Methods and compositions for purifying adeno-associated viral particles or adenovirus

Abstract

The present invention relates to compositions and methods for lysing cells and isolating and / or purifying adeno-associated viral particles or adenoviral particles using a detergent selected from the group consisting of alkyl dimethyl amine oxides.

Description

TECHNICAL FIELD

[0001] The present invention relates to compositions and methods for lysing cells and thus purifying adeno-associated virus particles or adenoviruses using a detergent selected from an alkyl dimethyl amine oxide. BACKGROUND

[0002] Adeno-associated viruses (AAV) as well as adenoviruses (AdV) have been characterized and developed as efficient viral vectors for delivering genes in vitro in cultured cells as well as in vivo for delivering genes. AAV is meanwhile the leading platform for in vivo delivery of gene therapies. AdV is used for gene therapy as well as for use as a vaccine. 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) capable of forming a T-shaped secondary structure and serving as the origin of genome replication, a rep region encoding four overlapping replication proteins Rep78, Rep68, Rep52, and Rep40, and a cap region encoding three structural proteins VP1, VP2, and VP3, and assembly-activating protein (AAP). Naturally isolated serotypes 1-9 of AAV virus share the genomic structure, although these serotypes can show different tissue tropisms. Since AAVs appear to be non-pathogenic, showing efficient transduction and stable expression, they are considered to be one of the most promising gene delivery vectors. Adenoviruses (AdV) are medium-sized, non-enveloped viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. Most AdVs used for gene therapy and vaccine development originate from serotype 2 (Ad2) and serotype 5 (Ad5).

[0003] AAV vectors can be produced in various cell lines in adherent or suspension cell culture format using transient transfection or co-infection methods. Depending on the specific serotype and time of production, virus particles including intact, partial, and empty species can be secreted from the cells into the culture medium or contained inside the cells in various ratios.

[0004] Initially, stable AAV producer cells are generated by transfection and selection of human cells (such as HeLa or HEK293 cells) with rAAV transfer vectors containing ITR cassettes and packaging constructs containing Rep and Cap. Production of recombinant AAV vectors (rAAVs) is then achieved by infection with helper viruses that provide the helper functions, such as adenovirus (AdV). After identification of the AdV genes required for AAV vector packaging, helper virus-free methods were established using double or triple transfection protocols consisting of two or three plasmids including helper plasmids instead of helper viruses. This system is widely used for research and drug development. In addition, the development of baculovirus expression vectors provides another method for the production of rAAV viruses in insect Sf9 cells. These different techniques have shown to be able to produce sufficient amounts of rAAV viruses for use in laboratories and clinical trials.

[0005] AdV vectors can also be produced from HEK293 or PER.C6 cells or derivatives in adherent or suspension cell culture. Due to the large genome of 26-45 kb, it is not feasible to reconstitute the necessary viral genes into several plasmids and production of recombinant AdV has been achieved by single infection with the same viral vector.

[0006] A cell lysis step is usually 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 widely used. However, for certain serotypes (e.g. AAV2), viral particles tend to bind tightly to insoluble cellular components, thus limiting the efficiency of certain cell lysis reagents in virus release.

[0007] Current methods to release viruses, especially cell-associated viruses, are often time-consuming, e.g. like physical lysis methods are difficult to scale up, or can introduce unwanted and difficult to remove impurities, e.g. Triton X-100. Degradation products of Triton X-100 are octylphenol, an ecotoxicant with proven estrogenic effects. It would therefore be advantageous to have effective reagents and methods to lyse cells and isolate and / or purify viruses, such as AAV and AdV, from cells producing said viruses, wherein said methods would not necessarily use ecologically problematic reagents. SUMMARY

[0008] The inventors have surprisingly found that certain groups of ecologically friendly detergents are particularly suitable for releasing cell-associated viruses. In combination with salts, such as sodium chloride, they are even more effective than the known procedures using Triton X100.

[0009] Thus, the present application relates to a method for lysing cells encapsulating adeno-associated virus (AAV) particles or adenovirus particles, said method being performed by contacting a suspension of said cells with an effective amount of a composition comprising an alkyl dimethyl amine oxide to promote cell lysis and release of said AAV particles from said cells, wherein said viral particles remain unaffected.

[0010] Typically, such a lysis method is part of a method for purifying viruses, in which method further separation or purification steps are performed in addition to the lysis step.

