Method and composition for purifying adeno-associated virus particles or adenovirus

Abstract

The present invention relates to compositions and methods for lysing cells and isolating and / or purifying adeno-associated virus particles or adenovirus particles using detergents selected from the group of alkyldimethylamine oxides.

Description

[Technical Field]

[0001] The present invention relates to compositions and methods for lysing cells and thereby purifying adeno-associated virus particles or adenoviruses using a washing agent selected from the group of alkyldimethylamine oxides. [Background technology]

[0002] Adeno-associated viruses (AAVs) and adenoviruses (AdVs) have been characterized and developed as potent viral vectors for delivering genes in vitro and in vivo into cultured cells. AAVs, on the other hand, are the primary platform for in vivo delivery of gene therapy. AdVs are used for gene therapy and as vaccines. AAV is a small, non-enveloped virus containing a single-stranded DNA genome of approximately 4.7 kb, forming a T-shaped secondary structure and capable of acting as a starting point for genome replication. It consists of one rep region encoding four duplicated replication proteins, Rep78, Rep68, Rep52, and Rep40, as well as two reverse-ended terminal repeats (ITRs) of one cap region encoding three structural proteins, VP1, VP2, and VP3, and the assembly activation protein (AAP). While naturally isolated serotypes 1–9 of AAV viruses share a common genomic structure, these serotypes may exhibit different tissue-specific characteristics. Because AAVs appear nonpathogenic and exhibit efficient transduction and stable expression, they are considered one of the most promising gene delivery vehicles. Adenoviruses (AdVs) are standard-sized, non-enveloped viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. Most AdVs used in gene therapy and vaccine development are derived from serotypes 2 (Ad2) and 5 (Ad5).

[0003] 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 the specific serotype and production cycle, viral particles containing complete, partial, and empty forms are secreted from outside the cell into the culture medium or contained inside the cell in varying proportions.

[0004] Initially, stable AAV-producing cells were generated by transfection and selection of human-derived cells, such as HeLa or HEK293 cells, containing an rAAV transfer vector with an ITR cassette and a packaging construct containing Rep and Cap. Subsequently, the production of recombinant AAV vectors (rAAV) was achieved by infection with an auxiliary virus, such as adenovirus (AdV), which provides helper function. After identifying the AdV gene required for AAV vector packaging, a helper virus-free method was established using a double or triple transfection protocol consisting of two or three plasmids containing a constructed helper plasmid instead of an auxiliary virus. This system is widely used in research and drug discovery. In addition, the development of baculovirus expression vectors provides another method for producing rAAV virus in insect Sf9 cells. These various techniques have been shown to produce rAAV virus of sufficient quality for laboratory and clinical use.

[0005] AdV vectors can also be produced in adherent or suspension cell culture formats by HEK293 or PER.C6 cells or derivatives. Due to its large genome of 26–45 kb, the reconstruction of essential viral genes into a small plasmid is not feasible, and recombinant AdV production has been achieved through single infection with the same viral vector.

[0006] The cell lysis step generally requires the release of viral particles into the supernatant during cell collection. Typical cell lysis reagents, such as Triton X-100, Tween 20, and NaCl, are widely used in this application. However, for certain serotypes (e.g., AAV2), viral particles tend to be tightly associated with insoluble cellular components, thus limiting the efficiency of the cell lysis reagent in terms of viral release.

[0007] Current methods for releasing viruses, particularly cell-associated viruses, are often time-consuming, such as physical lysis methods which are difficult to reduce or enlarge, or they may introduce unwanted and difficult-to-remove impurities, such as Triton X-100. The degradation products of Triton X-100 are octylphenol, an ecotoxin with demonstrated estrogenic effects. Therefore, it would be preferable to have a reagent-based method that is effective for lysing and isolating viruses such as AAV and AdV from the cells in which they are produced, and / or purifying them, so that the method does not require the use of ecologically questionable reagents.

[0008] The inventors have surprisingly found that a certain group of ecologically friendly cleaning agents are particularly suitable for releasing cell-associated viruses. When combined with salts such as sodium chloride, they are even more effective than known procedures using Triton X 100. [Overview of the project]

[0009] Therefore, the present invention relates to a method for lysing cells surrounding adeno-associated virus (AAV) particles or adenovirus particles by contacting a suspension of said cells with an effective amount of a composition containing alkyldimethylamine oxide, which promotes cell lysis and release of AAV or AdV particles from the cells, thereby leaving the virus particles unaffected. Typically, this lysis method is part of a method for purifying the virus, thereby allowing for further isolation or purification steps in addition to the lysis step.

