WO2025027058A1 - Polishing for aav particles using anion exchange chromatography with improved elution system - Google Patents

Abstract

The present invention relates to methods for polishing and enrichment of full adeno-associated virus particles by anion exchange chromatography using basic amino acids in the elution buffer.

Description

Polishing for AAV particles using anion exchange chromatography with improved elution system

The present invention relates to methods for polishing and enrichment of full adeno-associated virus particles by anion exchange chromatography using basic amino acid as additives in the elution buffer.

Adeno-associated virus (AAV) have been characterized and developed as a potent viral vector to deliver genes in vitro in cultured cells and in vivo. AAV is meanwhile a leading platform for in vivo delivery of gene therapies. AAV is a small, non-enveloped virus containing a single-stranded DNA genome of approximately 4.7 kb, consisting of two inverted terminal repeats (ITRs) that are capable of forming T-shape secondary structure and acting as origins of genome replication, one rep region that encodes four overlapping replication proteins, Rep78, Rep68, Rep52, and Rep40, and one cap region that encodes three structural proteins, VP1 , VP2, and VP3, and an assembly activating protein (AAP). Naturally isolated serotypes 1-9 of the AAV viruses share the genomic structure although these serotypes may display different tissue tropism. As the AAVs seem to be nonpathogenic, show an efficient transduction and a stable expression, they are regarded as being one of the most promising gene delivery vehicles.

AAV vectors can be produced in various cell lines in adherent or suspension cell culture formats using transient transfection or co-infection methods. Depending on specific serotypes and production times, viral particles including full, partial, and empty species can be secreted out of cells into culture medium or contained inside cells at various ratios.

Initially, stable AAV producer cells were generated by transfection and selection of human-derived cells, like HeLa or HEK293 cells, with an rAAV transfer vector containing the ITR cassette and a packaging construct containing Rep and Cap. Production of recombinant AAV vectors (rAAV) was then achieved by infection with auxiliary viruses such as adenoviruses (AdV) that provide the helper function. After identification of AdV genes required for AAV vector packaging, a helper virus-free method was established using a duo or triple transfection protocol consisting of two or three plasmids including a constructed helper plasmid instead of an auxiliary virus. This system is widely used in research and drug development. In addition, development of baculovirus expression vectors provides another method to produce rAAV viruses in insect Sf9 cells. These different technologies are shown to be able to produce enough rAAV viruses for use in laboratories and clinical trials.

A cell lysis step is generally required at harvest to release viral particles into the supernatant. For this application, typical cell lysis reagents such as Triton X-100, Tween 20, and NaCI are broadly utilized.

After cell lysis, the AAVs need 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 chromatography or in addition to chromatography. Final steps in AAV purification typically involve concentration and diafiltration into suitable excipient buffer composition and sterile filtration.

Although there is an increasing demand for viral vectors as vehicles in gene therapy, current manufacturing processes suffer from purification methods that simultaneously scale to meet the production demands but also reduce the impurity burden. Due to their ability to induce dividing and non-dividing tissues, patient safety and the control for cell specific applications, adeno- associated virus (rAAV) is an attractive vector in that field. However, producing these viruses while increasing efficiency and lowering manufacturing costs is a difficult task for downstream processing.

A major challenge in current AAV processes is the production of nongenome containing particles. Although their mechanism of action is not fully understood, empty rAAV capsids are treated as a major process impurity that can hamper safety and efficacy of the final formulated drug. Independent of being produced in mammalian or insect cell lines, resulting rAAV feed streams typically contain a level of 10-90% empty capsids that need to be removed further downstream. A typical downstream scheme consists of two steps; an initial affinity capture step to capture all rAAV particles from the feed stream and remove other process related impurities and a subsequent polish step to separate full of empty rAAV particles. Full capsids have been explored to have higher charge density due to the negatively charged genome that they carry. And even though this negatively charged genome changes the isoelectric point (pl) just slightly (empty: pl=5.9 vs. full: pl=6.3), these attributes are enough that ion exchange chromatography, in particular anion exchange chromatography (AEX) has been widely explored for this type of application (Wang, Dan; Tai, Phillip W. L.; Gao, Guangping (2019): Adeno-associated virus 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).

To overcome the challenges of separation efficiency in chromatography steps and generate two distinct elution peaks, the process conditions such as pH, salts and additives often need to be individually fine-tuned for each rAAV serotype purification. Several solutions have been widely discussed in the literature such as the use of ammonium and various salts from the family of quaternary alkyl ammonium salts (QAAS) (Hua Yang, S. K. (2021 ). Waters: Anion-Exchange Chromatography for Determining Empty and Full Capsid Contents in Adeno-Associated Virus. Retrieved from https://www.waters.com/webassets/cms/library/docs/720006825en.pdf) as well as divalent cations (Wang et al., C. (2019, December). Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2. Molecular Therapy: Methods & Clinical Development Vol. 15. DOI: 10.1016/j.omtm.2019.09.006).

Both solutions are suitable to enhance the quantification of viral capsids on the analytical level. However, the acceptance in a scalable drug manufacturing process remains questionable, as QAAS such as tetraethyl ammonium enhance the stabilizing hydrophobic interactions between viral capsid proteins, show a strong background adsorption (260-280nm) and possess serios toxic effects (Pubchem. (2021 , Mai 29).

Tetramethylammonium, retrieved from https://pubchem.ncbi.nlm.nih.gov/compound/Tetraethylammonium). Divalent cations, such as Mg²⁺, are known to stabilize non-enveloped viruses under thermal stress but are well-known cofactors for enzymes as well. Furthermore, using soluble metals in process buffers leads to the introduction of additional process steps like extensive washing scenarios to avoid the generation of insoluble metal hydroxides, lowering process economics (Gagnon et al., P. (2021 , May). Removal of empty capsids from adeno-associated virus preparations by multimodal metal affinity chromatography. Journal of Chromatography A 1649. DOI:

10.1016/j. chroma.2021 .462210). These factors, that hamper efficacy in the final drug formulations, remain a risk that should be avoided.

In WO04113494 methods are disclosed for separating empty and full particles by single or multiple anion- (AEX) and or cation exchange (CEX) steps.

In WO22159679 it is disclosed that separation of filled recombinant virus particles from empty or partially filled recombinant virus particles in a feed composition can be increased in an anion exchange chromatography media by equilibrating the anion exchange chromatography media with a buffer comprising an anionic compound, e.g., a weak acid or a salt thereof, such as citric acid or a salt thereof prior to contacting the anion exchange media with the feed composition comprising the virus particles and wherein the feed composition further comprises a predetermined amount of an anionic compound, e.g., a weak acid or a salt thereof. The equilibration buffer can also comprise an amino acid. The effect of the method described in WO2215679 is that by choosing a loading/equilibration buffer as defined above the binding of the empty AAV particles to the chromatography matrix is prohibited or reduced.

Nevertheless, there is still the need for an efficient and simple method that provides for purification of target AAVs from process related impurities as well as for separation of empty and full capsids.

The inventors have surprisingly found that the use of certain additives in the elution buffer can be notably efficient in enriching full capsids in an anion exchange chromatography step for AAV polishing while removing empty ones. In this case the effect is based on the finding that when using an elution buffer comprising the basic amino acids as additives, empty and partially filled AAV particles which are bound to the chromatography matrix in parallel to the filled AAV particles desorb and elute easier and thus prior to the filled AAV particles.

The present invention is thus directed to a method for enriching full adeno associated virus (AAV) particles in a sample comprising full and empty adeno-associated virus (AAV) particles by contacting them with an anion exchange chromatography matrix, binding at least full AAV particles to the anion exchange chromatography matrix and using an elution buffer comprising at least one basic amino acid additive.

Embodiments In a preferred embodiment the method of the present invention comprises the steps of a) contacting the sample comprising the empty and full AAV particles with a chromatography matrix comprising anion exchange groups so that full AAV particles are bound to the chromatography matrix b) optionally washing the chromatography matrix eluting full AAV particles which bound to the chromatography matrix with an elution buffer comprising one or more basic amino acids.

In one embodiment the sample is a clarified lysate that underwent at least one filtration and/or centrifugation step before contacting the chromatography matrix.

In a preferred embodiment the sample is a pre-purified lysate that has been pre-purified by a chromatography step.

