[go: up one dir, main page]

WO2025008397A1 - Procédés et compositions pour purifier des particules de virus adéno-associés - Google Patents

Procédés et compositions pour purifier des particules de virus adéno-associés Download PDF

Info

Publication number
WO2025008397A1
WO2025008397A1 PCT/EP2024/068725 EP2024068725W WO2025008397A1 WO 2025008397 A1 WO2025008397 A1 WO 2025008397A1 EP 2024068725 W EP2024068725 W EP 2024068725W WO 2025008397 A1 WO2025008397 A1 WO 2025008397A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
composition
aav
formula
compounds
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/068725
Other languages
English (en)
Inventor
Alice ANTONELLO
Sarah LECHAT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck Patent GmbH
Original Assignee
Merck Patent GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck Patent GmbH filed Critical Merck Patent GmbH
Publication of WO2025008397A1 publication Critical patent/WO2025008397A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
    • C11D1/66—Non-ionic compounds
    • C11D1/72—Ethers of polyoxyalkylene glycols
    • C11D1/721—End blocked ethers
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/48—Medical, disinfecting agents, disinfecting, antibacterial, germicidal or antimicrobial compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material

Definitions

  • the present invention relates to compositions and methods for lysing cells and thus purifying adeno-associated virus particles using a detergent selected from the group of non-ionic surfactants, namely secondary fatty alcohol ethoxylates.
  • Adeno-associated virus have been characterized and developed as a potent viral vector to deliver genes in vitro in cultured cells and also 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).
  • ITRs inverted terminal repeats
  • 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.
  • rAAV recombinant AAV vectors
  • AdV adenoviruses
  • 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.
  • development of baculovirus expression vectors provides another method to produce rAAV viruses in insect Sf9 cells.
  • a cell lysis step is generally required at harvest to release viral particles into the supernatant.
  • typical cell lysis reagents such as TritonTM X-100, TweenTM 20, and NaCI are broadly utilized.
  • serotypes e.g., AAV2
  • viral particles tend to be tightly associated with insoluble cellular components thus limit the efficiency of certain cell lysis reagents in terms of virus release.
  • TritonTM X-100 A degradation product of TritonTM X-100 is 4-tert octylphenol, which mimics the hormone estradiol and leads to harmful effects to the endocrine system of aquatic organisms.
  • OPE octylphenol ethoxylates
  • Annex XIV is a list of banned substances in the Ell.
  • OPE products including TritonTM X-100 detergent
  • ECHA European Chemicals Agency
  • the inventors have surprisingly found that a certain group of sustainable detergents is especially suitable for releasing cell associated viruses.
  • the present invention is thus directed to a method for lysing cells enclosing adeno-associated virus (AAV) particles by contacting a suspension of said cells with an effective amount of a composition comprising one or more compounds of Formula I to promote cell lysis and release of the AAV particles from the cells whereby the viral particles remain unaffected.
  • AAV adeno-associated virus
  • an effective amount is preferably an amount that after contacting with the suspension of cells results at a final overall concentration of compounds according to Formula I above their CMC.
  • this lysis method is part of a method for purifying viruses whereby further isolation or purification steps are performed in addition to the lysis step.
  • the present invention is further directed to a method for purifying viral particles of an adeno-associated virus (AAV) from a sample comprising cells enclosing the viral particles by a) contacting a suspension of the cells and the viral particles with an effective amount of a composition comprising one or more compounds of Formula I to promote cell lysis and release of the AAV particles from the cells b) isolating and/or purifying the AAV particles.
  • AAV adeno-associated virus
  • the suspension is contacted with an effective amount of a composition comprising one or more compounds of Formula I and a salt like sodium chloride either as a combination or subsequently.
  • a salt like sodium chloride is added subsequently, the suspension is typically incubated with the composition for a certain time, preferably 1 to 2 hours, prior to the addition of the one or more salts like sodium chloride.
  • the composition comprises a C11-C15 secondary alcohol ethoxylate according to CAS 68131-40-8 and/or a C12- C14 secondary alcohol ethoxylate according to CAS 84133-50-60.