[0011] The present application also relates to a method for purifying adeno-associated virus (AAV) or adenovirus (AdV) viral particles from a sample comprising cells encapsulating viral particles, said method being performed by:

[0012] a) contacting a suspension of said cells and said viral particles with an effective amount of a composition comprising an alkyl dimethyl amine oxide to promote cell lysis and release of AAV or AdV particles from said cells,

[0013] b) isolating and / or purifying said AAV or AdV particles.

[0014] In a preferred embodiment, said suspension is contacted with an effective amount of a composition comprising an alkyl dimethyl amine oxide and a salt, such as sodium chloride.

[0015] In a very preferred embodiment, said composition comprises LDAO (lauryl dimethyl amine-N-oxide) and / or TDAO (tetradecyl dimethyl amine-N-oxide) and sodium chloride and / or potassium chloride. Especially preferred is a composition comprising LDAO (lauryl dimethyl amine-N-oxide) and / or TDAO (tetradecyl dimethyl amine-N-oxide) and sodium chloride.

[0016] In another preferred embodiment, in step a) said cells are contacted with said composition for 30 to 180 minutes, preferably 60 to 90 minutes.

[0017] In another embodiment, said viral particles are AAV, especially AAV2 or AAV9 serotype.

[0018] In one embodiment, said composition is an aqueous solution comprising an alkyl dimethyl amine oxide and a salt, such as sodium chloride. Preferably, said composition contains only water, one or more alkyl dimethyl amine oxides, sodium chloride and optionally a buffer component.

[0019] In a preferred embodiment, the concentration of the alkyl dimethyl amine oxide in the composition is such that the concentration in the mixture with the cells is between 1% and 4% (w / w).

[0020] In another preferred embodiment, the concentration of the salt (such as sodium chloride) in the composition is such that the concentration in the mixture with the cells is between 0.05 mol / l and 1 mol / l.

[0021] In one embodiment, step b) is performed by filtration or centrifugation.

[0022] In one embodiment, the method of the application comprises one or more of the following steps:

[0023] - clarification,

[0024] - filtration,

[0025] - dialysis / diafiltration,

[0026] - tangential flow filtration,

[0027] - treatment with a nuclease, such as an RNAse and / or a DNAse,

[0028] - treatment with chloroform,

[0029] - ion exchange chromatography,

[0030] - affinity chromatography,

[0031] - hydrophobic interaction chromatography,

[0032] - centrifugation,

[0033] - PEG precipitation.

[0034] The application also relates to a composition comprising one or more alkyl dimethyl amine oxides and a salt (such as sodium chloride).

[0035] In a preferred embodiment, the composition is an aqueous solution comprising 5% to 30% (w / w) TDAO and / or LDAO and 1 to 6 mol / l NaCl.

[0036] In a preferred embodiment, the composition has a pH between 6 and 9.

[0037] The application also relates to a kit comprising the composition of the application and a nuclease. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 AAV2 titers after cell lysis with single reagents are shown. More details can be found in Example 1.

[0039] Figure 2 AAV2 titers after cell lysis with single and double reagents are shown. More details can be found in Example 2.

[0040] Figure 3 AAV2 genome titers after cell lysis with NaCl and NaCl / TDAO are shown. More details can be found in Example 4.

[0041] Figure 4 Percentage of intact virus particles (vp) which is the ratio of genome titers to physical titers is shown. More details can be found in Example 4.

[0042] Figure 5 AAV9 titers after cell lysis with single and double reagents are shown. More details can be found in Example 5.

[0043] Figure 6 Percentage of intact virus particles (vp) which is the ratio of genome titers to physical titers is shown. More details can be found in Example 5. DETAILED DESCRIPTION

[0044] Alkyl dimethyl amine oxides suitable for lysing virus production cells are amphiphilic, charged amine oxides coupled to saturated hydrocarbon chains of different lengths. Preferably, the length of the saturated hydrocarbon chain is between 8 and 18 carbon atoms. In a preferred embodiment, the alkyl dimethyl amine oxide is selected from the group consisting of dimethyl decyl amine oxide, dimethyl undecyl amine oxide, dimethyl dodecyl amine oxide (LDAO), dimethyl tridecyl amine oxide and dimethyl tetradecyl amine oxide (TDAO).

[0045] Table 1 shows the properties of selected alkyl dimethyl amine oxides.