In a very preferred embodiment the chromatography step for pre-purifying the lysate is an affinity chromatography step.

In another embodiment, in a) the sample comprises empty and full AAV capsids that bind to a chromatography matrix comprising anion exchange groups while remaining process related impurities flow through the matrix.

In another embodiment, in c) the elution buffer has a conductivity higher than the conductivity of the sample in step a).

In another embodiement the chromatography matrix is a resin, membrane or monolith.

In a preferred embodiment the chromatography matrix comprises strong anion exchange groups, preferrably TMAE groups. In a very preferred embodiment the anion exchange groups are attached to the chromatography matrix via polymer chains, also called tentacles, which are made by grafting monomers comprising an anion exchange group to the base material of the matrix. In some embodiments the polymer chains also comprise monomers which do not comprise an anion exchange group but e.g. a hydrophobic group so that the resulting chromatography matrix is an anion exchanche hydrophobic mixed mode matrix.

In a preferred embodiment, in c) said basic amino acids are arginine and/or lysine.

In another preferred embodiment the concentration of the basic amino acids in the elution buffer is between 2 and 800 mM.

In another embodiment, in step c), the elution buffer has a pH equal to the sample loading buffer, preferably between 8 and 10, preferably around 9.

The elution buffer may be applied as linear gradient, preferably increasing in conductivity and in parallel molarity of the basic amino acids while maintaining a constant pH.

In another embodiment the majority of empty AAV capsids elute of the anion exchange chromatography matrix together with other impurities prior to the majority of full AAV particles.

In another very preferred embodiment of AAVs polished with the method of the present invention, full AAV particles undergo an enrichment of at least 100%.

In another embodiment the AEX chromatography step can be repeated one or more times, typically 2, 3 or 4 times. Definitions

Before describing the present invention in detail, it is to be understood that this invention is not limited to specific compositions or process steps, as such may vary. It must be noted that, as used in this specification and the appended claims, the singular form "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 the like.

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 is related. The following terms are defined for purposes of the invention as described herein.

Adeno-associated virus (AAV) is a member of the Parvoviridae family. The AAV genome is composed of a linear single-stranded DNA molecule which contains approximately 4.7 kilobases (kb) and consists of two major open reading frames encoding the non-structural Rep (replication) and structural Cap (capsid) proteins. Flanking the AAV coding regions are two cis-acting inverted terminal repeat (ITR) sequences, approximately 145 nucleotides in length, with interrupted palindromic sequences that can fold into hairpin structures that function as primers during initiation of DNA replication. In addition to their role in DNA replication, the ITR sequences have been shown to be necessary for viral integration, rescue from the host genome, and encapsulation of viral nucleic acid into mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158:97-129).

Multiple serotypes of AAV exist and offer varied tissue tropism. Known serotypes include, for example, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11 .

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 they are devoid of the virus structural genes, thereby eliminating the natural host cell responses to virus infection, e.g., interferon-mediated responses. In addition, wild-type viruses have never been associated with any pathology in humans.

According to the present invention scAAV are also within the group of AAVs. Self-complementary adeno-associated vectors (scAAV) are viral vectors engineered from the naturally occurring adeno-associated virus (AAV) for use in gene therapy. ScAAV is termed "self-complementary" because the coding region has been designed to form an intramolecular double-stranded DNA template.

Thus, in some embodiments, by an "AAV " is meant a vector or virus derived from an adeno-associated virus serotype, including without limitation, 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 of the AAV wild-type genes deleted in whole or part, e.g., the rep and/or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication, and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences that provide for replication and packaging (e.g., functional ITRs) of the virus. The ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g., by the 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, with AAV-2 derived ITRs. Additionally, by an "AAV " is meant the protein shell or capsid, which provides an efficient vehicle for delivery of vector nucleic acid to the nucleus of target cells.

The term "AAV" as used herein is intended to also encompass recombinant AAV. Adeno-associated virus (AAV) are herein also called viruses, viral particles, or viral vectors.

As used herein, the term “cell” or "cell line" refers to a single cell or to a population of cells capable of continuous or prolonged growth and division in vitro. In some embodiments, e.g., the terms "HEK293 cells", "293 cells" or their grammatical equivalents are used interchangeably here and refer to the host/packing cell line used in the methods disclosed herein.

Suitable cells and cell lines have been described for use in production of AAVs and AdVs. 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 desirable host cells are selected from among any mammalian species, including, without limitation, A549, WEHI, 3T3, 10T1/2, BHK, MDCK, COS 1 , COS 7, BSC 1 , BSC 40, BMT 10, VERO, WI38, HeLa, a HEK 293 cell, Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cells derived from mammals including human, monkey, mouse, rat, rabbit, and hamster.

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

In addition to the expression cassette, the cells contain the sequences which drive expression of AAVs in the cells (cap sequences) and rep sequences of the same source as the source of the AAV ITRs found in the expression cassette, or a cross-complementing source. The AAV cap and rep sequences may be independently selected from different AAV parental sequences and be introduced into the host cell in a suitable manner known to one in the art. While the full-length rep gene may be utilized, it has been found that smaller fragments thereof, i.e. , the rep78/68 and the rep52/40 are sufficient to permit replication and packaging of the AAV.

The cells also require helper functions to package the AAV of the invention. Optionally, these helper functions may be supplied by a herpesvirus. In another embodiment, the necessary helper functions are each provided from a human or non-human primate adenovirus source, such as are available from a variety of sources, including the American Type Culture Collection (ATCC), Manassas, Va. (US).

During the manufacturing of AAVs, a percentage of capsids might not incorporate any of the transgenes and are referred to as empty capsids, empty AAVs or empty AAV particle. Additionally, capsids that contain fragments of the transgene are called partial capsids, partial AAVs or partial AAV particles. These undesired product-related impurities are co-produced with the full capsids or full AAVs which contain the full length of the desired transgene.

Purification means to increase the degree of purity of a target molecule, in this case the AAVs, e.g., by removing one or more impurities, especially it means the enrichment of full AAVs in a sample while other impurities and empty AAVs are removed.

The term "impurity" or “contaminant” as used herein, refers to any foreign or objectionable molecules or species, including a biological macromolecules such as DNA, RNA, one or more host cell proteins, nucleic acids, endotoxins, lipids, impurities of synthetic origin like detergents, partial and/or empty AAVs or AdVs as well as one or more additives which may be present in a sample containing the viral particles to be purified and thus to be separated from one or more of the impurities. As used herein, and unless stated otherwise, the term “sample” refers to any composition or mixture that contains AAVs. 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. The sample may also include diluents, buffers, detergents, and contaminating species, debris and the like that are found mixed with the target molecule. The sample may be "partially purified" (i.e. , having been subjected to one or more purification steps, such as filtration steps) or may be obtained directly from a host cell producing the AAVs, e.g., the sample may comprise harvested cell culture fluid.

The terms "purifying", “enriching”, "separating," or "isolating," as used interchangeably herein, refer to increasing the degree of purity of the full target AAVs from a composition or sample comprising the full target AAVs and one or more impurities.

The term "chromatography" refers to any kind of technique which separates an analyte of interest (e.g., a full target AAV) from other molecules present in a sample. Usually, the full target AAV is separated from other molecules as a result of differences in rates at which the individual molecules of the mixture bind to and/or migrate through a chromatography matrix under the influence of a moving phase.

The term "matrix" or "chromatography matrix" are used interchangeably herein and refers to a solid phase, also called stationary phase, through which the sample migrates in the course of a chromatographic separation. The matrix typically comprises a base material and ligands covalently bound to the base material. The matrix of the present invention comprises or consists e.g., of particles, a membrane or a monolith.

A “ligand” is a functional group that is part of the chromatography matrix, typically it is attached to the base material of the matrix, and that determines the binding properties 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 interactions groups, metal affinity groups, affinity groups, bio affinity groups, and mixed mode groups (combinations of the aforementioned). It is also possible that one ligand has more than one binding I interaction property. The matrix of the present invention comprises at least anion exchange groups.