  • step a) the cells are contacted with the composition for 30 to 180 minutes, preferably for 60 to 120 minutes.
  • the viral particles are AAVs, especially an AAV2, AAV5 or AAV9 serotype.
  • the composition is an aqueous solution comprising one or more compounds of Formula I and a salt like sodium chloride.
  • the composition contains only water, one or more compounds of Formula I, sodium chloride and optionally buffer components.
  • the concentration of the one or more compounds of Formula I in the composition is such that the concentration in the mixture with the cells is between 0.1 % and 4%, preferably between 0.25% to 1 % (w/w).
  • the concentration of the salt like sodium chloride in the composition is such that the concentration in the mixture with the cells is between 0.05 and 1 mol/l, preferably between 0.1 and 1 mol/l.
  • step b) is performed by filtration or centrifugation.
  • the method of the present invention comprises one or more of the following steps:
  • nuclease e.g. RNase and/or DNase
  • the present invention is further directed to a composition comprising one or more compounds of Formula I and a salt like sodium chloride.
  • the composition is an aqueous solution comprising 5 to 30 % (w/w) of a C11-C15 secondary alcohol ethoxylate according to CAS 68131-40-8 and/or a C12-C14 secondary alcohol ethoxylate according to CAS 84133-50-60 and 1 to 6 mol/l of a salt like NaCI, preferably NaCI.
  • composition further comprises a nuclease, preferably a salt tolerant nuclease, most preferred WT Vibrio salmonicida EndA nuclease.
  • the composition has a pH between 6 and 9.
  • the present invention is further directed to a kit comprising the composition of the present invention as well as a nuclease.
  • Figure 1 shows AAV2 viral genome titers after cell lysis with the different detergents. TweenTM 20 and TritonTM X-100 are benchmark comparisons. The lysis procedure included a nuclease and a subsequent step with 0.5 M NaCI. Further details can be found in Example 1 .
  • Figure 2 shows the plot static surface tension as a function of secondary alcohol ethoxylate concentration. This plot is employed to determine the critical micelle concentration of the detergent (CMC). Further details can be found in Example 2.
  • Figure 3 shows the percentage of viable HEK 293 adherent cells after the lysis.
  • TweenTM 20 and TritonTM X-100 are benchmark comparison, the condition without detergent is the control.
  • the lysis procedure included Benzonase® endonuclease and no NaCI was added to the amount present in the cell culture media. The values were calculated based on the total and viable cells counted by the Vi-CELL (Beckmann Coulter). Further details can be found in Example 3.
  • Figure 4 shows the percentage of viable HEK 293 adherent cells after the lysis.
  • TweenTM 20 and TritonTM X-100 are benchmark comparison, the condition without detergent is the control.
  • the lysis procedure included Benzonase® salt tolerant endonuclease and NaCI was added to reach a total concentration of 0.5 M. The values were calculated based on the total and viable cells counted by the Vi-CELL (Beckmann Coulter). Further details can be found in Example 3.
  • Figure 5 shows the percentage of viable HEK 293 suspension cells after the lysis.
  • TweenTM 20 and TritonTM X-100 are benchmark comparison, the condition without detergent is the control.
  • the lysis procedure included Benzonase® endonuclease and no NaCI was added to the amount present in the cell culture media. The values were calculated based on the total and viable cells counted by the Vi-CELL (Beckmann Coulter). Further details can be found in Example 3.
  • Figure 6 shows the percentage of viable HEK 293 suspension cells after the lysis.
  • TweenTM 20 and TritonTM X-100 are benchmark comparison, the condition without detergent is the control.
  • the lysis procedure included Benzonase® salt tolerant endonuclease and NaCI was added to reach a total concentration of 0.5 M. The values were calculated based on the total and viable cells counted by the Vi-CELL (Beckmann Coulter). Further details can be found in Example 3.
  • Figure 7 shows AAV2 viral genome titers after HEK 293 adherent cell lysis with the different detergents. TweenTM 20 and TritonTM X-100 are benchmark comparisons. The lysis procedure included a Benzonase® nuclease and no NaCI was added to the amount present in the cell culture media. Further details can be found in Example 3.
  • Figure 8 shows AAV2 viral genome titers after HEK 293 adherent cell lysis with the different detergents. TweenTM 20 and TritonTM X-100 are benchmark comparisons. The lysis procedure included a Benzonase® salt tolerant endonuclease and NaCI was added to reach a total concentration of 0.5 M. Further details can be found in Example 3.