[0046] Chemical name Alkyl chain length CAS MW g / mol CMC *mM CMC wt% Biodegradable within 28 days N,N-dimethyloctylamine N-oxide C8 2605-78-9 173,3 150 2,60 100% N,N-dimethyldecylamine N-oxide C10 2605-79-0 201,35 15 0,30 97% N,N-dimethyldodecylamine N-oxide synonym: lauryldimethylamine oxide (LDAO) C12 1643-20-5 229,40 1,7 0,039 95% N,N-dimethyltetradecylamine N-oxide (TDAO) C14 3332-27-2 257,46 0,268 0,0069 88% N,N-dimethylhexadecylamine N-oxide C16 7128-91-8 285,52 0,025 0,00071 <88% N,N-dimethyloctadecylamine N-oxide C18 2571-88-2 313,56

[0047] Table 1

[0048] These compounds are preferably used at concentrations higher than their critical micelle concentration. The critical micelle concentration (CMC) is defined as the concentration of a detergent above which micelles form and all additional detergent added to the system goes into the micelles. The CMC value for a given detergent in a given medium depends on the temperature, the pressure, and (sometimes strongly) on the presence and concentration of other surface active substances and electrolytes.

[0049] Examples of salts (such as sodium chloride) are compounds comprising a metal cation such as K + , Na + , Li + , Mg 2+ , Ca2+ and an anionic component such as F - , SO4 2- , HPO4 2- , acetate, CI - . Preferred are salts comprising a monatomic ion. Particularly preferred are chloride salts, such as sodium chloride and potassium chloride, with sodium chloride being most preferred.

[0050] Adeno-associated virus (AAV) is a member of the Parvoviridae family. The AAV genome consists of a linear single-stranded DNA molecule containing approximately 4.7 kilobases (kb) and composed of two major open reading frames encoding nonstructural Rep (replication) proteins 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 an interrupted palindrome sequence that can fold into a hairpin structure that acts as a primer 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 encapsidation of viral nucleic acid into mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158:97-129).

[0051] There are multiple serotypes of AAV and they provide different tissue tropisms. Known serotypes include, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11.

[0052] Vectors derived from AAV are particularly attractive for the delivery of genetic material because they are able to infect (transduce) a wide variety of non-dividing and dividing cell types, including muscle fibers and neurons, and they lack viral structural genes, thereby eliminating natural host cell responses to viral infection, such as interferon-mediated responses. Moreover, the wild-type virus has never been associated with any pathology in humans.

[0053] According to the present invention, scAAV is also within the AAV group. Self-complementary adeno-associated viral vectors (scAAV) are viral vectors engineered from naturally occurring adeno-associated virus (AAV) for use in gene therapy. ScAAV are termed "self-complementary" because the coding region has been designed to form an intramolecular double-stranded DNA template.

[0054] Accordingly, in some embodiments, "AAV" means 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. An AAV vector can have one or more of the AAV wild-type genes (e.g., rep and / or cap genes) deleted in whole or in part, but retains functional flanking ITR sequences. Functional ITR sequences are necessary for rescue, replication, and packaging of AAV virions. Accordingly, an AAV vector is defined herein as at least including those sequences provided for replication and packaging of the virus (e.g., functional ITRs). The ITR need not be a wild-type nucleotide sequence and can be altered, e.g., by insertion, deletion, or substitution of nucleotides, so long as the sequence provides for functional rescue, replication, and packaging. In one embodiment, the vector is an AAV-9 vector, which has AAV-2-derived ITRs. "AAV" also refers to the protein shell or capsid, which provides an efficient vehicle for delivery of the vector nucleic acid to the nucleus of a target cell.

[0055] The term "AAV" as used herein is intended to also encompass recombinant AAV.

[0056] Adenoviruses (AdV) are medium-sized (90-100 nm), nonenveloped (without an external lipid bilayer) viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. They have a broad vertebrate host; in humans, more than 50 different adenovirus serotypes (belonging to seven species (A-G)) have been found to cause a wide range of diseases.

[0057] Methods of making recombinant AdV and packaging them into suitable host cells are known in the art. They are used for gene therapy and as vaccines to express foreign antigens. Adenoviral vectors can be replication-defective; certain essential viral genes are deleted and replaced by a cassette expressing an exogenous therapeutic gene. Replication-competent (oncolytic) vectors are used for cancer gene therapy. Oncolytic vectors are engineered to preferentially replicate in cancer cells and destroy the cancer cells by the natural process of lysis of viral replication. Numerous clinical trials have shown that replication-defective adenoviral vectors and replication-competent adenoviral vectors are safe and have therapeutic activity.