Examples of anion exchange groups are ionic or ionisable groups, like

-NR7R8 or -N+R7R8R9, in which

R7 and R8, independently of one another, H, alkyl having 1-5 C atoms and

R9 alkyl having 1 -5 C atoms with the proviso that in case of -NR7R8R9, R7 and R8 cannot be H,

- guanidinium

Anion exchange groups can be subdivided into strong anion exchange groups, such as quaternary ammonium groups like trimethylammonium (- CH₂N(CH₃)3⁺) or triethylammonium (-H2N(CH2CH3)3⁺) as well as weak anion exchange groups, such as ammonium (-NH3⁺), N,N diethylamino or diethylaminoethyl DEAE. The matrix may additionally comprise further other types of ligands so that the matrix is a mixed mode matrix. Such ligands may e.g., have hydrophobic interaction groups, such as phenyl, butyl, propyl, hexyl. The groups may be part of the base material, they may also be part of a ligand. One ligand may comprise one or several different anion exchange groups and/hydrophobic interaction groups. According to the present invention, the term anion exchange chromatography matrix covers matrices which only comprise one or more types of anion exchange groups as well as matrices comprising one or more types of anion exchange groups in combination with other types of functional groups like hydrophobic interaction groups, i.e. , mixed mode matrices.

The ligands can be attached to the base material of the matrix by any type of covalent attachment. Covalent attachment can for example be performed by directly bonding the functional groups to suitable residues on the base material like OH, NH2, carboxyl, phenol, anhydride, aldehyde, epoxide, or thiol etc. It is also possible to attach the ligands via suitable linkers. It is also possible to generate the matrix by polymerizing monomers comprising the ligands and a polymerizable moiety. Examples of matrices generated by polymerization of suitable monomers are polystyrene, polymethacrylamide or polyacrylamide-based matrices generated by polymerizing suitable styrene or acryloyl monomers.

In another embodiment the chromatography matrix can be generated by grafting the ligands onto the base material or from the base material. For grafting from processes with controlled free-radical polymerisation, such as, for example, the method of atom-transfer free-radical polymerisation (ATRP), are suitable. A very preferred one-step grafting from polymerisation reaction of acrylamides, methacrylates, acrylates, methacrylates etc. which are functionalized e.g. with ionic, hydrophilic or hydrophobic groups can be initiated by cerium(IV) on a hydroxyl-containing support, without the support having to be activated.

The term "anion exchange matrix" is thus used herein to refer to a chromatography matrix which carries at least anion exchange groups. That means it typically has one or more types of ligands that are positively charged under the chromatographic conditions used, such as quaternary amino groups.

When the chromatography matrix is used in a chromatographic separation it is typically used in a separation device, also called housing, as a means for holding the matrix. 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 a person skilled in the art. They typically comprise cylindrical tubes or cartridges filled with the stationary phase as well as filters and/or means for fixing the stationary phase in the tube or cartridge and optionally connections for solvent delivery to and from the tube or cartridge. The size of the chromatography column varies depending on the application, e.g. analytical or preparative. In one embodiment the column or generally the separation device is a single use device.

In certain embodiments, the housing comprises a housing unit, wherein the housing unit comprises

(a) an inlet and an outlet;

(b) a fluid flow path between the inlet and the outlet; and

(c) a chromatography matrix within the housing unit.

In one embodiment the chromatography matrix comprises a support member comprising a plurality of pores extending through the support member; and a non-self-supporting macroporous cross-linked gel comprising macropores having an average size of 10 nm to 3000 nm, said macroporous gel being located in the pores of the support member; wherein said macropores of said macroporous cross-linked gel are smaller than said pores of said support member; wherein the pores of the support member are substantially perpendicular to the fluid flow path.

In certain embodiments, the invention relates to a fluid treatment device comprising a plurality of housings, especially housing units, wherein each housing unit comprises

(a) an inlet and an outlet;

(b) a fluid flow path between the inlet and the outlet; and

(c) a chromatography matrix within the housing unit

In certain embodiments, especially if the chromatography matrix is a membrane, the chromatography matrix is arranged in the housing in a substantially coplanar stack of substantially coextensive sheets, a substantially tubular configuration, or a substantially spiral wound configuration.

A "buffer" is a solution that resists changes in pH by the action of its acidbase conjugate components. Various buffers which can be employed depending, for example, 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, as well as combinations of these.

According to the present invention the term “buffer” or “solvent” is used for any liquid composition that is used to load, wash, elute, re-equilibrate, strip and/or sanitize the chromatography matrix.

According to the present invention the term “additive” is used for a chemical substance that is added to a buffer that is used as elution buffer in a chromatographic separation. The basic amino acid additives to be used according to the present invention increase the separation and enrichment of viral particles, especially full AAVs, when performing the chromatographic separation on a chromatography matrix comprising anion exchange groups compared to otherwise the same chromatographic separation with the same elution buffer which does not comprise the one or more additives. Separation and enrichment of full AAV particles is achieved by the elution buffer comprising the one or more additives causing desorption of the empty AAV particles prior to desorption of the full AAV particles. Additives to be used in the present invention are basic amino acids. Basic amino acids are amino acids with a basic side chain at neutral pH. Preferred amino acids are amino acids with an isoelectric point above 7. Preferably, such basic amino acids comprise an imidazole ring, a guanidino- or an amino-group in the variable side chain. Examples of basic amino acids are histidine, arginine and lysine. In a preferred embodiment the basic amino acid is arginine and/or lysine. The amino acids used in the method of the present invention can be D and/or L amino acids, they can be natural or non-natural amino acids.

Equilibration of a chromatography matrix is done with an equilibration buffer prior to the loading of the sample to prepare the matrix for the loading of the sample and the next chromatographic purification. Typically, the equilibration buffer is identical to the loading buffer.

When “loading” a chromatography column in bind and elute mode, the sample or composition comprising the target molecule and one or more impurities is loaded onto a chromatography column. In preparative chromatography, the sample is preferably loaded directly without the addition of a loading buffer. If the sample needs to be adjusted to loading conditions, the sample is mixed with a loading buffer. The loading buffer has a composition, a conductivity and/or pH such that at least the full AAV particles are bound to the stationary phase while ideally all remaining impurities are not bound and flow through the column. Depending on the loading and equilibration buffer it might happen that also empty and partially filled AAV particles are bound to the chromatography matrix. Typically, the loading buffer, if used, has the same or similar composition as the equilibration buffer used to prepare the column for loading.

The final composition of the sample loaded on the column is called feed. The feed may comprise the sample and the loading buffer.

By “wash” or "washing" a chromatography matrix is meant passing an appropriate 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 prior to eluting the target molecule. Additionally, wash steps can be used to reduce levels of residual detergents, enhance viral clearance and/or alter the conductivity carryover during elution.

To "elute" a molecule (e.g., the target AAVs) from a matrix means that the molecule is removed therefrom. Elution may take place by altering the solution conditions such that a buffer different from the loading and/or washing buffer competes with the molecule of interest for the ligand sites on the matrix or alters the equilibrium of the target molecule between stationary and mobile phase such that it favours that the target molecule is preferentially present in elution buffer.

A non-limiting example is to elute a molecule from an anion exchange resin by altering the ionic strength of the buffer surrounding the anion exchange material such that the buffer competes with the molecule for the charged sites on the ion exchange material.

Elution can be performed by using a buffer gradient. A gradient typically starts with an elution buffer similar to the equilibration buffer and is then, in the course of the gradient, changed so that some of its properties, like pH, conductivity, composition, differ more and more from the equilibration buffer. This causes elution conditions which at the beginning provide adequate compound retention so that all compounds will not immediately elute from a chromatography column. Compounds that are retained less elute first. By further changing the elution buffer in the course of the gradient compounds that are retained stronger on the matrix will then also elute from the matrix. An elution gradient thus ideally separates the target molecule from its impurities by causing elution at different elution buffer composition.

There are two main gradient designs:

-Linear gradient - a linear change of a least one property like pH or conductivity in the elution buffer

-Step gradient - a stepwise change in least one property like pH or conductivity in the elution buffer

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. an AAV) contained in a sample along with one or more impurities is intended to flow through a chromatography matrix, which usually binds one or more impurities, where the target molecule usually does not bind (i.e. , flows through) and is eluted from the chromatograph matrix with the loading buffer.

The terms "bind and elute mode" and "bind and elute process", as used herein, refer to a separation technique in which at least one target molecule contained in a sample (e.g., an AAV) binds to a suitable chromatography matrix and is subsequently eluted with a buffer different from the loading buffer.