  • Figure 9 shows AAV5 viral genome titers after HEK 293 suspension cell lysis with the different detergents. TweenTM 20 and TritonTM X-100 are benchmark comparisons. The lysis procedure included a Benzonase® nuclease and no NaCI was added to the amount present in the cell culture media. Further details can be found in Example 3.
  • Figure 10 shows AAV5 viral genome titers after HEK 293 suspension cell lysis with the different detergents. TweenTM 20 and TritonTM X-100 are benchmark comparisons. The lysis procedure included a Benzonase® salt tolerant endonuclease and NaCI was added to reach a total concentration of 0.5 M. Further details can be found in Example 3.
  • Figure 11 shows AAV2 viral genome titers after Sf-RVN® cell lysis with the different detergents. TweenTM 20 and TritonTM X-100 are benchmark comparisons. The lysis procedure included a Benzonase® nuclease and no NaCI was added to the amount present in the cell culture media. Further details can be found in Example 3.
  • Figure 12 shows AAV2 viral genome titers after Sf-RVN® cell lysis with the different detergents. TweenTM 20 and TritonTM X-100 are benchmark comparisons. The lysis procedure included a Benzonase® salt tolerant endonuclease and NaCI was added to reach a total concentration of 0.5 M. Further details can be found in Example 3.
  • Figure 13 Infectivity of AAV2 after the lysis of HEK293 adherent cells with detergents.
  • the lysis procedure included a Benzonase® nuclease and no NaCI was added to the amount present in the cell culture media. The infectivity was determined with the transduction unit assay.
  • Figure 14 Infectivity of AAV2 after the lysis of HEK293 adherent cells with detergents.
  • the lysis procedure included a Benzonase® salt tolerant endonuclease and NaCI was added to reach a total concentration of 0.5 M.
  • the infectivity was determined with the transduction unit assay.
  • Surfactants are defined as “surface active molecules”.
  • surfactant refers to compounds that lower the surface tension between two liquids or between a liquid and a solid. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents, and dispersants.
  • a detergent is defined as a surfactant or a mixture containing one or more surfactants having cleaning properties in dilute solutions. Detergents especially have the ability to permeabilize lipid membranes.
  • Micelles are defined as “surfactant aggregates formed in solution, which exist in equilibrium with the molecules or ions from which they are formed”.
  • the critical micelle concentration (CMC) is defined as “relatively small range of concentrations separating the limit below which virtually no micelles are detected and the limit above which virtually all additional surfactant molecules form micelles”.
  • CMC critical micelle concentration
  • IUPAC. 2008a “Critical Micelle Concentration.” The IUPAC Compendium of Chemical Terminology 1077:2014. / IUPAC. 2008b. “Micelle.” The IUPAC Compendium of Chemical Terminology 1077:3889.). Above the CMC virtually all additional detergents added to the system go to micelles.
  • the value of the CMC for a given detergent in a given medium depends on temperature, pressure, and (sometimes strongly) on the presence and concentration of other surfaceactive substances and electrolytes.
  • Secondary alcohol alkoxylates contain an ethylene oxide chain attached to a secondary alcohol.
  • the secondary alcohol alkoxylates to be used in the method of the present invention are secondary alcohol ethoxylates made from secondary alcohols with 11 to 15 carbons and carrying 7 to 15 ethylene oxide units.
  • An especially preferred group of such secondary alcohol ethoxylates is shown in Formula II comprising 9 ethylene oxide units.
  • Such compounds are commercially available as TergitolTM 15-S-9, The Dow Chemical Company or Deviron® 13-S9, Merck KGaA, Germany.
  • These compounds are preferably used at concentrations above their critical micelle concentration.
  • salts like sodium chloride are salts comprising a metal cation like K + , Na + , Li + , Mg 2+ , Ca 2+ and an anionic component like F SO4 2 ; HPO4 2 ; acetate, Cl’.
  • Preferred are salts comprising monoatomic ions.
  • chloride salts like sodium chloride and potassium chloride, whereby sodium chloride is most preferred.
  • Adeno-associated virus 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.
  • ITR inverted terminal repeat
  • ITR sequences 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).
  • 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 are able to infect (transduce) a wide variety of nondividing 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.
  • wild-type viruses have never been associated with any pathology in humans.
  • scAAV are also within the group of AAVs.
  • Self-complementary adeno-associated vectors 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.