[0010] The present invention relates to a method for purifying viral particles from a sample containing cells surrounding adeno-associated virus (AAV) or adenovirus (AdV) viral particles, a) Contacting a suspension of cells and virus particles with an effective amount of a composition containing alkyldimethylamine oxide, which promotes cell lysis and release of AAV or AdV particles from the cells. b) Isolating and / or purifying AAV or AdV particles. This further relates to the aforementioned method.

[0011] In a preferred embodiment, the suspension is brought into contact with an effective amount of a composition containing alkyldimethylamine oxide and a salt such as sodium chloride. In a highly preferred embodiment, the composition comprises LDAO (lauryldimethylamine-N-oxide) and / or TDAO (tetradecyldimethylamine-N-oxide) and sodium chloride and / or potassium chloride. Particularly preferred is a composition comprising LDAO (lauryldimethylamine-N-oxide) and / or TDAO (tetradecyldimethylamine-N-oxide) and sodium chloride. In another preferred embodiment, in step a), the cells are brought into contact with the composition for 30 to 180 minutes, preferably 60 to 90 minutes.

[0012] In another embodiment, the viral particle is AAV, and in particular, AAV2 or AAV9 serotype. In one embodiment, the composition is an aqueous solution containing an alkyldimethylamine oxide and a salt such as sodium chloride. Preferably, the composition contains only water, one or more alkyldimethylamine oxides, sodium chloride, and optionally a buffering component. In a preferred embodiment, the concentration of alkyldimethylamine oxide in the composition is such that the concentration in the mixture containing cells is between 1% (w / w) and 4% (w / w). In another preferred embodiment, the concentration of a salt, such as sodium chloride, in the composition is such that the concentration in the mixture containing cells is between 0.05 mol / l and 1 mol / l.

[0013] In one embodiment, step b) is carried out by filtration and centrifugation. In one embodiment, the method of the present invention comprises one or more of the following steps: - Clarification - filtration - Dialysis and diafiltration - Tangential flow filtration - Treatment using nucleases, e.g., RNase and / or DNase. - Treatment using chloroform - Ion exchange chromatography - Affinity chromatography - Hydrophobic interaction chromatography - Centrifugal separation - PEG precipitation.

[0014] The present invention is further directed to compositions comprising one or more alkyldimethylamine oxides and salts such as sodium chloride. In a preferred embodiment, the composition is an aqueous solution containing 5% (w / w) to 30% (w / w) TDAO and / or LDAO and 1 mol / l to 6 mol / l NaCl. In a preferred embodiment, the composition has a pH between 6 and 9. The present invention is further directed to kits comprising the composition and nuclease of the present invention. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 shows the AAV2 titer after cell lysis using a single reagent. Further details can be found in Example 1. [Figure 2] Figure 2 shows the AAV2 titer after cell lysis using single and double reagents. Further details can be found in Example 2. [Figure 3] Figure 3 shows the AAV2 genome titer after cell lysis using NaCl and NaCl / TDAO. Further details can be found in Example 4. [Figure 4] Figure 4 shows the percentage of complete virus particles (vp) as the ratio of genome titer to physical titer. Further details can be found in Example 4. [Figure 5] Figure 5 shows the AAV9 titer after cell lysis using single and double reagents. Further details can be found in Example 5. [Figure 6] Figure 6 shows the percentage of complete virus particles (vp) as the ratio of genome titer to physical titer. Further details can be found in Example 5.

[0016] Alkyldimethylamine oxides suitable for lysing cells producing virus are amphiphilic and are charged amine oxides that bind to saturated hydrocarbon chains of various lengths. Preferably, the length of the saturated hydrocarbon chain is carbon atoms between 8 and 18. In a preferred embodiment, the alkyldimethylamine oxide is selected from the group consisting of dimethyldecylamine oxide, dimethylundecylamine oxide, dimethyldodecylamine oxide (LDAO), dimethyltridecylamine oxide, and dimethyltetradecylamine oxide (TDAO).

[0017] Table 1 shows the characteristics of the selected alkyldimethylamine oxides.

Table 1

[0018] These compounds are preferably used at concentrations above their critical micelle concentration. The critical micelle concentration (CMC) is defined as the concentration of the detergent above which the micellar form and all additional detergents added to the system become micelles. The value of the CMC for a given detergent in a given medium depends on temperature, pressure, as well as (sometimes strongly) the presence and concentration of other surfactants and electrolytes.