[0058] Adenoviruses (AdV) and adeno-associated viruses (AAV) are also referred to herein as viruses, viral particles, or viral vectors.

[0059] As used herein, the term "cell" or "cell line" refers to a single cell or group of cells that are capable of growing and dividing serially or for a prolonged period of time in vitro. In some embodiments, for example, the terms "HEK293 cells," "293 cells," or their grammatical equivalents are used interchangeably herein and refer to a host / packaging cell line used in the methods disclosed herein.

[0060] Suitable cells and cell lines for producing AAV and AdV have been described. The cells themselves can 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 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, HEK 293 cells, Saos, C2C12, L cells, HT1080, HepG2, and primary fibroblast, hepatocyte, and myoblast cells derived from mammals including humans, monkeys, mice, rats, rabbits, and hamsters.

[0061] Generally, the expression cassette is composed of at least a 5' AAV inverted terminal repeat (ITR), a nucleic acid sequence encoding a desirable therapeutic, immunogen, or antigen operably linked to regulatory sequences that direct its expression, and a 3' AAV ITR. In one embodiment, the 5' ITR and / or the 3' ITR of AAV serotype 2 is used. However, the 5' ITR and 3' ITR from other suitable sources can be selected. It is this expression cassette that is packaged into the capsid protein to form the AAV virus or particle.

[0062] In addition to the expression cassette, the cell contains sequences that drive AAV expression in the cell (cap sequences) and rep sequences that have the same source or cross-complementary source as the AAV ITRs found in the expression cassette. The AAV cap and rep sequences can be independently selected from different AAV parental sequences and are introduced into the host cell in a suitable manner known to those skilled in the art. While full-length rep genes can be used, it has been found that smaller fragments thereof (i.e., rep78 / 68 and rep52 / 40) are sufficient to allow replication and packaging of AAV.

[0063] The cell also requires helper functions in order to package the AAV of the present application. Optionally, these helper functions can be provided by a herpes virus. In another embodiment, the necessary helper functions are each provided from a human or non-human primate adenovirus source, such as can be obtained from a variety of sources, including the American Type Culture Collection (ATCC), Manassas, Va. (US).

[0064] A buffer or buffered solution is a solution with a certain pH used to prevent changes in pH. Examples of buffers are CO2 / HCO3 (carbonate), phosphate, HEPES, PIPES, ACES, BES, TES, MOPS and TRIS.

[0065] During the manufacture of AAV, a certain percentage of the capsids can not incorporate any transgene and are referred to as empty capsids or empty AAV. In addition, capsids containing fragments of the transgene are referred to as partial capsids or partial AAV. These undesired product-related impurities are produced in addition to the full-length, full capsids or full AAV containing the desired transgene. The same terminology can be used for AdV.

[0066] Purification means increasing the purity of the target molecule (in this case, AAV or AdV), for example by removing or reducing the amount of one or more impurities.

[0067] The term "impurity" or "contaminant" as used herein refers to any foreign or harmful molecule or species, including biological macromolecules such as DNA, RNA, one or more host cell proteins, nucleic acids, endotoxins, lipids, impurities of synthetic origin such as detergents, partial and / or empty AAV or AdV and one or more additives that can be present in a sample containing viral particles that are to be purified and thus are to be separated from one or more impurities.

[0068] A bioreactor is any vessel or tank in which cells can be cultured. Culturing is typically performed under suitable conditions (such as a suitable temperature, etc.) and with a suitable medium that supports cell growth / culturing. The skilled person knows suitable culturing conditions for supporting or maintaining cell growth / culturing.

[0069] The present application is based on the finding that certain types of compositions comprising at least an alkyl dimethyl amine oxide based detergent are especially suitable for lysing cells and liberating viral particles. AAV or AdV vectors can be produced in a variety of cell lines using transient transfection, infection or co-infection methods in adherent or suspension cell culture formats. Depending on the specific serotype and time of production, viral particles including intact, partial and empty species can be secreted into the culture medium from the cells or contained inside the cells in various ratios. A cell lysis step is often required at harvest to release viral particles into the supernatant. Sometimes, viral particles tend to be tightly bound to insoluble cell components, which limits the efficiency of certain cell lysis reagents in virus release.

[0070] It has been found that alkyl dimethyl amine oxides alone or in combination with a salt such as sodium chloride are especially suitable for facilitating cell lysis and isolation and / or purification of viral particles.