  • 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.
  • 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.
  • the vector is an AAV-9 vector, with AAV-2 derived ITRs.
  • 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.
  • AAV as used herein is intended to also encompass recombinant AAV.
  • 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.
  • 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.
  • 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.
  • prokaryotic e.g., bacterial
  • 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.
  • 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
  • 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.
  • ITR inverted terminal repeat
  • 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.
  • 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.
  • helper functions in order to package the AAV of the invention.
  • these helper functions may be supplied by a herpesvirus.
  • 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).
  • ATCC American Type Culture Collection
  • US Manassas, Va.
  • a buffer or a buffer solution is a solution of a certain pH used to prevent pH changes.
  • buffers are CO2/HCO3 (carbonate), phosphate, HEPES, PIPES, ACES, BES, TES, MOPS and TRIS.
  • capsids might not incorporate any of the transgenes and are referred to as empty capsids or empty AAVs. Additionally, capsids that contain fragments of the transgene are called partial capsids or partial AAVs. 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 or reducing the amount of one or more impurities.
  • impurity 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 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.
  • 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 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.
  • a bioreactor is any vessel or tank in which cells can be cultured. Incubation is typically done under suitable conditions like suitable temperature etc and with a suitable medium for supporting the growth/culturing of the cells. A person skilled in the art is aware of suitable incubation conditions for supporting or maintaining the growth/culturing of cells.
  • the present invention is based on the finding that a certain type of composition comprising at least one or more compounds of Formula I as a detergent is especially suitable for lysing cells and setting free viral particles.
  • AAV vectors can be produced in various cell lines in adherent or suspension cell culture formats using transient transfection, infection, or coinfection 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.
  • a cell lysis step is generally required at harvest to release viral particles into the supernatant. Sometimes, the viral particles tend to be tightly associated with insoluble cellular components which limits the efficiency of certain cell lysis reagents in terms of virus release.
  • the production of cells comprising AAVs is known to a person skilled in the art.
  • 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.
  • suitable seeding numbers before transfection are 0.5 to 2 e6 viable cells per ml.
  • adherent HEK 293 cells typically a lower seeding density is required.
  • 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.
  • adherent or suspension human embryonic kidney (HEK293) cells are transfected with a double or triple DNA plasmid polyethylenimine (PEI) co-precipitation.
  • PEI polyethylenimine
  • the present disclosure provides a method for manufacturing an AAV based viral vector comprising the steps of (i) culturing cells in a bioreactor, (ii) transfecting the cells with plasmids to enable production of the AAV particles, (iii) contacting a mixture of the cells and the viral particles with an effective amount of a composition comprising one or more compounds of Formula I and optionally a salt like sodium chloride to promote cell lysis and release of the viral particles from the cells iv) isolating and/or purifying the viral particles.
  • the cells are lysed, and the viral particles harvested.
  • the cells are dissociated from the bioreactor before the cell lysis process is initiated.
  • the cells are lysed in situ.
  • the cells are contacted with a composition comprising one or more compounds of Formula I and optionally a salt like sodium chloride.
  • the salt like sodium chloride can be added together with one or more compounds of Formula I or in a subsequent step.
  • the lysis solution is added to the bioreactor comprising the suspension of the cells so that a mixture of the cell suspension and the composition is generated.
  • Incubation of the mixture comprising the cells and the composition comprising one or more compounds of Formula I and optionally a salt like sodium chloride is done typically for 30 to 180 minutes, preferably for an incubation time between 60 and 120 minutes. Shorter and longer times may also be appropriate.