[0019] Examples of salts such as sodium chloride are K + , Na + , Li + , Mg 2+ , Ca 2+ metal cations such as, and F - , SO4 2- , HPO4 2- , acetate, Cl - salts containing anionic components such as. Preferred are salts containing monoatomic ions. Particularly preferred are chloride salts such as sodium chloride and potassium chloride, where sodium chloride is most preferred.

[0020] 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 consisting of two major open reading frames encoding non-structural Rep (replication) and structural Cap (capsid) proteins. Flanking both sides of the AAV coding region are two cis-acting inverted terminal repeat (ITR) sequences approximately 145 nucleotides in length and palindromic sequences that can fold into hairpin structures that function as primers during the initiation of DNA replication. In addition to their role in DNA replication, the ITR sequences have been shown to be required for virus assembly, rescue from the host genome, and capsid formation of the viral nucleic acid into mature virions (Muzyczka, (1992) Cur. Top. Microbiol. Immunol. 158:97-129). Multiple serotypes of AAV exist, exhibiting diverse tissue-specific characteristics. Known serotypes include, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11.

[0021] Vectors derived from AAV are particularly attractive for delivering genetic material because they can infect (transduce) a wide variety of non-mitotic and mitotic cell types, including muscle fibers and neurons, and because they lack genes in the viral structure, thereby eliminating the natural host cell response to viral infection, such as the interferon-mediated response. In addition, wild-type viruses have never been associated with any pathological conditions in humans.

[0022] According to the present invention, scAAV is also within the group of AAVs. Self-complementary adeno-associated vectors (scAAVs) are viral vectors modified from naturally occurring adeno-associated viruses (AAVs) for gene therapy. ScAAVs are given the term "self-complementary" because their coding regions are designed to form an intramolecular double-stranded DNA template.

[0023] Therefore, in some embodiments, “AAV” means a vector or virus derived from adeno-associated virus serotypes, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, and AAV-11. An AAV vector may have one or more AAV wild-type genes, e.g., rep and / or cap genes, deleted in whole or in part, but may retain functional bilateral ITR sequences. Functional ITR sequences are necessary for the rescue, replication, and packaging of AAV virions. Therefore, an AAV vector is defined herein as containing at least those sequences (e.g., functional ITRs) that provide for the replication and packaging of the virus. The ITRs do not have to be wild-type nucleotide sequences and may be modified, e.g., by nucleotide insertions, deletions, or substitutions, as long as the sequences provide functional rescue, replication, and packaging. In one embodiment, the vector is an AAV-9 vector having an ITR derived from AAV-2. Furthermore, "AAV" refers to a protein shell or capsid that provides an efficient vehicle for the delivery of vector nucleic acids to the nucleus of target cells. When the term "AAV" is used herein, it is intended to encompass recombinant AAVs as well.

[0024] Adenoviruses (AdV) are standard-sized (90-100 nm), non-enveloped viruses (lacking an outer lipid bilayer) with an icosahedral nucleocapsid containing a Japanese strand DNA genome. They have a wide range of vertebrate hosts; in humans, more than 50 distinct viral serotypes belonging to 7 species (A-G) have been found to cause a wide range of diseases. Methods for preparing recombinant AdV and packaging them into suitable host cells are known in the art. They are used for gene therapy and as vaccines expressing foreign antigens. Adenovirus vectors can be replication-defective; some essential viral genes are deleted and replaced with a cassette expressing a foreign therapeutic gene. Replication-eligible (oncolytic) vectors are employed in cancer gene therapy. Oncolytic vectors are modified to preferentially replicate in cancer cells and to destroy cancer cells through the natural process of lytic viral replication. Numerous clinical trials have indicated that replication-defective and replication-qualified adenovirus vectors are safe and possess therapeutic activity. Adenoviruses (AdV) and adeno-associated viruses (AAV) are also referred to herein as viruses, viral particles, or viral vectors.

[0025] When used herein, the terms “cell” or “cell line” refer to a single cell or population of cells capable of continuous or sustained growth and division in vitro. In some embodiments, for example, the terms “HEK293 cell,” “293 cell,” or their grammatical equivalents refer to a host / packing cell line used interchangeably herein and in the manner disclosed herein. Suitable cells and cell lines are described for the production of AAV and AdV. The cells themselves may be selected from any biological organism, encompassing prokaryotic (e.g., bacterial) cells as well as eukaryotic cells, including insect cells, yeast cells, and mammalian cells. The specifically desired host cells are selected from any mammal, but are not limited to, 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 cell, HT1080, HepG2, and primary fibroblasts, hepatocytes, and myoblasts derived from mammals including humans, monkeys, mice, rats, rabbits, and hamsters.