[0071] Production of cells comprising AAV or AdV is known to those skilled in the art. Typically, the selected cells are expanded in a suitable medium under suitable conditions in a bioreactor. The cells can be grown as adherent or suspension cultures. For example, in suspension cultures of HEK293 cells, a suitable seeding number prior to transfection is 0.5 to 1.1 e6 viable cells / ml.

[0072] Suitable transduction methods are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV or AdV with the cells in a suitable medium and screening those cells carrying the DNA of interest using conventional techniques such as Southern blotting and / or PCR or by using selectable markers.

[0073] Transfection can be performed using any technique known in the art, including but not limited to electroporation, lipofection, for example with lipofectamine, cationic polymers and cationic lipids. Any suitable transfection medium can be used. In one embodiment of the transfection method, adherent or suspension human embryonic kidney (HEK293) cells are transfected with triple DNA plasmid polyethylenimine (PEl) co-precipitation.

[0074] In embodiments, the present disclosure provides a method for manufacturing an AAV or AdV based viral vector comprising the steps of: (i) culturing cells in a bioreactor, (ii) transfecting the cells with a plasmid to enable production of AAV particles or infecting the cells with an AdV vector to expand / produce the same AdV particles, (iii) contacting the mixture of the cells and the viral particles with an effective amount of a composition comprising an alkyl dimethyl amine oxide and optionally a salt such as sodium chloride to facilitate cell lysis and release of viral particles from the cells, iv) isolating and / or purifying the viral particles.

[0075] After a suitable virus production period following transfection or infection, the cells are lysed and the viral particles are harvested. In some embodiments, the cells are dissociated from the bioreactor prior to the start of the cell lysis process. In some embodiments, the cells are lysed in situ.

[0076] According to the present invention, for lysis, the cells are contacted with a composition comprising an alkyl dimethyl amine oxide and optionally a salt such as sodium chloride. Preferably, the lysis solution is added to the bioreactor comprising the cell suspension, thereby creating a mixture of the cell suspension and the composition.

[0077] The incubation of the mixture comprising the cells and the composition containing the alkyl dimethylamine oxide and optionally a salt, such as sodium chloride, is typically performed for 30 to 180 minutes, preferably for an incubation time between 60 minutes and 90 minutes. Shorter and longer times can also be suitable.

[0078] The pH of the mixture during the incubation can vary in a wide range. For example, it can be between pH 4 and pH 10, typically between pH 6 and pH 9.

[0079] The temperature of the mixture during the incubation can also vary in a wide range. For example, it can be between 20 °C and 37 °C.

[0080] To obtain an effective amount of the composition comprising the alkyl dimethylamine oxide to promote cell lysis and release of AAV particles from the cells, the concentration of the alkyl dimethylamine oxide in the composition is such that it is effective to induce 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. To this end, the concentration of the alkyl dimethylamine oxide in the final mixture with the cells is preferably higher than 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 less than the volume of the cell culture.

[0081] Thus, a suitable concentration of the alkyl dimethylamine oxide, especially TDAO, in the composition is between 10% and 30% (w / w). A suitable concentration of the salt, such as sodium chloride, in the composition to be added to the cell suspension is between 1 mol / l and 6 mol / l, typically about 3 to 5 mol / l, so that the final concentration in the mixture with the cell suspension is between 0.05 mol / l and 1 mol / l.

[0082] In a preferred embodiment, the used detergent is TDAO (N,N-dimethyltridecylamine N-oxide) and / or LDAO, either as a single component or as a mixture with NaCl.

[0083] In a very preferred embodiment, the composition is an aqueous solution. It can also comprise one or more buffers.

[0084] It has been found that the composition as defined above is effective to induce lysis of the cells and, especially, to effectively separate viral particles that tend to adhere to the cells and cell debris from the cells.

[0085] Subsequently, the released AAV or AdV can be isolated and / or purified after incubation with the lysis composition. This can be done with any method. Typically, it is done by one or more method steps including filtration and / or centrifugation.

[0086] In one embodiment, the mixture is filtered through a filter that removes macromolecular contaminants and cell debris, but allows the AAV to pass through.

[0087] In one embodiment, the released viral particles can be isolated and purified from the cell culture medium using clarification. Clarification can be a microfiltration process in which relatively large 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.

[0088] Tangential flow filtration can be used to concentrate the mixture of purified viral particles and remove salts and proteins. Tangential flow filtration (TFF) refers to a generally rapid and efficient method for filtering or purifying a solution containing a target product and / or impurities during which the solution or liquid flows parallel to the filtration membrane.

