Volatilization (0.1M Tris, 2M NaCl, pH10.6. 20MV) CIP (1M NaOH, 20MV) Re-equilibration B (25 mM sodium acetate, 25 mM phosphate, 500 mM NaCl, pH 8.5, 30 MV) Re-equilibration A (25 mM sodium acetate, 25 mM phosphate, 120 mM NaCl, pH 5.3, 30 MV)

[0144] Fractions were taken for the flow-through, wash, elution, and stripping steps, and then each sample was stored at -80°C for further analysis. Figure 2 shows a chromatogram of AAV2 from a capture run using step gradient elution on a 1 mL volume mixed-mode membrane, where the equilibration buffer consisted of a combination of salts and 120 mM NaCl and was at pH 5.3, and the elution buffer contained 500 mM NaCl and the same constituent salts and was at pH 8.5.

[0145] Example 3 To improve HCP removal in chromatographic processes, pH variation has been investigated as an improving parameter for cation exchange chromatography in the field of antibody purification for HCP removal. Therefore, a capture run was performed with increased pH for the loading buffer and the same process steps applied for the previous capture run, and the chromatogram is reported in FIG.

[0146] Figure 3 shows a chromatogram of AAV2 from a capture run using step gradient elution on a 1 mL volume mixed-mode membrane aimed at improving HCP clearance. Operating conditions: pH 6.5, 120 mM NaCl, where the elution buffer contains 500 mM NaCl and the same constituent salts, and is at pH 8.5.

[0147] The HCP amounts assessed in the analyzed fractions confirmed what was observed from the silver stained gel, where AAV2-related impurity bands were detected in the flow-through and especially in the elution fraction. HCP levels determined by ELISA resulted in a 1.5 log reduction in the elution fraction, however, this cannot be considered a meaningful HCP removal.

[0148] Example 4 A 1 mL Natrix® CH membrane device was loaded with 13 mL of HEK293 cell rAAV2 crude lysate (treated and clarified with Benzonase®; diluted 1:10 in equilibration buffer). Elution was performed under a constant conductivity of 150 mM Na+ and a linear pH gradient from pH 5.3 to 10.

[0149] Figure 4 shows chromatograms of AAV2 from a capture run using linear gradient elution on a 1 mL volume mixed-mode membrane aimed at improving HCP clearance and intact rAAV2 capsid on step 1. Binding conditions: pH 5.3, 150 mM NaCl, where the elution buffer contains 150 mM NaCl and the same constituent salts and is at pH 10.

[0150] In this example, the majority of the HCPs were found in 98% of the flow-through fraction, with 3% co-eluting with 30% of empty rAAV2 capsids in the first peak. In the second peak, a fraction of highly pure rAAV2 can be detected. The log removal of HCPs was approximately 3, and two fractions contained nearly 70% of intact rAAV2.

Claims

1. A method for purifying adeno-associated virus (AAV) particles, comprising contacting a sample containing the AAV particles with a mixed-mode cation exchange chromatography matrix.

2. The method according to claim 1, comprising the following steps: a) Contacting a sample containing AAV particles with a mixed-mode cation exchange chromatography matrix. b) Optionally, wash the chromatography matrix. c) Elute the AAV particles bound to the chromatography matrix in step a) with the elution buffer.

3. The method according to claim 2, wherein in step c), the elution buffer has a pH higher than the pH of the sample in step a).

4. The method according to claim 1, comprising recovering AAV particles that flow through a chromatography matrix without binding to it.

5. The method according to claim 1, wherein the chromatography matrix is ​​a membrane or a monolith.

6. The method according to claim 1, wherein the chromatography matrix is ​​a hydrogel membrane.

7. The method according to claim 1, wherein the sample comprises empty and complete AAV capsids, and the empty AAV particles are partially or completely eluted prior to the complete AAV particles.

8. The method according to claim 1, wherein the sample is crude lysate.

9. The method according to claim 2, wherein the elution in step c) is carried out on a linear pH gradient from a pH between 4 and 6.5 to a pH between 9 and 11, while maintaining the conductivity at a constant level.

10. The method according to claim 2, wherein in one fraction of the AAV eluted in step c), the HCP is reduced by a logarithmic decrease of 2 log or more compared to the sample to which it was applied in step a), and the percentage of complete AAV exceeds 90%.

11. The following is a method for purifying AAV particles from a sample containing cells encapsulated with AAV particles: a) To lyse cells, b) Isolating and / or purifying the AAV particles by contacting them with a mixed-mode cation exchange chromatography matrix in accordance with the method described in claim 1.

12. The method according to claim 11, wherein cell lysis is performed using a surfactant selected from the group of alkyldimethylamine oxides and optionally sodium chloride.

13. A method according to claim 11 or 12, comprising one or more of the following steps: - Clarification - Filtration - Dialysis / Dialysis Filtration - Tangential flow filtration - Treatment using nucleases, e.g., RNase and / or DNase - Treatment using chloroform - Ion exchange chromatography - Affinity chromatography - Hydrophobic interaction chromatography - Centrifugal separation - PEG precipitation.

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