  • the pH of the mixture during the incubation can vary in broad range. It can for example be between pH 4 and pH 10, typically it is between pH 6 and pH 9.
  • the temperature of the mixture during incubation can also vary in a broad range. It can for example be between 20 and 37°C.
  • concentration of the one or more compounds of Formula I in the composition is such that it effectively induces cell lysis, that means that after the incubation with the composition under suitable conditions as described above, at least 80%, preferably 100% of the cells are lysed.
  • the overall concentration of the one or more compounds of Formula I in the final mixture with the cells is preferably above its CMC.
  • the concentration in the mixture with the cells is typically between 0.1 % and 4% (w/w), preferably between 0.25% (w/w) and 1 % (w/w).
  • the volume of the composition that is added is smaller than the volume of the cell culture.
  • suitable concentrations of the one or more compounds of Formula I in the composition are between 10 and 30% (w/w).
  • suitable concentrations of a salt like sodium chloride to be added to the cell suspension are between 1 and 6 mol/l, typically around 3 to 5 mol/l, so that the final concentration in the mixture with the cell suspension is between 0.05 and 1 mol/l.
  • a solution of sodium chloride and/or salts like sodium chloride can be added to the cell suspension simultaneously with one or more compounds of Formula I or it can be added subsequently, after a certain incubation time only with the detergent, preferably between 1 and 2 hours.
  • the detergent used is a C11-C15 secondary alcohol ethoxylate according to CAS 68131-40-8 and/or a C12-C14 secondary alcohol ethoxylate according to CAS 84133-50-60, either as single component or as a mixture, e.g., with a salt like NaCI, preferably NaCI.
  • the composition is an aqueous solution. It might also comprise one or more buffers.
  • the preferred pH is between 6 and 9. It has been found that the composition as defined above effectively induces lysis of the cells and that especially viral particles which tend to stick to the cells and the cellular debris can be effectively separated from the cells.
  • the released AAVs can then be isolated and/or purified. This can be done with any methods. Typically, it is done by one or more method steps including filtration and/or centrifugation.
  • the mixture is filtered through a filter that removes large molecule contaminants and cellular debris but that permits AAVs to pass therethrough.
  • the released viral particles can be separated and purified from the cell culture medium using clarification.
  • Clarification can be a microfiltration process in which relatively larger components such as lysed cells and/or impurities are removed from a solution.
  • Clarification filters include depth filtration, charged depth filtration and similar microfiltration techniques.
  • Tangential flow filtration can be used to concentrate the mixture of purified viral particles and to remove salts and proteins.
  • Tangential flow filtration 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.
  • 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.
  • the isolation and/or purification of the AAVs typically includes one or more of the following process steps:
  • nuclease e.g. RNase and/or DNase
  • 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.
  • the endonuclease is a genetically engineered endonuclease from Serratia marcescens that is sold under the name Benzonase® (EMD Millipore).
  • the nuclease is a salt tolerant nuclease, preferably WT Vibrio salmonicida EndA nuclease. Said nuclease is for example described in Altermark B, et al., FEBS J. 274 (2007) 252-63. And Niiranen et al., FEBS J. 275 (2008) 1593-1605.
  • ion exchange chromatography is applied for further purification.
  • This can for example be an anion (AEX) or a cation exchange (CEX) capture chromatography step.
  • AEX anion
  • CEX cation exchange
  • Such a step is used, e.g., to separate the viral particles from host cell proteins, host cell DNA, host cell lipids, detergents, and other process-related impurities.
  • the principles of cation and anion exchange chromatography are well known in the art.
  • the sample is loaded, and the column is washed with a loading buffer. Finally, an elution buffer is used to elute the sample of interest off the column, and fractions containing the sample are collected.
  • Suitable chromatographic methods are hydrophobic interaction chromatography, size exclusion chromatography or affinity chromatography. Instead of bind-elute mode, also flow-through mode can be suitable.
  • PEG polyethylene glycol
  • Chloroform can be added to dissolve lipids and inactivate abundant proteins.