[0089] Centrifugation can be, for example, low speed centrifugation to remove larger particles such as cell debris. This can be performed, for example, at 10000 g to 12000 g for 10 to 30 minutes. The released viral particles can be found in the supernatant.

[0090] Isolation and / or purification of AAV typically comprises one or more of the following process steps:

[0091] - clarification,

[0092] - filtration,

[0093] - dialysis / diafiltration,

[0094] - tangential flow filtration,

[0095] - treatment with a nuclease, such as an RNAse and / or DNAse,

[0096] - treatment with chloroform,

[0097] - ion exchange chromatography,

[0098] - affinity chromatography,

[0099] - hydrophobic interaction chromatography,

[0100] - centrifugation,

[0101] - PEG precipitation.

[0102] 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, simultaneously or after lysis. The nuclease can be one that degrades both DNA and RNA. In one embodiment, the endonuclease is a genetically engineered endonuclease from Serratia marcescens (Mukai et al., 2008) sold under the name Benzonase® (EMD Millipore). Serratia marcescens

[0103] In various embodiments, ion exchange chromatography is applied for further purification. This can be, e.g., anion exchange (AEX) or cation exchange (CEX) capture chromatography steps. This step is used, e.g., to separate host cell proteins, host cell DNA, host cell lipids, detergents and other process-related impurities from the virus particles. The principles of cation exchange chromatography and anion exchange chromatography are well known in the art. The sample is loaded and the column is washed with loading buffer. Finally, the target sample is eluted from the column using elution buffer and fractions containing the sample are collected.

[0104] Other suitable chromatographic methods are hydrophobic interaction chromatography, size exclusion chromatography or affinity chromatography. Instead of a bind-elute mode, a flow-through mode can also be suitable.

[0105] It is also possible to precipitate AAV or AdV with polyethylene glycol (PEG). For this, PEG is added to the virus sample. The molecular weight of the PEG is typically between about 3,000 g / mol and about 15,000 g / mol. The concentration of PEG in the precipitation solution can be adjusted as needed, which can be, e.g., about 5% (w / w).

[0106] Chloroform can be added to solubilize lipids and inactivate a large number of proteins. Virus particles are not affected by chloroform, which can then be separated by phase separation and / or centrifugation.

[0107] Salt, such as sodium chloride, can be removed from the virus particles by dialysis. The virus-containing solution can be dialyzed against water or another solution, depending on the impurities to be removed.

[0108] Typical AAV purification methods include clarification, concentration, and diafiltration using tangential flow filtration, chromatographic purification by using affinity chromatography and ion exchange chromatography. In some methods, ultracentrifugation and gradient ultracentrifugation are used instead of or in addition to chromatography. The final step in AAV purification typically includes concentration and diafiltration into a suitable excipient buffer composition and sterile filtration.

[0109] ​The present application also relates to a composition for use in the method of the present application, said composition comprising an aqueous solution of an alkyl dimethyl amine oxide and sodium chloride. The concentration of alkyl dimethyl amine oxide in said composition is typically higher than 1 % (w / w), preferably between 10 % (w / w) and 30 % (w / w), such that when the composition is added to a suspension comprising cells to be lysed, the final concentration of alkyl dimethyl amine oxide can be adjusted to be higher than 0.1 % (w / w), preferably between 1 % and 4 % (w / w).

[0110] In a preferred embodiment, the used detergent is TDAO (N,N-dimethyltridecylamine N-oxide) and / or LDAO, either as a single component or as a mixture with NaCl. Most preferred is a mixture of TDAO with NaCl.

[0111] In a very preferred embodiment, said composition is an aqueous solution. It can also comprise one or more buffers.

[0112] The concentration of salt, such as sodium chloride, in said composition is typically between 1 mol / l and 6 mol / l, preferably about 3 to 5 mol / l, such that the final concentration in the mixture with the cell suspension is between 0.05 mol / l and 1 mol / l.

[0113] In a preferred embodiment, said composition is made of water, one or more alkyl dimethyl amine oxides, sodium chloride and / or potassium chloride and optionally a buffer only.

[0114] The present application also relates to a kit comprising a composition of the present application as described above and a nuclease, preferably an endonuclease, most preferably benzonase®. Said kit typically comprises two containers, such as bottles, with said two components, but it can also comprise additional components and thus additional containers.