The term “ionic density” as used herein, refers to number of ions per unit of volume or mass of a given separation material, more particularly, the number of ions of given type (e.g., positive ions or negative ions) per unit volume or mass of separation material. Usually, the number of ions is estimated titrating the given separation material. Moreover, the amount of the ions is given in equivalents (eq) per mass or volume unit for separation material. The term “conductivity" as used herein, refers to an inherent property of most materials, that quantifies how strongly it resists or conducts electric current. In aqueous solutions, such as buffers, the electrical current is carried by charged ions. The conductivity is determined by the number of charged ions, the amount of charge they carry and how fast they move. Hence, for most aqueous solutions, the higher the concentration of dissolved salts, the higher the conductivity. Raising the temperature enables the ions to move faster, hence increasing the conductivity. Typically, the conductivity is defined in room temperature, if not otherwise indicated. The basic unit of conductance is 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. Therefore, the values are estimated in mS/cm.

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 levels of contamination before and after the process, so an increment of 1 corresponds to a reduction in concentration by a factor of 10. In general, an n-log reduction means that the concentration of remaining contaminants is only 10-n times that of the original. For example, a 0-log reduction is no reduction at all, while a 1 -log reduction corresponds to a reduction of 90 percent from the original concentration, and a 2-log reduction corresponds to a reduction of 99 percent from the original concentration.

A membrane as chromatographic matrix can be distinguished from particlebased chromatography by the fact that the interaction between a solute, e.g., the target AAVs or contaminants, and the matrix does not take place in the dead-ended pores of a particle, but mainly in the through-pores of the membrane. Exemplary types of membranes are flat sheet systems, stacks of membranes, microporous polymer sheets with incorporated cellulose, polystyrene, or silica-based membranes as well as radial flow cartridges, hollow fiber modules and hydrogel membranes. Preferred are hydrogel membranes. Such membranes comprise a membrane support and a hydrogel formed within the pores of said support. The membrane support provides mechanical strength to the hydrogel. The hydrogel determines the properties of the final product, like pore size and binding chemistry.

The membrane support can consist of any porous membrane like polymeric membranes, ceramic based membranes and woven or non-woven fibrous material. Suitable polymeric materials for membrane supports are cellulose or cellulose derivatives as well as other preferably inert polymers like polyethylene, polypropylene, polybutylenterephthalate or polyvinylidene- difluoride.

The hydrogels can be formed through in-situ reaction of one or more polymerizable monomers with one or more crosslinkers and/or one or more cross-linkable polymers to form a cross-linked gel that has preferably macropores. Suitable polymerizable monomers include monomers containing vinyl or acryl groups. Preferred are monomers comprising an additional functional group that either directly forms the ligand of the matrix or is suitable for attaching the ligands. Suitable crosslinkers are compounds containing at least two vinyl or acryl groups. Further details about suitable membrane supports, monomers, crosslinkers etc. as well as suitable production conditions can be found in WO04073843 and WO2010/027955.

Especially preferred are membranes made of an inert, flexible fiber web support comprising in assembly within and around the fiber web support a porous polyacrylamide hydrogel with quaternary ammonium groups (strong anion exchange groups), like Natrix® Q Chromatography membrane, Merck KGaA, Germany.

Depending on the membrane device used, the respective processes are conducted by different operating principles like dead-end operation, crossflow operation and radial flow operation systems. Dead-end operation is preferred. Examples of suitable membranes to be used in the method of the present invention are

- Membranes with a polyethersulfone (PES)-based support and a crosslinked polymeric coating, functionalized with quaternary ammonium groups (strong anion exchange groups), like Mustang® Q, Pall.

- Membranes made of stabilized reinforced cellulose, comprising a hydrogel with quaternary ammonium groups (strong anion exchange groups) or comprising DEAE groups (diethylaminoethyl, weak ion exchange groups), like Sartobind® membranes, Sartorius.

- Membranes made of a fine fiber non-woven scaffold comprising a hydrogel with quaternary ammonium groups (strong anion exchange groups), like 3M^TM Emphaze™ AEX Hybrid Purifier, 3M.

- Membranes made of an inert, flexible fiber web support comprising within and around the fiber web support a porous polyacrylamide hydrogel with quaternary ammonium groups (strong anion exchange groups), like Natrix® Q Chromatography membrane, Merck KGaA, Germany.

A monolith or a monolithic sorbent, similar to a membrane, has through- pores, like interconnected channels, so that liquid can flow from one side of the monolith, through the monolith, to the other side of the monolith.

Since the mobile phase is flowing through these through-pores, molecules to be separated are transported by convection rather than by diffusion. Due to their structure, monolithic sorbents show flow rate independent separation efficiency and dynamic capacity.

The monolith is typically formed in situ from reactant solutions and can have any shape or confined geometry, typically with frit-free construction, which guarantees convenience of operation. Preferably, monolithic materials have a binary porous structure, mesopores and macropores. The micron-sized macropores are the through-pores and ensure fast dynamic transport and low backpressure in applications; mesopores contribute to sufficient surface area and thus high loading capacity.

The monoliths can be made of organic, inorganic or organic/inorganic hybrid materials. Preferred are organic polymer-based monoliths.

The synthesis of organic polymer monoliths is typically done by a one-step polymerization providing a tuneable porous structure with tailored functional groups. Generally, a pre-polymerization mixture consisting of the monomers, crosslinkers, porogenic solvents, and initiators in an appropriate ratio is polymerized in a suitable container, also called mold, determining the format of the monolith. Polymerization is typically initiated by heating, use of UV radiation, microwave or y-ray radiation in the presence of initiators. After reaction for the prescribed time at an appropriate temperature, the resulting material is typically washed with solvents to remove unreacted components and porogenic solvents.

Suitable organic polymers are polymethacrylates, polyacrylamides, polystyrenes, polyurethanes, etc., like Poly(methacrylic acid-ethylene dimethacrylate), Poly(glycidyl methacrylate-ethylene dimethacrylate) or Poly(acrylamide-vinylpyridine-N,N'-methylene bisacrylamide).

Inorganic monoliths can be made of silica or other inorganic oxides. Preferably they are made of silica. Silica monoliths are normally prepared via a sol-gel method with phase separation. This mainly includes hydrolysis, condensation, and polycondensation of silica precursors. Typically, tetraethoxysilane (TEOS) or tetramethylorthosilicate (TMOS) is distributed in a suitable solvent in the presence of a porogen (e.g. polyethylene glycol) (PEG)), followed by the addition of a catalyst, acid or base, or a binary catalyst, acid and base in sequence. After reaction for a prescribed time, the resulting gel-like product is washed with solvents to remove unreacted precursor, porogen, and catalyst, followed by the proper post treatment, typically a heat treatment.

The monoliths can be modified with suitable functional groups, preferably at least ion exchange groups, to generate the targeted interaction with the sample comprising the target molecule and thus the targeted separation.

Typically, the monoliths are contained in a housing like a column.

Particle-based resins intended for liquid chromatography are normally comprised of particles that are packed together in a tubular cylinder called column to form a bed. The packed bed shows a distinct space between the particles, so called void volume, which mainly defines the liquid fluid permeability and hydrodynamic properties of the packed bed.

The particles typically consist of a cross-linked polymer matrix in spherical, bead-like or granular shape with relatively uniform size for improved chromatographic and hydrodynamic characteristics of the packed bed. They can have a dense structure with discrete or very small pores but usually exhibit a porous multichannel or reticular structure forming an inner pore volume and additional surface area inside the particle. The particle surface area can be modified with a variety of functional groups suitable for chromatography applications either by using functional monomers for the backbone-polymer structure, coupling of functional groups to the particle surface directly of via ligands or short polymers structures (grafts).

Membranes and monoliths can also be produced by 3D printing processes.

Particulate base materials can be prepared, for example, from organic polymers. Organic polymers of this type can be polysaccharides, such as agarose, dextranes, starch, cellulose, etc., or synthetic polymers, such as poly(acrylamides), poly(methacrylamides), poly(acrylates), poly(methacrylates), hydrophilic substituted poly(alkyl allyl ethers), hydrophilic substituted poly(alkyl vinyl ethers), poly(vinyl alcohols), poly(styrenes) and copolymers of the corresponding monomers. These organic polymers can preferably also be employed in the form of a crosslinked hydrophilic network. This also includes polymers made from styrene and divinylbenzene, which can preferably be employed, like other hydrophobic polymers, in a hydrophilized form.