  • the virus particles are not affected by the chloroform which can later be separated by phase separation and/or centrifugation.
  • Salts like 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 impurity that shall be removed.
  • Typical AAV purification processes include clarification, concentration and diaf iltration using tangential flow filtration, chromatography 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 involves concentration and diafiltration into suitable excipient buffer composition and sterile filtration.
  • the present invention is further directed to a composition to be used in the method of the invention comprising an aqueous solution of one or more compounds of Formula I and sodium chloride.
  • concentration of the one or more compounds of Formula I in the composition is typically above 1 % (w/w), preferably between 10% (w/w) and 30% (w/w) so that when the composition is added to the suspension comprising the cells to be lysed, the final concentration of the one or more compounds of Formula I can be adjusted to being above 0.1 % (w/w), preferably between 0.25 and 1 % (w/w).
  • the detergent used is a C11-C15 secondary alcohol ethoxylate according to CAS 68131-40-8 and/or a C12-C14 secondary alcohol ethoxylate according to CAS 84133-50-60, either as single component or as a mixture with salts like NaCI, preferably NaCI.
  • the composition is an aqueous solution. It might also comprise one or more buffers.
  • the concentration of the salt like sodium chloride in the composition is typically between 1 and 6 mol/l, preferably around 3 to 5 mol/l, so that the final concentration in the mixture with the cell suspension is between 0.05 and 1 mol/l.
  • the composition is only made of water, one or more compounds of Formula I, sodium chloride and/or potassium chloride and optionally a buffer.
  • the present invention is also directed to a kit comprising the composition of the invention as described above and a nuclease, preferably an endonuclease, e.g., Benzonase®.
  • the kit typically comprises two containers, e.g., bottles, with the two components but it may also comprise further components and thus further containers.
  • Another advantage of the present invention is that the detergent and optionally a salt like sodium chloride that is used for cell lysis and viral release can be removed using tangential flow filtration. In tangential flow filtration, ultrafiltration membranes can be used to purify and concentrate viral particles and perform diafiltration to remove detergent.
  • AAV2 were produced via Ambr® 15 Advanced Microbioreactor system (Sartorius). HEK293 cells were seeded with 1.0*10 6 cells/mL.
  • the cells grew 24 hours until they reached 2.0*10 6 cells/mL. At this point the cells were transfected with a triple DNA plasmid polyethylenimine (PEI) co-precipitation.
  • PEI polyethylenimine
  • the cells were lysate with the detergents and the nuclease, Benzonase® (Merck KGaA Darmstadt, Germany).
  • TritonTM X-100 TritonTM X-100
  • TweenTM 20 secondary alcohol ethoxylate
  • the samples were centrifuged post lysis, and the supernatants were removed and stored at -70 °C for the essays.
  • the critical micelle concentration of the C11-C15 secondary alcohol ethoxylate is determined at different temperatures and in different media.
  • the CMC is determined by measuring the static surface tension of several surfactant solutions at different concentrations. The static surface tension values are plotted in function of the logarithm of the surfactant concentration. The CMC corresponds to the concentration point (or narrow range) at which a sharp change of the slope occurs. Further details of the method are provided in the guideline ISO 4311 (ISO 1979).
  • the static surface tension is measured by using a Force Tensiometer K100C (Kruss GmbH). The Wilhelmy plate was employed for the measurement.
  • Measurement temperature adjusted by a Peltier temperature controller. Measurement time: 300 s
  • the data are acquired until the standard deviation of the surface tension measured is less than 0.1 mN/m from its average value for 50 data points.
  • the solution is poured in the glass vessel of the force tensiometer K100C. The solution is allowed to reach the selected temperature. Just before starting the measurement, the Wilhelmy plate is flamed until glowing red to eliminate contaminations and assure a contact angle equal to zero.
  • This device is equipped with two automatic dispensers. One dispenses the liquid and the other extracts the same volume. Instead of preparing manually several solutions, only a highly concentrated solution is required as starting point. The device proceeds to execute progressive dilutions and measures the static surface tension of each one, plotting the data in a graph static surface tension vs concentration.