[0115] As can be seen from the examples, the method of the present application is very efficient. Using the method of the present application, at least 80 %, most preferably 100 % of the cells can be lysed while releasing the virus. Cell lysis can be detected, for example, via microscopic imaging. The number of virus particles released with the lysis method of the present application exceeds the number of virus particles released by using Triton X100 or sodium chloride or even a combination thereof. This is unexpected. The method and composition of the present application are especially suitable for AAV serotypes that tightly bind to cells.

[0116] For example, in an upstream method on AAV2 suspension, which comprises a cell lysis step using 0.5 % Triton X-100 (T100) or 0.5 M NaCl for 90 min, both at 37 °C, a significantly reduced virus titer was observed (Figure 1 ) in contrast, much higher virus titers can be achieved when using a lysis solution comprising 0.5 M sodium chloride and 1% TDAO for the same amount of time (see Figure 2 ).

[0117] Another advantage of the present invention is that tangential flow filtration can be used to remove the detergents and optionally salts (such as sodium chloride) used for cell lysis and virus release. In tangential flow filtration, ultrafiltration membranes can be used to purify and concentrate the virus particles and diafiltration can be performed to remove the detergents.

[0118] The present invention is further illustrated by the following figures and examples, however, the invention is not limited thereto.

[0119] The entire disclosure of all applications, patents and publications, cited above and below, as well as the entire disclosure of U.S. Provisional Patent Application 63 / 024,643, filed May 14, 2020, are hereby incorporated by reference. Examples

[0120] The following examples are representative of actual applications of the present invention.

[0121] Example 1 - Comparative Example

[0122] An AAV2 suspension upstream process was performed including a cell lysis step using various concentrations of various single agents (such as Triton X-100 (T100) or TDAO) or 0.5 M NaCl, all at 37°C. Samples were centrifuged at different time points after lysis at about 13000 g for 5 min and the supernatant was removed and stored at -70°C for assays. Physical titers were measured by enzyme-linked immunosorbent assay (ELISA) using an AAV2 titration ELISA kit (Progen) following the manufacturer’s protocol, and genome titers were measured by quantitative PCR (qPCR) targeting specific sequences within the transgene. All reagents and assay protocols were from MilliporeSigma unless otherwise stated. In all conditions tested, 0.5 M NaCl was the only agent able to release significant amounts of AAV2 at physical titers of e10 vp / mL and genome titers at levels of e9 gc / mL Figure 1 ). Moreover, a time-dependent effect from 30 min to 90 min after lysis was observed for all 3 single agents, and a concentration-dependent effect from 0.5% to 1% for both single detergents Figure 1 ). However, none of the single agents showed satisfactory AAV release levels typically at physical titers of e11 vp / mL and genome titers of e10 gc / mL.

[0123] InFigure 1 Each bar represents the mean of 2 dilutions x 2 assay wells or 3 assay wells, and error bars are standard deviations.

[0124] Example 2

[0125] The same method as in Example 1 was performed, but NaCl was combined with different detergents.

[0126] It was demonstrated that the addition of 1% TDAO to 0.5 M NaCl, but not 1% Triton X-100, promoted a 3-9-fold increase in AAV2 dissociation ( Figure 2 ). Furthermore, a longer lysis time of 3 h was evaluated as equivalent compared to the shorter time of 90 min ( Figure 2 ).

[0127] In Figure 2 Each bar represents the mean of 2 dilutions x 2 assay wells or 3 assay wells, and error bars are standard deviations.

[0128] Example 3

[0129] Cell lysis performed according to the general procedure of Example 1 was performed with different reagents and compared in terms of turbidity reduction and lysis efficiency. Notably, the combination of 1% TDAO and 0.5 M NaCl resulted in faster and more complete cell lysis compared to single reagent - 0.5 M NaCl or 0.5% Triton X-100 alone (Table 2).

[0130]

[0131] Table 2

[0132] Each turbidity in Table 2 is the mean of 1-3 bioreactors x 2 measurements. PT = post-transfection. The turbidity of HEK293-derived cells was about 300-400 NTU and of HEK293T-derived cells was about 500-600 NTU prior to lysis. Cell counts were performed by ViCell XR prior to lysis and at 30 min post-lysis, and % cell lysis was calculated as the ratio of lysed cells to total cells prior to lysis.