Alternatively, inorganic materials, such as silica, zirconium oxide, titanium dioxide, aluminium oxide, etc., can be employed as particulate base materials. It is equally possible to employ composite materials, i.e. , for example, particles which can themselves be magnetised by copolymerisation of magnetisable particles or of a magnetisable core. It is also possible to use core shell materials whereby the shell, i.e. at least the surface or a coating, has OH groups.

However, preference is given to the use of hydrophilic base materials which are stable to hydrolysis or can only be hydrolysed with difficulty since the materials according to the invention should preferably withstand alkaline cleaning or regeneration at e.g., basic pH over an extended use duration. The base matrix may consist of irregularly shaped or spherical particles, whose particle size can be between 2 and 1000 pm. Preference is given to average particle sizes between 3 and 300 pm, in a most preferred embodiment the average particle size is between 20 - 63 pm.

The particulate base material may be in the form of non-porous or preferably porous particles. The average pore sizes can be between 2 and 300 nm. Preference is given to pore sizes between 5 and 200 nm, most preferred average pore size is between 40 - 110 nm.

In a very preferred embodiment, the particulate base material is formed by copolymerisation 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 cyclo-'hexane-'dimethanol monovinyl ether and divinylethyleneurea (1 ,3-divinylimidazolin-2-one) as crosslinking agent. An example of a suitable commercially available vinylether based base material is Eshmuno®, Merck KGaA, Germany.

In a preferred embodiment the polymer to be used as a particulate matrix in the method of the present invention is derivatised by graft polymerisation with tentacle-like structures, which can in turn carry the corresponding ligands or be functionalised by means of the latter. The grafting is preferably carried out in accordance with EP 0 337 144 page 12 example 8 or US 5453186 page 9 example 8 using N-(2-Trimethylammoniumethyl)- acrylamide and/or another monomer carrying suitable functional groups. The polymerisation catalyst employed is cerium(IV) ions, since this catalyst forms free-radical sites on the surface of the base material, from which the graft polymerisation of the monomers is initiated.

The polymerisation is terminated by termination reactions involving the cerium salts. For this reason, the (average) chain length can be influenced by the concentration ratios of the base material, the initiator and the monomers. Furthermore, uniform monomers or also mixtures of different monomers can be employed; in the latter case, grafted copolymers are formed.

Suitable monomers for the preparation of the graft polymers and further details about the grafting procedure are e.g., disclosed in WO 2007/014591 , EP 0337 144, especially page 12, example 8 and US 5453186 page 9, example 8.

Preferably the matrix is derivatised with cationic groups by graft polymerisation whereby the resulting chains that are grafted onto the base material have a length of between 2 and 100, preferably 5 and 60, in particular between 10 and 30 monomer units, each unit typically carrying one cationic group. The matrix might carry additional other functional groups like hydrophobic or hydrophilic groups in addition to the anion exchange groups but in any case, it has anion exchange groups.

Preferred particulate matrices are matrices with weak anion exchange and/or strong anion exchange groups, e.g., with trimethylammoniumethyl (TMAE) groups, like Eshmuno® Q, Merck KGaA, Germany.

The base material may equally also be in the form of fibres, hollow fibres or coatings.

The present invention provides methods of separating or purifying AAVs. This means that one or more AAVs can be separated from one or more other AAVs, from empty or full AAVs and/or from other impurities in a sample. Preferably at least one full AAV is separated from at least one empty AAV and/or one or more impurities. This is done by a chromatographic separation on an anion exchange chromatography matrix which comprises at least one type of anion exchange groups and optionally further functional groups like hydrophobic groups whereby the elution buffer comprises a certain additive.

By the methods of the invention target AAVs can be separated, enriched, isolated and/or purified enabling an efficient separation. In particular aspects of the invention, targeted full AAVs can be separated from empty AAVs and impurities in one chromatographic step. A high-resolution separation can be achieved and different rAAV species can be separated and isolated. The one or more additive that is part of the elution buffer results in a modification of the retention of different AAV species, especially of the retention of empty and full AAVs, whereby the presence of one or more additives in the elution buffer causes a decrease in the retention of empty AAV particles in comparison to the filled AAV particles of the same type. The production of cells comprising AAVs is known to a person skilled in the art. Typically, the selected cells are expanded in suitable culture media in a bioreactor under suitable conditions. The cells may be grown as adherent or suspension culture. For example, in suspension culture of HEK293 cells suitable seeding numbers before transfection are 0.5 to 1 .1 e6 viable cells per ml.

Suitable methods for the transduction are known in the art. In one embodiment, cells can be transduced in vitro by combining a rAAV with the cells, e.g., in appropriate media, and screening for those cells harboring the DNA of interest using conventional techniques such as Southern blots and/or PCR, or by using selectable markers.

Transfection can be performed using any of the techniques known in the art, including but not limited to electroporation, lipofection, e.g. with a lipofectamine, cationic polymers and cationic lipids. Any suitable transfection media may be used. In one embodiment of the transfection process, adherent or suspension human embryonic kidney (HEK293) cells are transfected with a triple DNA plasmid polyethylenimine (PEI) coprecipitation.

After a suitable virus production period post transfection or infection, the cells are lysed, and the viral particles 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.

Suitable methods for lysing cells are known to a person skilled in the art.

Preferably, for lysis, the cells are contacted with a composition comprising a detergent. After incubation with the lysis composition the released AAVs can then be isolated and/or purified, whereby at least one chromatographic purification on an anion exchange matrix is included.

In an embodiment, the present disclosure provides a method for manufacturing an AAV based viral vector comprising the steps of (i) culturing cells in a bioreactor or flask, (ii) transfecting the cells with plasmids to enable production of the AAV particles, (iii) lysing the mixture of the cells and the viral particles to release the viral particles from the cells (iv) isolating and/or purifying the viral particles whereby step (v) comprises a chromatographic purification on a chromatography matrix comprising anion exchange groups whereby the elution buffer comprises one or more additives selected from the group of basic amino acids, in a preferred embodiment arginine and/or lysine.

Optionally, after lysis the obtained mixture is first filtered or centrifuged.

In one embodiment the mixture is filtered through a filter that removes large molecule contaminants and cellular debris but that permits AAVs to pass therethrough.

In one embodiment, the released viral particles can be separated and purified from the cell culture medium using clarification. Clarification can be a centrifugation and/or microfiltration process in which relatively larger components such as lysed cells and/or impurities are removed from a solution. Clarification filters include depth filtration, charged depth filtration and similar microfiltration techniques. The resulting sample is a clarified lysate, also called clarified sample.

Centrifugation can for example be a low-speed centrifugation to remove larger particles like cellular debris. This can be for example done at 10000 to 12000 g for 10 to 30 minutes. The released viral particles can be found in the supernatant.

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

In one embodiment the sample is a pre-purified lysate. A pre-punfication step is used to remove the majority of impurities, such as host cell proteins and others. Pre-punfication comprises chromatographic methods, such as affinity chromatography, to produce a capture eluate with reduced impurities and a mixture of AAV particles. In a preferred embodiment, after lysis, the mixture of the cells and the viral particles is first clarified by filtration and/or centrifugation and then pre-purified by affinity chromatography. Afterwards, the pre-purified sample is further purified on a chromatography matrix comprising anion exchange groups whereby the elution buffer comprises one or more additives selected from the group of basic amino acids, in a preferred embodiment arginine and/or lysine.