  • Method 3 AAV2 and AAV5 release from HEK 293 and Sf-RVN cells by cell
  • HEK293 suspension cells were selected to produce AAV5, HEK293 adhesion cells for AAV2 and Sf-RVN® cells for AAV2. Each cell line was cultivated according to their type with the following procedures.
  • Suspension HEK 293 cells (clone AC2) were seeded on Day 0 in a 500 mL baffled vented Erlenmeyer flask with 1.5x10 6 vc/mL in 125 mL of proprietary cell culture media (Virus Express Excell HEK293 CD Viral Vector Medium, Merck KGaA Darmstadt Germany). On Day 1 the cells reached a VCD > 3 x 10 6 vc/mL and they were transfected with a triple DNA plasmid and polyethylenimine (PEI). The cells were monitored and let grow for two days. Harvest took place on Day 4.
  • PEI polyethylenimine
  • Sf-RVN® cells were cultivated with the proprietary cell culture media (EXCELL CD Insect Cell Culture Medium, Merck KGaA Darmstadt Germany) in 1 L bioreactor (Applikon). The seeding density was 2x10 6 vc/mL in 750 mL medium. The cells were infected with Baculovirus vectors at the time of seeding. Cells were cultivated for 4 days and harvested.
  • NaCI content NaCI already present in the cell culture media (125 mM) in the experiments with Benzonase® endonuclease (Merck KGaA Darmstadt Germany; in the experiments with Salt tolerant Benzonase® endonuclease (Merck KGaA Darmstadt Germany) NaCI was added to reach a concentration of 500 mM.
  • virus-containing supernatant was clarified by centrifugation.
  • the supernatant was analyzed to determine genome titer (viral genome copies/mL) by digital PCR.
  • the infectivity of AAV2 produced by HEK293 adhesion cells was tested with the transduction unit assay. Before the infectivity evaluation, the residual detergent was removed from the samples using a detergent removal column (Pierce).
  • Example 1 AAV2 titer determination after cell lysis
  • the AAV2 genome titers were measured with ddPCR (droplet digital PCR) after the lysis steps and the centrifugation.
  • Figure 1 the genome titers after the lysis with the different detergents are reported.
  • the critical micelle concentration is determined according to Method 2.
  • the surface tension values measured for each dilution are plotted in a graph static surface tension vs concentration.
  • the concentration axis is in logarithmic base.
  • the critical micelle concentration is the concentration (or the narrow range of concentrations) corresponding to the beginning of the micelle self-assembly. From this concentration (or narrow range) on, an increase of surfactant concentration does not affect the surface tension and the plot of surface tension vs concentration is a horizontal line.
  • the CMC corresponds to the concentration point (or narrow range) at which a sharp change of the slope occurs.
  • the CMC of the secondary alcohol ethoxylate in Milli-Q® water at 23.9 °C is equal to 49 mg/L.
  • Example 3 AAV2 and AAV5 release from HEK 293 and Sf-RVN cells by cell lysis
  • the cell lysis efficacy of the detergents was evaluated by counting the total and living cells in each sample. This determination was performed automatically with the Vi-CELL for both HEK293 cell lines and manually evaluating microscope images for the Sf-RVN® cells. With all the cell lines, the lysis of most of the cells was evident from the microscope images, confirming the effective lysis performance of the secondary alcohol ethoxylate, comparable to or better than the benchmarks, TritonTM X-100 and TweenTM 20.
  • HEK293 cells adhered and suspension after lysis are shown in Figure 3, 4, 5, 6.
  • the percentage is calculated based on the automated counting performed by the software, so should be considered as a trend with the actual viable percentage likely to be lower because the software may count lysed cells as still viable.
  • the percentage cell viability data confirmed as well that the secondary alcohol ethoxylate lyses the cells comparably well if not better than the benchmarks, TritonTM X-100 and TweenTM 20. Comparing the experiments with different NaCI concentration, the increase of NaCI concentration increased the lysis efficiency also without detergent due to the osmosis.
  • the genome titer was determined with digital PCR. Figures 7, 8, 9, 10,11 ,12 display that the secondary alcohol ethoxylate was as efficient as the benchmarks in releasing capsids (data not shown) and the viral genome titers were comparably high, at all NaCI concentrations.
  • the AAV Following cellular release and purification, the AAV must be functional to be an effective product.
  • the infectivity of the AAV is a critical quality requirement. The infectivity was determined with a transduction unit assay and the results demonstrate that the secondary alcohol ethoxylate preserves the infectivity of the AAV. ( Figure 13 and 14)