[0026] Generally, an expression cassette consists of at least a 5' AAV reverse-terminal repeat (ITR), a desired therapeutic immunosource or antigen-encoding nucleic acid sequence operably ligated to a regulatory sequence that directs its expression, and a 3' AAV ITR. In one embodiment, a 5' and / or 3' ITR of AAV serotype 2 is used. However, a 5' and / or 3' ITR may be selected from another suitable source. This expression cassette is packaged in a capsid protein to form an AAV virus or particle.

[0027] In addition to the expression cassette, the cell contains a sequence that drives AAV expression in the cell (cap sequence) and a rep sequence from the same source as the AAV ITR found in the expression cassette, or from complementary sources. The AAV cap and rep sequences may be independently selected from different AAV parent sequences and introduced into the host cell in a manner known to those skilled in the art. While full-length rep genes may be available, their smaller fragments, namely rep78 / 68 and rep52 / 40, are sufficient to allow for AAV replication and packaging.

[0028] The cells also require helper functions to package the AAV of the present invention. Optionally, these helper functions may be supplied by herpesviruses. In another embodiment, the required helper functions are each supplied from a human or primate non-human animal adenovirus source, which can be obtained from a variety of sources, including, for example, the American Type Culture Collection (ATCC), Manassas, Va. (US). A buffer or buffer solution is a solution of a certain pH used to prevent changes in pH. Examples of buffers include CO2 / HCO3 (carbonate), phosphate, HEPES, PIPES, ACES, BES, TES, MOPS, and TRIS.

[0029] During the production of AAVs, a percentage of capsids may not incorporate any of the transgenes and are referred to as empty capsids or empty AAVs. In addition, capsids containing fragments of the transgenes are called partial capsids or partial AAVs. These undesirable impurities are produced along with the complete capsids or complete AAVs containing the full length of the desired transgene. The same terminology may be used for AdVs. Purification means increasing the purity of the target molecule, in this case AAV or AdV, by removing or reducing the amount of one or more impurities.

[0030] The term “impurity” or “contaminant” as used herein means any foreign or objectionable molecule or species, including biological macromolecules such as DNA, RNA, one or more host cell proteins, nucleic acids, endotoxins, lipids, synthetic impurities such as detergents, partial and / or empty AAV or AdV, and one or more additives that may be present in a sample containing viral particles, which are purified and thus separated from one or more impurities. A bioreactor is any vessel or tank in which cells can be cultured. Incubation is typically carried out under suitable conditions, such as a suitable temperature, and with a suitable medium to support cell growth / culture. Those skilled in the art are aware of suitable incubation conditions for supporting or maintaining cell growth / culture.

[0031] The present invention is based on the finding that certain types of compositions comprising at least an alkyldimethylamine oxide-based washing agent are particularly suitable for lysing cells and setting them free of viral particles. AAV or AdV vectors can be produced in various cell lines in adherent cell culture formats or suspension cell culture formats using transient transfection, infection, or co-infection methods. Depending on the specific serotype and production cycle, viral particles, including complete, partial, and empty species, can be secreted from outside the cell into the culture medium and contained inside the cell in varying proportions. The cell lysis step generally requires the release of viral particles into the supernatant during cell collection. Sometimes, viral particles tend to be tightly associated with insoluble cellular components, limiting the efficiency of certain cell lysis reagents in terms of viral release. Alkyldimethylamine oxide, either alone or in combination with salts such as sodium chloride, was found to be particularly suitable for promoting cell lysis and the isolation and / or purification of viral particles.

[0032] The production of cells containing AAV or AdV is known to those skilled in the art. Typically, selected cells are expanded in a bioreactor under suitable conditions and in a suitable cell medium. The cells may be grown as adherent cultures or suspension cultures. For example, in suspension culture of HEK293 cells, a suitable seeding rate before transfection is 0.5–1.1 e6 viable cells per ml. Suitable methods for transduction are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV or AdV with cells, for example in a suitable culture medium, and screening those cells containing DNA of interest using conventional techniques such as Southern blotting and / or PCR, or by using selectable markers.