The isolation and/or purification of the AAVs typically includes one or more of the following process steps:

- Clarification

- filtration

- dialysis/ diafiltration

- tangential flow filtration

- treatment with nuclease, e.g., RNase and/or Dnase

- treatment with chloroform

- ion exchange chromatography

- affinity chromatography - hydrophobic interaction chromatography

- centrifugation

- PEG precipitation

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

In the anion exchange chromatography step, the target AAVs are separated from at least one empty AAV and/or one impurity in a sample, whereby the sample comprising the AAVs is brought into contact with the chromatography matrix. Contact times are usually in the range from 6 seconds to 24 hours. It is advantageous to work in accordance with the principles of liquid chromatography by passing the liquid through a chromatography column or other type of housing which contains the anion exchange chromatography matrix. The liquid can run through the column or housing merely through its gravitational force or be pumped through by means of a pump. An alternative method is batch chromatography, in which the separation material is mixed with the liquid by stirring or shaking for as long as the AAVs need to be able to bind to the separation material. It is likewise possible to work in accordance with the principles of the chromatographic fluidized bed by introducing the liquid to be separated into, for example, a suspension comprising the chromatography matrix, where the chromatography matrix is selected so that it is suitable for the desired separation owing to its high density and/or a magnetic core.

The chromatographic process is run in the bind and elute mode, so that the target AAVs bind to the chromatography matrix when being contacted with the chromatography matrix. The chromatography matrix with the bound target AAVs can optionally but preferably subsequently be washed with one or more wash buffers, which preferably have the same ionic strength and the same pH as the liquid in which the sample comprising the AAVs is brought into contact with the chromatography matrix. The wash buffer removes substances which do not bind to the chromatography matrix. Further washing steps with other suitable buffers may follow without desorbing the target AAVs. The desorption of the bound AAVs is carried out by elution with an elution buffer. The elution buffer differs from the wash buffer and the feed. It differs in at least one of the following properties:

- molarity of basic amino acid additives

- the conductivity

- the pH.

In any case it comprises at least one basic amino acid, in a preferred embodiment arginine and/or lysine.

AAVs can thus be obtained in the eluent. AAVs usually have a purity above 70%, e.g., of 70 percent to 99 percent, preferably above 85%, e.g., 85 percent to 99 percent, particularly preferably above 90%, e.g., 90 percent to 99 percent, after desorption.

Unexpectedly, it has been found, that the use of additives, such as basic amino acids, in the elution buffer can efficiently separate empty AAVs from full AAVs. When preferably using gradient elution with an elution buffer comprising increasing amounts of at least one basic amino acid, in a preferred embodiment arginine or lysine, empty AAV particles elute prior to full AAVs from the anion exchange chromatography matrix. That means empty AAV particles elute with an elution buffer comprising less basic amino acid additives and preferably lower conductivity compared to full AAV which only elute with an elution buffer having a higher basic amino acid concentration and preferably a higher conductivity. With the method of the invention, empty and full AAVs can be separated by peak fractionation with high resolution and almost baseline separation. Preferably, the chromatographic separation is performed by equilibrating and loading the matrix as well as optionally washing the loaded matrix with a buffer having a conductivity below 10 mS/cm and a pH between 8 and 10. Said buffer may comprises 0 to 125 mM of at least one basic amino acid. Elution is done by linear or stepwise gradient elution with a molarity increase in a range from 0 -1 M of at least one basic amino acid, in a preferred embodiment arginine and/or lysine, while the pH is kept at a constant level in the range of 8 to 10. In parallel to the increase in molarity of the basic amino acid during the elution the conductivity of the elution buffer increases in a range from 1 to 50 mS/cm. If a condition increases in a range it means that this condition alters from a certain starting value within this range to another, higher value in the range. The starting value and the higher value can be located somewhere within the range and need not be identical with the limits. Typically, the elution buffer has the same pH as the equilibration- and loading buffer and the gradient elution starts with an elution buffer having the same conductivity and amino acid content as the equilibration- and loading buffer.

In general, every buffer substance known to a skilled person in art can be used to generate pH and conductivity stable aqueous solutions.

Examples of suitable buffers to be used, are listed in Table 1 for equilibration and in Table 2 for elution. If pH and conductivity need to be adjusted, concentrations of buffers might change.

Table 1 : Chemicals used for preparation of equilibration buffer

Table 2: Chemicals used for preparation of elution buffer

The increase in conductivity of the elution buffer can be achieved by increasing the amount of basic amino acid additives and by optionally additionally adding salt to generate a linear or step mode gradient in conductivity. The salt may be selected from the group consisting of: NaCI, KCI, sulfate, formate and acetate or mixtures thereof; preferably NaCI. For some amino acids like arginine and lysine it is typically not required to add salt to achieve a sufficient increase in conductivity but for some other amino acids, the addition of salt is preferred to achieve sufficient increase in conductivity.

The term “column volume” refers to the volume inside of a packed column not occupied by the chromatography matrix. This volume includes both the interstitial volume (volume outside of the matrix) and the own internal porosity (pore volume) of the matrix.

In a preferred embodiment, for elution, the applied linear gradient lasts around 30 to 50 column volume (CV) plus an additional hold step at target elution buffer for at least 20 CV.

In another preferred embodiment the AAV recovery rates are above 30%, preferably above 60%.

The basic amino acid additive range in the elution buffer is between 0 and 1 M, where in a preferred embodiment it is between 0 and 800 mM.

The conductivity range of the elution buffer is between 1 mS/cm and 50mS/cm, where in the more preferred embodiment is conductivity range between 2 and 40 mS/cm. The pH range is between pH 8 and 10, where in the more preferred embodiment the pH range is between pH 8,5 and 9,5, in a very preferred embodiment around pH 9. Very preferred the pH is kept constant or close to a constant value during loading, washing and elution.

In one embodiment, the method of the present invention comprises equilibrating the anion exchange chromatography media with a buffer comprising an anionic compound, e.g., a weak acid or a salt thereof, such as citric acid, acetic acid or succinic acid or a salt thereof prior to contacting the anion exchange media with the feed composition comprising the virus particles and wherein the feed composition optionally also comprises a predetermined amount of an anionic compound, e.g., a weak acid or a salt thereof. The equilibration buffer as well as the feed may comprise the anionic compound such as a weak acid or salt thereof, such as citric acid or citrate at a concentration of from about 0.5mM to about 15mM, from about 1 mM to about 10mM, preferably from about 1.5mM to about 7.5mM.

The equilibration buffer as well as the feed can also comprise an amino acid e.g., a natural or non-natural amino acid. For example, the equilibration buffer and optionally the feed may comprise a predetermined amount of histidine, lysine and/or arginine. The concentration of the amino acid is preferably between 25 and 125 mM. The effect choosing a loading/equilibration buffer comprising a weak acid or salt thereof as well as optionally an amino acid is that binding of the empty AAV particles to the chromatography matrix is prohibited or reduced. Further details can be found in WO22159679.

A "weak” acid is a compound that has an acid dissociation constant of less than about 10 ~⁴.

The method of the present invention can be applied for all rAAV and wt serotypes without a need for previous process steps. Preferably, clarified lysate (nuclease treated and clarified) or pre-purified lysate can be applied directly to the chromatography matrix.

In a very preferred embodiment, a sample, e.g., in form of a clarified or prepurified lysate which has been treated with a nuclease like Benzonase® (Merck KGaA, Germany) and has been clarified as well as optionally previously purified using another chromatographic method, is applied to a matrix comprising anion exchange groups at a pH between 8 and 10 and a conductivity between 2 and 7 mS/cm. Optionally the loaded matrix is washed with wash buffer with a pH between 8 and 10 and a conductivity between 2 and 7 mS/cm.

Elution is performed by applying a linear gradient from a molarity of amino acid additives starting with 0 to 100 mM of e.g. arginine and/or lysine to a concentration between 450 and 850 mM arginine and/or lysine. This equals a conductivity between 2 and 7 mS/cm, depending on the sample conductivity, to a conductivity between 30 and 40 mS/cm at constant pH in a range between 8 and 10. Conductivity can additionally be adjusted by adding salts to the elution buffer.

Elution preferably results in two fractions depending on the concentration of amino acid additives and/or conductivity reached during this step. Fraction 1 elutes from the matrix in the concentration range from 0 to 150 mM basic amino acid additive like arginine and/or lysine and comprises the majority of empty rAAV particles. Fraction 2 elutes from the matrix in the concentration range between 150 and 300 mM basic amino acid additive like arginine and/or lysine and comprises a highly enriched portion of full rAAV.