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des compositions et des procédés de lyse de cellules et d'isolement et/ou de purification de particules de virus adéno-associés à l'aide d'un détergent choisi dans le groupe des éthoxylates d'alcools gras secondaires.
PCT/EP2024/068725 2023-07-05 2024-07-03 Procédés et compositions pour purifier des particules de virus adéno-associés Pending WO2025008397A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP23306130 2023-07-05
EP23306130.8 2023-07-05

Publications (1)

Publication Number Publication Date
WO2025008397A1 true WO2025008397A1 (fr) 2025-01-09

Family

ID=87934015

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/068725 Pending WO2025008397A1 (fr) 2023-07-05 2024-07-03 Procédés et compositions pour purifier des particules de virus adéno-associés

Country Status (1)

Country Link
WO (1) WO2025008397A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020023612A1 (fr) * 2018-07-24 2020-01-30 Voyager Therapeutics, Inc. Systèmes et méthodes de production de formulations de thérapie génique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020023612A1 (fr) * 2018-07-24 2020-01-30 Voyager Therapeutics, Inc. Systèmes et méthodes de production de formulations de thérapie génique

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ALTERMARK B ET AL., FEBS J., vol. 274, 2007, pages 252 - 63
BERA A ET AL: "Enhanced oil recovery by nonionic surfactants considering micellization, surface, and foaming properties", PETROLEUM SCIENCE, vol. 14, no. 2, 8 April 2017 (2017-04-08), pages 362 - 371, XP036242329, ISSN: 1672-5107, [retrieved on 20170408], DOI: 10.1007/S12182-017-0156-3 *
IUPAC: "Critical Micelle Concentration", THE IUPAC COMPENDIUM OF CHEMICAL TERMINOLOGY 1077:2014. / IUPAC, no. 84133-50-60, 2008
MICELLE, THE IUPAC COMPENDIUM OF CHEMICAL TERMINOLOGY, vol. 1077, pages 3889
MUZYCZKA, CURR. TOP. MICRO. IMMUNOL., vol. 158, 1992, pages 97 - 129
NIIRANEN ET AL., FEBS J., vol. 275, 2008, pages 1593 - 1605

Similar Documents

Publication Publication Date Title
US20240287467A1 (en) Methods for purification of recombinant aav vectors
JP6165752B2 (ja) アデノ随伴ウイルスの産生のための細胞株
WO2019241535A2 (fr) Chromatographie d'échange d'anions pour la production de vaa recombinants
EP3787771A1 (fr) Procédé de clarification évolutif pour la production d'aav recombinants
WO2016128407A1 (fr) Purification de particules de virus adéno-associé recombinant par chromatographie échangeuse d'anions en plusieurs étapes
US20230183656A1 (en) Methods and compositions for purifying adeno associated virus particles or adenoviruses
WO2025008397A1 (fr) Procédés et compositions pour purifier des particules de virus adéno-associés
EP4602172A1 (fr) Procédés de chromatographie pour la purification de capsides d'aav
KR20250036743A (ko) 재조합 aav 입자를 생산하는 방법
WO2024206396A1 (fr) Procédés de production de virus adéno-associé recombiné (raav)
WO2025252480A1 (fr) Procédé de purification d'adn plasmidique
RU2588387C2 (ru) Улучшенные способы очистки векторов на основе рекомбинантного aav
HK1227432A1 (en) Improved methods for purification of recombinant aav vectors
HK1227432A (en) Improved methods for purification of recombinant aav vectors

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24739508

Country of ref document: EP

Kind code of ref document: A1