[0033] Transfection may be carried out using any technique known in the art, but is not limited to, electroporation, lipofection, and, for example, lipofectamine, cationic polymers, and cationic lipids. Any suitable transfection medium may be used. In one embodiment of the transfection process, adherent or suspension human lung-kidney (HEK293) cells are transfected using triple DNA plasmid polyethyleneimine (PEI) coprecipitation.

[0034] In one embodiment, the present disclosure provides a method for producing an AAV or AdV-based viral vector, comprising the steps: (i) culturing cells in a bioreactor; (ii) transfecting the cells with a plasmid to enable the production of AAV particles, or infecting the cells with an AdV vector to amplify / produce the same AdV particles; (iii) contacting a mixture of cells and viral particles with an effective amount of a composition comprising alkyldimethylamine oxide and optionally a salt such as sodium chloride to promote cell lysis and release of viral particles from the cells; and (iv) isolating and / or purifying the viral particles.

[0035] After a suitable period of virus production following transfection or infection, the cells are lysed and the virus particles are collected. 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.

[0036] In accordance with the present invention, for dissolution, the cells are brought into contact with a composition comprising alkyldimethylamine oxide and optionally a salt such as sodium chloride. Preferably, the dissolution solution is added to a bioreactor containing the cell suspension so that a mixture of the cell suspension and the composition is produced. The incubation of the mixture of cells and alkyldimethylamine oxide and optionally a salt such as sodium chloride is typically carried out for an incubation time between 30 and 180 minutes, preferably between 60 and 90 minutes. Shorter and longer times may also be appropriate.

[0037] The pH of the mixture during incubation can vary over a wide range. It could be, for example, between pH 4 and pH 10, and typically between pH 6 and pH 9. The temperature of the mixture during incubation can also vary over a wide range. For example, it could be between 20°C and 37°C.

[0038] To have an effective amount of a composition containing alkyldimethylamine oxide that promotes cell lysis and the release of AAV particles from cells, the concentration of alkyldimethylamine oxide in the composition is such that it effectively induces cell lysis, meaning that at least 80%, preferably 100%, of the cells are lysed after incubation using the composition under the preferred conditions as described above. For this reason, the concentration of alkyldimethylamine oxide in the final mixture containing cells is preferably above its CMC. For TDAO, N,N-dimethyltridecylamine N-oxide, the concentration in the mixture containing cells is typically between 0.1% (w / w) 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.

[0039] As a result, the preferred concentration of alkyldimethylamine oxide, particularly TDAO, in the composition is between 10% (w / w) and 30% (w / w). The preferred concentration of salts such as sodium chloride in the composition added to the cell suspension is between 1 mol / l and 6 mol / l, typically between approximately 3 mol / l and 5 mol / l, so that the final concentration in the mixture having the cell suspension is between 0.05 mol / l and 1 mol / l.

[0040] In a preferred embodiment, the detergent used is TDAO, N,N-dimethyltridecylamine N-oxide, and / or LDAO, either as a single component or as a mixture with NaCl. In a very preferred embodiment, the composition is an aqueous solution. It may also contain one or more buffering agents.

[0041] It was found that the composition defined above effectively induces cell lysis, and that viral particles and cellular debris, which tend to adhere to cells, can be effectively separated from the cells. Following incubation with the soluble composition, the released AAV or AdV is then isolated and / or purified. This can be done by any method. Typically, it is carried out by one or more method steps including filtration and / or centrifugation.

[0042] In one embodiment, the mixture is filtered through a filter that removes macromolecular contaminants and cellular debris but allows AAV to pass through. In one embodiment, released viral particles may be separated from the cell culture medium and purified using clarification. Clarification can be a microfiltration process in which relatively larger components, such as lysed cells and / or impurities, are removed from the solution. Clarification filters include deep filtration, charged deep filtration, and similar microfiltration techniques.

[0043] Tangential flow filtration can be used to concentrate a mixture of purified virus particles, as well as to remove salts and proteins. Tangential flow filtration (TFF) generally refers to a rapid and efficient method for filtering or purifying a solution containing a target product and / or impurities, during which the solution or liquid stream flows parallel to the filter membrane. Centrifugation may be a slow centrifugation method to remove larger particles, such as cell debris. This can be done, for example, for 10-30 minutes at 10,000-12,000 g. The released viral particles may be found in the supernatant.