The preferred buffer composition is a buffer with a concentration of more than 500 mM to about 1 M of the basic amino acid depending of the solubility of the basic amino acid, most preferred around 800 mM amino acid additive with a wide elution gradient range from 0 to 1 M, preferably 0 to 800 mM, additive concentration, but not limited to. With the method of the present invention, full AAVs can be enriched efficiently. Anion exchange chromatography in combination with an elution buffer system containing basic amino acids, can be used as a method to capture rAAV from pre-purified lysate with very high recoveries of full rAAVs in the eluate and independent of the serotype and expression host. Typically, full rAAVs can be enriched by 100% or more. The reduction or removal of empty particles provides for reduction of the overall process steps as no separate, additional process step is needed for this.

The enrichment of full AAV capsids can be calculated using UV absorbance as quotient of the integrated relative peak area measured at 260 nm divided by the integrated relative peak area measured at 280 nm. Values between 0.6 to 0.7 indicate empty AAVs whereas values between 1 .3 and 1.4 show full AAV particles. (Wagner, C.; Innthaler, B.; Lemmerer, M.; Pletzenauer, R.; Birner-Gruenberger, R. Biophysical Characterization of Adeno- Associated Virus Vectors Using Ion-Exchange Chromatography Coupled to Light Scattering Detectors. Int. J. Mol. Sci. 2022, 23, 12715. https://doi.org/ 10.3390/ijms232112715) In the enriched elution fractions generated with the method of the present invention this quotient reaches values from 1.24 to 1 .41 which means that the majority of AAV particles in this fraction contain DNA and so are considered as full particles. In not enriched fractions this quotient is typically below 1 . This shows that a single purification step with an anionic interaction matrix capture and basic amino acid additive elution was able to enrich the amount of full AAVs significantly.

In a preferred embodiment, the chromatography matrix is a particulate resin, most preferred a polymeric resin to which the anion exchange groups are attached via polymer chains made of monomers carrying an anion exchange group which are grafted to the polymeric base material. The nature of the chromatography matrix used (i.e., strong and/or weak anion exchangers, optionally additional hydrophobic groups) and the conditions of additive and salt concentration, buffer used, and pH, will vary on the AAV capsid variant (i.e., AAV capsid serotype or pseudo-type). While the known AAV capsid variants all share features such as size and shape, they differ in fine details of molecular topology and surface charge distribution. Hence, while all capsid variants are expected to be amenable to enrichment by anion exchange chromatography optimal methods can be determined in a systematic manner using chromatography resin and buffer screening experiments, different conditions will be required for each AAV capsid variant to achieve efficient full AAV particle isolation. The determination of such conditions is readily apparent to the skilled artisan.

Due to the high recovery rate of the method of the present invention it is also possible to repeat the method of the present invention two or more times which further reduces the content of empty AAV particles in the purified sample.

The present invention is further illustrated by the following figures and examples, however, without being restricted thereto.

Examples

The following examples represent practical applications of the invention.

Example 1 : Comparison of additives influence on resolution in FLD- HPLC

Empty and full AAV capsids (rAAV2, Sirion Biotech) are measured in FLD (Em 350nm, Ex 280nm) - HPLC (Ultimate3000, Thermo) on a non-porous strong anion-exchange column (BioSAX Agilent, ID 4.6 x 50 mm, #5190- 2468) at pH 9. Using an increase of additive concentration through gradient elution its influence on peak resolution between full and empty rAAV capsids is measured. Explicitly, arginine and lysine are being compared to tetramethyl ammonium chloride, representing the current state of the art. Table 1: HPLC method with gradient and buffer components at pH 9. Find additives listed in Table 2.

Time % A % B Amount of

[min] 70 mM BTP 70 mM BTP + additive

800 mM [mM] additive

0.0 100 0 0

1.0 87.5 12.5 100

2.5 87.5 12.5 100

12.5 62.5 37.5 300

12.6 10 90 720

14.6 10 90 720

14.7 100 0 0

20.0 100 0 0 Table 2: List of chemicals, which are screened in gradient elution (see table 1). The resolution between the peaks of empty rAAV2 capsids is determined for comparison of efficacy.

Name HPLC results

Ammonium chloride 0.6 min resolution, nearly baseline separation

Tetramethyl ammonium 2 min resolution, nearly baseline chloride separation

Trimethyl ammonium chloride 1.7 min resolution, nearly baseline hydrochloride separation

Tetraethyl ammonium 0.9 min resolution, no baseline chloride monohydrate separation

Arginine hydrochloride 1 .0 min resolution, almost baseline separation

Lysine 1.7 min resolution, nearly baseline separation

A: Tetramethyl ammonium chloride

Tetramethyl ammonium chloride is used as an elution substance in buffer B (70 mM BTP + 800 mM Tetramethyl ammonium chloride) to observe its influence on the resolution of full and empty rAAV2 capsids. Both samples are diluted in buffer A (70 mM BTP) to a physical titer of 2E11 vp/mL (determined via ELISA and ddPCR) and 20 pL of each sample are injected onto the column.

Table 3: Peak integration using software Chromeleon version 7.2.10 on

HPLC run. empty AAV2

Volume Ret. Time Rel. Area Height

Sample Area [counts*min]

[pL] [min] [%] [counts] empty 20 6.99 95.4 4,168,765 4,289,663 full 20 6.98 11.4 409,530 393,038 full AAV2

Volume Ret. time Rel. Area Height

Sample Area [counts*min]

[pL] [min] [%] [counts] empty 20 8.54 4.6 200,069 239,767 full 20 9.00 84.4 3,022,348 4,138,039

The sample consisting of empty rAAV2 particles only showed a main peak at 6.99 min retention time with a relative area of 95.4 % (see figure 1 ), meanwhile the sample containing full rAAV2 capsids showed a main peak at 9 min retention time with a relative area of 84.4% (data shown in Table 3), indicating that the two samples contain at least two significantly different species of rAAV2. The peak of the full rAAV2 sample showing up at 6.98 min retention time with a relative area of 11 .4% represents the portion of empty rAAV2 capsids, which were not sufficient separated during the purification performed by the supplier.

When using tetramethyl ammonium chloride the resolution between the main peaks resulting from empty and full rAAV2 capsid containing samples is roughly around 2 min.

B: Arginine

Arginine is used as an elution substance in buffer B (70 mM BTP + 800 mM Arginine) to observe its influence on the resolution of full and empty rAAV2 capsids. Both samples are diluted in buffer A (70 mM BTP) to a physical titer of 2E11 vp/mL (determined via ELISA and ddPCR) and 20 pL of each sample are injected onto the column.

Table 4: Peak integration using software Chromeleon version 7.2.10 on HPLC run. empty AAV2

Volume Ret. Time Rel. Area Height

Sample Area [counts*min]

[pL] [min] [%] [counts] empty 20 5.37 100.0 548,645 1 ,060,534 full 20 5.54 8.0 52,043 144,653 full AAV2

Volume Ret. time Rel. Area Height

Sample Area [counts*min]

[pL] [min] [%] [counts] empty 20 n.a. n.a. n.a. n.a. full 20 6.04 81.1 530,150 758,945

The sample consisting of empty rAAV2 particles only showed a main peak at 5.3 min retention time with a relative area of 100 % (see figure 2), meanwhile the sample containing full rAAV2 capsids showed a main peak at 6.2 min retention time with a relative area of 81.1 % (data shown in Table 4), when using an Arginine gradient as mobile phase for elution.

When using Arginine the resolution between the main peaks resulting from empty and full rAAV2 capsid containing samples is roughly around 55 sec.

C: Lysine

Lysine is used as an elution substance in buffer B (70 mM BTP + 800 mM Lysine) to observe its influence on the resolution of full and empty rAAV2 capsids. Both samples are diluted in buffer A (70 mM BTP) to a physical titer of 2E11 vp/mL (determined via ELISA and ddPCR) and 20 pL of each sample are injected onto the column.

Table 5 Peak integration using software Chromeleon version 7.2.10 on HPLC run. empty AAV2

Volume Ret. Time Rel. Area Height

Sample Area [counts*min]

[pL] [min] [%] [counts] empty 20 6.11 100.0 9,227,094 7,992,488 full 20 6.15 7.5 894,179 769,203 full AAV2

Volume Ret. time Rel. Area Height

Sample Area [counts*min]

[pL] [min] [%] [counts] empty 20 n.a. n.a. n.a. n.a. full 20 7.82 47.6 5,709,057 8,941 ,434

The sample consisting of empty rAAV2 particles only showed a main peak at 6.11 min retention time with a relative area of 100 % (see figure 3), meanwhile the sample containing full rAAV2 capsids showed a main peak at 7.82 min retention time with a relative area of 47.6% (data shown in Table 5), when using a lysine gradient as mobile phase for elution.