[0133] Example 4

[0134] To investigate the optimal concentration of TDAO in combination with 0.5 M NaCl, AAV2 dissociation experiments were performed using 0% to 4% TDAO and including an additional 60 min lysis time. As Figure 3As shown in FIG. 2, and as previously described, it was confirmed that the addition of TDAO to 0.5 M NaCl increased AAV2 dissociation by at least 3-5 fold, and the optimal concentration of TDAO in combination with 0.5 M NaCl was identified to be 2%. Furthermore, the optimal lysis time of 90 min was confirmed based on genome titer as previously described. When the % of intact virus particles (vp) was calculated as the ratio of genome titer to physical titer, it was noted that 2% TDAO also promoted more release of intact virus particles over time due to an unknown mechanism, with intact VP% of about 100% at 90 min post-lysis Figure 4 ).

[0135] In Figure 3 , each bar represents the average of 3 assay wells, and error bars are standard deviations. In Figure 4 , each bar represents only one data from one experiment.

[0136] Example 5

[0137] The AAV9 suspension upstream process was performed as in Example 1, but using both single and dual agents in combination with NaCl.

[0138] In contrast to the AAV2 results in Figure 1 , single detergents TDAO or Triton X-100 enhanced AAV9 dissociation by 2-5 fold compared to 0.5 M NaCl alone Figure 5 ). However, similar to the AAV2 results in Figure 2 , the combination of 1% TDAO and 0.5 M NaCl promoted AAV9 dissociation by 2-17 fold compared to the corresponding single agent, although no benefit was observed for the dual agent with 2% TDAO compared to 1% TDAO Figure 5 ). These results demonstrate that, although AAV9 and AAV2 can interact with different cellular components in different ways, the combination of TDAO and NaCl can promote dissociation of AAVs of these different serotypes. Consistent between AAV2 and AAV9 as well was that the combination of TDAO and NaCl promoted more release of intact virus particles with much higher intact VP% compared to single agents Figure 4 and Figure 6 ). However, the underlying mechanism of this phenomenon is currently unknown.

[0139] In Figure 5 , each bar represents the average of 2 dilutions x 2 assay wells or 3 assay wells, and error bars are standard deviations. In Figure 6 , each bar represents only one data from one experiment.

Claims

1. A method for lysing cells encapsulating adeno-associated virus (AAV) particles, said method being performed by contacting a suspension of said cells with an effective amount of a composition comprising an alkyl dimethyl amine oxide, to promote cell lysis and release of said AAV particles from said cells, wherein: said composition comprising TDAO (tetradecyl dimethyl amine-N-oxide) and sodium chloride; the concentration of TDAO in the mixture of the suspension of said cells with an effective amount of the composition is comprised between 1% and 4% w / w; the concentration of sodium chloride in the mixture of the suspension of said cells with an effective amount of the composition is comprised between 0.05 mol / l and 1 mol / l.

2. A method for purifying viral particles of adeno-associated virus (AAV) from a sample comprising cells encapsulating viral particles, said method being performed by: a) contacting a suspension of said cells and said viral particles with an effective amount of a composition comprising an alkyl dimethyl amine oxide, to promote cell lysis and release of said viral particles from said cells, b) isolating and / or purifying said viral particles, wherein: said composition comprising TDAO (tetradecyl dimethyl amine-N-oxide) and sodium chloride; the concentration of TDAO in the mixture of the suspension of said cells and said viral particles obtained in step a) with an effective amount of the composition is comprised between 1% and 4% w / w; the concentration of sodium chloride in the mixture of the suspension of said cells and said viral particles obtained in step a) with an effective amount of the composition is comprised between 0.05 mol / l and 1 mol / l.

3. The method of claim 2, wherein, In step a), said cells are contacted with said composition for a time comprised between 30 and 180 minutes.

4. The method of claim 1 or claim 2, wherein, said viral particles are recombinant adeno-associated virus (AAV) particles.

5. The method of claim 1 or claim 2, wherein said composition is an aqueous solution comprising TDAO and sodium chloride.

6. The method of claim 1 or claim 2, wherein the concentration of said TDAO in said composition is comprised between 10% and 30% w / w.

7. The method of claim 1 or claim 2, wherein the concentration of sodium chloride in said composition is comprised between 1 mol / l and 6 mol / l.

8. The method of claim 2 or claim 3, wherein Step b) comprises a filtration and / or a centrifugation step.

9. The method of claim 2 or claim 3, wherein said method comprises one or more of the following steps: - clarification and filtration, - dialysis and diafiltration, - treatment with RNase and / or DNase, - treatment with chloroform, - ion exchange chromatography, - affinity chromatography, - hydrophobic interaction chromatography, - centrifugation, - PEG precipitation.

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