[0044] The isolation and / or purification of AAV typically involves one or more of the following process steps: - Clarification - filtration - Dialysis / Dialysis Filtration - Tangential flow filtration - Treatment using nucleases, e.g., RNase and / or DNase. - Treatment using chloroform - Ion exchange chromatography - Affinity chromatography - Hydrophobic interaction chromatography - Centrifugal separation - PEG precipitation.

[0045] In some embodiments, a nuclease, typically an endonuclease, is added to reduce the amount of host cell DNA, for example. It may be added directly to the mixture in the bioreactor before, during, or after lysis. The nuclease may degrade both DNA and RNA. In one embodiment, the endonuclease is a genetically modified endonuclease from Serratia marcescens, marketed under the name Bensonase® (EMD Millipore).

[0046] In various embodiments, ion exchange chromatography is applied for further purification. This may be, for example, an anion exchange chromatography (AEX) or cation exchange chromatography (CEX) step. Such steps are used, for example, to separate host cell proteins, host cell DNA, host cell lipids, washing agents, and impurities related to other processes. The principles of cation and anion exchange chromatography are well known in the art. The sample is packed, and the column is washed with the packing buffer. Finally, the elution buffer is used to elute the sample of interest from the column, and the fraction containing the sample is recovered.

[0047] Other suitable chromatographic methods include hydrophobic interaction chromatography, size exclusion chromatography, or affinity chromatography. Instead of the binding-elution mode, a flow-through mode may also be preferred. Precipitation of AAV or AdV using polyethylene glycol (PEG) is also feasible. Therefore, PEG is added to the viral sample. The molecular weight of PEG is typically about 3,000 g / mol to about 15,000 g / mol. The concentration of PEG in the precipitate solution can be adjusted as needed, for example, to approximately 5% (w / w).

[0048] Chloroform may be added to dissolve lipids and inactivate abundant proteins. Virus particles are unaffected by chloroform and can be subsequently separated by phase separation and / or centrifugation. Salts such as sodium chloride can be removed from virus particles by dialysis. Viruses in a solution can be dialyzed against water or another solution, depending on the impurities to be removed.

[0049] A typical AAV purification process includes clarification, concentration and dialysfiltration using tangential flow filtration, and chromatographic purification using affinity chromatography and ion exchange chromatography. In some processes, ultracentrifugation and gradient ultracentrifugation are used instead of or in addition to chromatography. The final step in AAV purification typically involves concentration and dialysfiltration to a suitable excipient buffer composition and sterile filtrate.

[0050] The present invention is further directed to compositions used in the methods of the present invention, comprising aqueous solutions of alkyldimethylamine oxide and sodium chloride. The concentration of alkyldimethylamine oxide in the composition is typically higher than 1% (w / w), preferably between 10% (w / w) and 30% (w / w), so that when the composition is added to a suspension containing cells to be lysed, the final concentration of alkyldimethylamine oxide is higher than 0.1% (w / w), preferably between 1% (w / w) and 4% (w / w).

[0051] In a preferred embodiment, the detergent used is TDAO, N,N-dimethyltridecylamine N-oxide, and / or LDAO, either as a single component or as a mixture with NaCl. Most preferably, it is a mixture of TDAO and NaCl. In a very preferred embodiment, the composition is an aqueous solution. It may also contain one or more buffering agents. The concentration of salts such as sodium chloride in the composition is typically between 1 mol / l and 6 mol / l, preferably between approximately 3 mol / l and 5 mol / l, such that the final concentration in the mixture having the cell suspension is between 0.05 mol / l and 1 mol / l. In a preferred embodiment, the composition consists only of water, one or more alkyldimethylamine oxides, sodium chloride and / or potassium chloride, and optionally a buffer.

[0052] The present invention is also directed to a kit comprising the composition of the present invention as described above, and a nuclease, preferably an endonuclease, most preferably Benzonase®. The kit typically comprises two containers, for example a bottle, having two components, but it may also comprise further components and thus further containers.

[0053] As the examples show, the method of the present invention is extremely efficient. Using the method of the present invention, at least 80%, most preferably 100%, of cells can be lysed while the virus is released. Cell lysis can be detected, for example, by microscopic imaging. The number of virus particles released using the lysis method of the present invention exceeds the number of virus particles released by Triton X100 or sodium chloride, or even a combination thereof. This is unpredictable. The method and composition of the present invention are particularly suitable for AAV serotypes that are strictly associated with cells.