The resolution has been improved by using lysine gradient as mobile phase, which results in roughly 1.5 min time difference between the main peaks resulting from empty and full rAAV2 capsid containing samples.

Experiment 2: Enrichment of full rAAV from feed lysate in FPLC

The findings are transferred to a preparative scale using a porous, strong anion exchange material with a mean particle size of 80 pm (Eshmuno® Q) in 5x50 mm Superformance column and a FPLC system (Akta™ Avant 25). All fractions are collected to be later analyzed vie ELISA and digital droplet PCR. The feedstock is a Benzonase® treated, clarified HEK293T cell lysate containing empty and full rAAV2 particles, which were pre-purified via affinity chromatography.

The Affinity capture eluate is adjusted in pH and conductivity by diluting 1 : 10 in Equilibration buffer A, see table 6.

Table 6 General method using the Akta™ system, for bind and elute separation of empty from full rAA V2 capsids on Eshmuno® Q. Buffer A (equilibration buffer) and B (elution buffer with increased conductivity) were used for a step elution. Steps were filled prior pumping on column.

Step Volumes Buffer

Equilibration 2 CV 70 mM BTP, pH 9.0, 3.5 mS/cm Load 20 mL pH and conductivity adjusted affinity capture eluate

Wash 5 CV 70 mM BTP, pH 9.0, 3.5 mS/cm

Elution 1 ) 0.42 CV gradient to A: 70 mM BTP, pH 9.0, 3.5

12.5% B mS/cm

2) 0.63 CV step B: 70 mM BTP, 800mM

12.5%B Arginine, pH 9.0, 36.0 mS/cm

3) 4.2 CV gradient

37.5% B

4) 4.5 CV step 90% B

Strip 5 CV 0.1 M Tris, 2 M NaCI

Re- 5 CV 70 mM BTP, pH 9.0, 3.5 mS/cm equilibration

Chromatogram showing the elution (see figure 4) and zoom-in (see figure 5) of rAAV2 (pre-purified via affinity capture) using a step elution on Eshmuno® Q. Buffer A consists of BTP and pH 9, whereby Arginine was added as buffer B. The elution steps finished after 10CV. Column dimensions: 5 mm i.d. x 50 mm length (CV = 1 mL). Flow rate: 0.33 mL/min (102 cm/h; residence time = 3 min).

Table 7: determined analysis data from ddPCR

Sample Capsids Capsids GC Purity

[total VP] [total GC] [%]

Load 4.48E+12 4.05E+11 9.0

Flow-Through N/D N/D N/A

Pool

Wash Pool N/D N/D N/A

Elution Pool 1.53E+12 N/D N/A

Elution Fraction 1.88E+11 1.03E+09 0.5

1 Elution Fraction 3.21 E+11 5.88E+10 18.3

2

Elution Fraction 6.94E+11 4.58E+10 6.6

3

Elution Fraction 2.40E+11 1.19E+10 5.0

4

In Table 7 the determined analysis data show that in fraction 2 (1 .C.8, see figure 5) an enrichment over 100% could be determined, where the purity increased from 9% to 18.3%.

Description of Figures

Figure 1 :

Tetramethylammonium chloride was used as elution substance (buffer B). Thin black line: empty AAV; bold grey line: full rAAV2; Injection volume 20p I of 2E11 vp/mL samples of empty and full rAAV2 (produced and pre-purified by Sirion Biotech GmbH) on HPLC system Ultimate3000, Thermo; column BioSAX Agilent (ID 4.6 x 50 mm, #5190-2468), detection FLD Em 350nm, Ex 280nm.

Figure 2:

Arginine was used as elution substance (buffer B). Thin black line: empty AAV; bold grey line: full rAAV2; Injection volume 20pl of 2E11 vp/mL samples of empty and full rAAV2 (produced and pre-purified by Sirion Biotech GmbH) on HPLC system Ultimate3000, Thermo; column BioSAX Agilent (ID 4.6 x 50 mm, #5190-2468), detection FLD Em 350nm, Ex 280nm.

Figure 3:

Lysine was used as elution substance (buffer B). Thin black line: empty AAV; bold grey line: full rAAV2; Injection volume 50pl of 2E11 vp/mL samples of empty and full rAAV2 (produced and pre-purified by Sirion Biotech GmbH) on HPLC system Ultimate3000, Thermo; column BioSAX Agilent (ID 4.6 x 50 mm, #5190-2468), detection FLD Em 350nm, Ex 280nm.

Figure 4:

Chromatogram showing the elution of rAAV2 (pre-purified via affinity capture) using a step elution on Eshmuno® Q. UV signal traces are displayed in dark grey (A280nm) and black (A260nm) lines and the conductivity signal trace in a light grey line. Fractions of interest for analysis are 1.C.7 (Elution 1 ), 1.C.8 (Elution 2), 1.C.9+10 (Elution 3) and 1.C.11 (Elution 4). Buffer A consists of BTP and pH 9, whereby Arginine was added as buffer B. The elution steps finished after 10CV. Column dimensions: 5 mm i.d. x 50 mm length (CV = 1 mL). Flow rate: 0.33 mL/min (102 cm/h; residence time = 3 min).

Figure 5:

Zoom in on figure 4 showing the elution of rAAV2 (pre-purified via affinity capture) using a step elution on Eshmuno® Q. UV signal traces are displayed in a dashed grey (A280nm) and black (A260nm) line, whereas the conductivity signal trace is a light grey line. Buffer A consists of BTP and pH 9, whereby Arginine was added as buffer B. The elution steps finished after 10CV. Column dimensions: 5 mm i.d. x 50 mm length (CV = 1 mL). Flow rate: 0.33 mL/min (102 cm/h; residence time = 3 min).

Claims

Claims

1 . A method for enriching full adeno-associated virus (AAV) particles by contacting a sample comprising full and empty AAV particles with a chromatography matrix comprising anion exchange groups so that full AAV particles bind to the matrix and eluting said particles with an elution buffer comprising one or more basic amino acids.

2. Method according to claim 1 whereby it comprises the steps of a) contacting the sample comprising the AAV particles with a chromatography matrix comprising anion exchange groups b) optionally washing the chromatography matrix c) eluting AAV particles which bound to the chromatography matrix with an elution buffer comprising one or more basic amino acids.

3. Method acording to claim 1 or 2, whereby the the sample is a clarified lysate that has been pre-purified by filtration and/or centrifugation.

4. Method acording to one or more of claims 1 to 3, whereby the sample is a pre-purified lysate that has been pre-purified by affinity chromatography.

5. Method according to one or more of claims 1 to 4, whereby in step c) the elution buffer has a conductivity higher than the conductivity of the sample in step a).

6. Method according to one or more of claims 1 to 5, whereby the chromatography matrix is a resin, membrane or a monolith.

7. Method according to one or more of claims 1 to 6, whereby the chromatography matrix comprises trimethylammoniumethyl (TMAE) groups.

8. Method according to one or more of claims 1 to 7, whereby the anion exchange groups are attached to the chromatography matrix via polymer chains which are made by grafting monomers comprising an anion exchange group to the base material of the chromatography matrix.

9. Method according to one or more claims 1 to 8, whereby the majority of empty AAV capsids elute prior to the majority of full AAV particles.

10. Method according to one or more claims 1 to 9, whereby the elution buffer has a pH equal to the loading buffer.

11 . Method according to one or more of claims 1 to 10, whereby said basic amino acid is arginine and/or lysine.

12. Methods according to one or more of claims 1 to 11 , whereby the the concentration of the basic amino acids in the elution buffer is between 2 and 800 mM.

13. Method according to one or more claims 1 to 12, where during elution the elution buffer is applied with a linear gradient.

14. Method acording to one or more of claims 1 to 13, whereby elution is performed with a linear conductivity gradient in the range between 2 and 40 mS/cm. while pH is kept at a constant level.

15. Method according to one or more of claims 2 to 14, whereby steps a) to c) are repeated two to four times.