[0054] For example, in AAV2 suspensions, upstream processes including a cell lysis step were observed to significantly reduce viral titer when using either 0.5% Triton X-100 (T100) or 0.5M NaCl, both at 37°C for 90 minutes (Figure 1). In contrast, much higher viral titers could be achieved when using lysis solutions containing 0.5M sodium chloride and 1% TDAO for the same duration (see Figure 2).

[0055] Another advantage of the present invention is that the washing agent and optionally salts such as sodium chloride used for cell lysis and virus release can be removed using tangential flow filtration. In tangential flow filtration, an ultrafiltration membrane may be used to perform dialysis to purify and concentrate the virus particles and to remove the washing agent.

[0056] The present invention is further illustrated by, but is not limited to, the following figures and examples. All applications, patents, and publications cited above and below, as well as the entire disclosure of U.S. Provisional Patent Application No. 63 / 024,643 filed on 14 May 2020, are incorporated herein by reference.

[0057] example The following examples illustrate practical applications of the present invention. Example 1 - Comparative Example Upstream processes of the AAV2 suspension, including cell lysis steps using various single reagents such as Triton X-100 (T100) or TDAO at various concentrations, or 0.5M NaCl, were all performed at 37°C. After lysis, the samples were centrifuged at 13000g for 5 minutes at different time points, the supernatant was removed, and the samples were stored at -70°C for assay. Physical titer was measured by enzyme-linked immunosorbent assay (ELISA) using the AAV2 Tiration ELISA kit (Progen), followed by the manufacturer's protocol, and genomic titer was measured by quantitative PCR (qPCR) targeting specific sequences within the transfer vector. All reagents and assay protocols were from MilliporeSigma unless otherwise specified. Of all the conditions tested, 0.5M NaCl was the only one capable of releasing significant amounts of AAV2 at physical titer levels of e10 vp / mL and genomic titer levels of e9 gc / mL (Figure 1). Furthermore, time-dependent effects from 30 to 90 minutes after dissolution were observed for all three single reagents, and concentration-dependent effects from 0.5% to 1% were observed for both single washing agents (Figure 1). However, none of the single reagents demonstrated satisfaction with AAV release at typical physical titers of e11 vp / mL and genomic titers of e10 gc / mL. In Figure 1, each bar represents the mean of 2 dilutions × 2 assay wells or 3 assay wells, and the error bars represent the standard deviation.

[0058] Example 2 The same process is carried out using NaCl with a different combination of detergents, as in Example 1. The addition of 1% TDAO to 0.5M NaCl, but without the addition of 1% Triton X-100, was demonstrated to promote a 3- to 9-fold increase in AAV2 dissociation (Figure 2). In addition, a longer dissolution time of 3 hours was evaluated as equivalent to a shorter time of 90 minutes (Figure 2). In Figure 2, each bar represents the mean of 2 dilutions × 2 assay wells or 3 assay wells, and the error bars represent the standard deviation.

[0059] Example 3 Cell lysis performed according to the general procedure of Example 1 was compared using different reagents in terms of turbidity reduction and lysis efficiency. Notably, the combination of 1% TDAO and 0.5% NaCl produced faster and more complete cell lysis compared to single reagents of 0.5M NaCl or 0.5% Triton X-100 alone (Table 2). [Table 2]

[0060] The turbidity values ​​in Table 2 are the average of 1-3 bioreactor × 2 measurements. PT = post-transfection. Before lysis, turbidity was 300-400 NTU for HEK293-derived cells and 500-600 NTU for HEK293T-derived cells. Cell counting was performed using ViCell XR before lysis and 30 minutes after lysis, and % cell lysis was calculated as the ratio of lysed cells to the total number of cells before lysis.

[0061] Example 4 To investigate the optimal concentration of TDAO in combination with 0.5M NaCl, AAV2 dissociation tests were performed using 0–4% TDAO, including an additional 60 minutes of lysis time. As shown in Figure 3, the addition of TDAO to 0.5M NaCl resulted in at least a 3–5-fold increase in AAV2 dissociation, as previously observed, and the optimal concentration of 2% TDAO in combination with 0.5% NaCl was identified. Furthermore, an optimal lysis time of 90 minutes was confirmed, as previously observed, based on genomic titer. When calculating % complete virus particles (vp) as the ratio of genomic titer to physical titer, it should be noted that 2% TDAO also promoted the release of complete virus particles over time, with ~100% complete VP at 90 minutes after lysis, due to an unknown mechanism (Figure 4). In Figure 3, each bar represents the mean of the three assay wells, and the error bar represents the standard deviation. In Figure 4, each bar represents only one data point from a single experiment.

[0062] Example 5 The upstream processes of the AAV9 suspension were carried out as in Example 1, but using both single reagents and dual reagents combined with NaCl. In contrast to the AAV2 results in Figure 1, either the single washing agent TDAO or Triton X-100 enhanced AAV9 dissociation by 2–5 times compared to 0.5M NaCl alone (Figure 5). However, no benefit was observed with the dual reagent using 2% TDAO compared to 1% TDAO, although, similar to the AAV2 results in Figure 2, the combination of 1% TDAO and 0.5M NaCl promoted a 2–17-fold increase in AAV9 dissociation compared to each single reagent (Figure 5). These results demonstrate that AAV9 and AAV2 may interact with different cellular components in different ways, but that the combination of TDAO and NaCl may promote AAV dissociation of these different serotypes. Furthermore, it has been consistent that the combination of TDAO and NaCl promoted the release of complete viral particles with a much higher percentage of complete VP between AAV2 and AAV9 compared to the use of a single reagent. Nevertheless, the underlying mechanisms behind such phenomena remain unknown. In Figure 5, each bar represents the mean of 2 dilutions × 2 assay wells or 3 assay wells, and the error bar represents the standard deviation. In Figure 6, each bar represents only one data point from a single experiment.

Claims

1. A method for lysing cells surrounding adeno-associated virus (AAV) particles, comprising contacting a suspension of the cells with an effective amount of a composition comprising alkyldimethylamine oxide and sodium chloride, which promotes cell lysis and the release of AAV particles from the cells.

2. A method for purifying viral particles from a sample containing cells surrounding adeno-associated virus (AAV) viral particles, a) Contacting a suspension of cells and virus particles with an effective amount of a composition containing alkyldimethylamine oxide and sodium chloride, which promotes cell lysis and release of virus particles from cells. b) Isolating and / or purifying the virus particles. by, The aforementioned method.

3. The method according to claim 2, characterized in that the composition comprises LDAO (lauryldimethylamine-N-oxide) and / or TDAO (tetradecyldimethylamine-N-oxide) and sodium chloride.

4. The method according to claim 2 or 3, characterized in that, in step a), the cells are brought into contact with the composition for 30 to 180 minutes.

5. The method according to any one of claims 2 to 4, characterized in that the virus particles are recombinant adeno-associated virus (AAV) particles.

6. The method according to any one of claims 2 to 5, characterized in that the concentration of alkyldimethylamine oxide in the mixture of the suspension of cells and virus particles and an effective amount of the composition obtained in step a) is between 1% (w / w) and 4% (w / w).

7. The method according to any one of claims 2 to 6, characterized in that the concentration of sodium chloride in the mixture of the suspension of cells and virus particles and an effective amount of the composition obtained in step a) is between 0.05 mol / l and 1 mol / l.

8. The method according to any one of claims 2 to 7, characterized in that the composition is an aqueous solution containing alkyldimethylamine oxide and sodium chloride.

9. The method according to any one of claims 2 to 8, characterized in that the concentration of alkyldimethylamine oxide in the composition is between 10% (w / w) and 30% (w / w).

10. The method according to any one of claims 2 to 9, characterized in that the concentration of sodium chloride in the composition is between 1 mol / l and 6 mol / l.

11. The method according to any one of claims 2 to 10, characterized in that step b) includes a filtration step and / or a centrifugal separation step.

12. The method of the present invention involves the following steps: - Clarification and filtration - Dialysis and diafiltration - Treatment using nucleases, e.g., RNase and / or DNase - Treatment using chloroform - Ion exchange chromatography - Affinity chromatography - Hydrophobic interaction chromatography - Centrifugal separation - PEG precipitation The method according to any one of claims 2 to 11, characterized by including one or more of the following.

13. A composition comprising one or more alkyldimethylamine oxides and sodium chloride, (i) For use in a method for lysing cells surrounding adeno-associated virus (AAV) particles, or (ii) The composition for use in a method for purifying viral particles from a sample containing cells surrounding adeno-associated virus (AAV) viral particles.

14. A composition for use in the method according to claim 11, characterized in that the composition is an aqueous solution containing 10% (w / w) to 30% (w / w) TDAO and / or LDAO and 1 mol / l to 6 mol / l NaCl.

15. A kit comprising the composition according to claim 13 or 14 and a nuclease.

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