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EP-4739783-A1 - METHODS AND COMPOSITIONS FOR PURIFYING ADENO ASSOCIATED VIRUS PARTICLES

EP4739783A1EP 4739783 A1EP4739783 A1EP 4739783A1EP-4739783-A1

Abstract

The present invention relates to compositions and methods for lysing cells and isolating and/or purifying adeno-associated virus particles using a detergent selected from the group of secondary fatty alcohol ethoxylates.

Inventors

  • ANTONELLO, Alice
  • LECHAT, Sarah

Assignees

  • Merck Patent GmbH

Dates

Publication Date
20260513
Application Date
20240703

Claims (15)

  1. 1 . 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 with with x = 7 to 15 and n + m = 8 to 12.
  2. 2. A method for purifying viral particles of 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 viral particles from the cells b) isolating and/or purifying the viral particles.
  3. 3. Method according to claim 2 characterized in that the composition comprises one or more compounds of Formula I and one or more salts selected from salts comprising a metal cation selected from K + , Na + , Li + , Mg 2+ , Ca 2+ and an anionic component selected from F SO4 2 ; HPO4 2 ; acetate, Cl- and/or that said salts are added to the suspension of the cells subsequent to said composition.
  4. 4. Method according to claim 2 or claim 3, characterized in that in step a) the cells are contacted with the composition for 30 to 180 minutes.
  5. 5. Method according to one or more of claims 2 to 4, characterized in that 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.
  6. 6. Method according to one or more of claims 2 to 5, characterized in that the overall concentration of the one or more compounds of formula I in the mixture obtained in step a) is between 0.1 and 4% (w/w).
  7. 7. Method according to one or more of claims 2 to 6, characterized in that the overall concentration of the one or more salts comprising a metal cation selected from K + , Na + , Li + , Mg 2+ , Ca 2+ and an anionic component selected from F SO4 2 ; HPO4 2 ; acetate, Cl’ in the mixture obtained in step a) is between 0.05 and 1 mol/l.
  8. 8. Method according to one or more of claims 2 to 7, characterized in that the composition is an aqueous solution comprising one or more compounds according to Formula I and sodium chloride.
  9. 9. Method according to one or more of claims 2 to 8, characterized in that the overall concentration of the one or more compounds according to Formula I oxide in the composition is between 10% and 30% (w/w).
  10. 10. Method according to one or more of claims 2 to 9, characterized in that the concentration of sodium chloride in the composition is between 1 and 6 mol/l.
  11. 11 . Method according to one or more of claims 2 to 10, characterized in that step b) comprises a filtration and/or centrifugation step.
  12. 12. Method according to one or more of claims 2 to 11 , characterized in that the method of the present invention comprises one or more of the following steps: - Clarification and filtration - dialysis and diafiltration - treatment with nuclease, e.g., RNase and/or DNase - treatment with chloroform - ion exchange chromatography - affinity chromatography - hydrophobic interaction chromatography - centrifugation - PEG precipitation
  13. 13. A composition comprising one or more compounds according to Formula I and a salt comprising a metal cation selected from K + , Na + , Li + , Mg 2+ , Ca 2+ and an anionic component selected from F SO4 2 ; HPO4 2 ; acetate, Cl’
  14. 14. A composition according to claim 13, characterized in that the composition is an aqueous solution comprising 10 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 NaCI.
  15. 15. A kit comprising the composition according to claim 13 or claim 14 as well as a nuclease.

Description

Methods and compositions for purifying adeno associated virus particles 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 (AAV) 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). Naturally isolated serotypes 1-9 of the AAV viruses share the genomic structure although these serotypes may display different tissue tropism. As the AAVs seem to be nonpathogenic, show an efficient transduction and a stable expression, they are regarded as being one of the most promising gene delivery vehicles. AAV vectors can be produced in various cell lines in adherent or suspension cell culture formats using transient transfection or co-infection methods. Depending on specific serotypes and production times, viral particles including full, partial and empty species can be secreted out of cells into culture medium or contained inside cells at various ratios. Initially, stable AAV producer cells were generated by transfection and selection of human-derived cells, like HeLa or HEK293 cells, with an rAAV transfer vector containing the ITR cassette and a packaging construct containing Rep and Cap. Production of recombinant AAV vectors (rAAV) was then achieved by infection with auxiliary viruses such as adenoviruses (AdV) that provide the helper function. After identification of AdV genes required for AAV vector packaging, a helper virus-free method was established using a duo or triple transfection protocol consisting of two or three plasmids including a constructed helper plasmid instead of an auxiliary virus. This system is widely used in research and drug development. In addition, development of baculovirus expression vectors provides another method to produce rAAV viruses in insect Sf9 cells. These different technologies are shown to be able to produce sufficient quantities of rAAV viruses for use in laboratories and clinical trials. A cell lysis step is generally required at harvest to release viral particles into the supernatant. For this application, typical cell lysis reagents such as Triton™ X-100, Tween™ 20, and NaCI are broadly utilized. However, for certain 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. Current methods of releasing viruses, especially cell-associated viruses, are often time consuming like e.g. physical lysis methods, are difficult to scale, or can introduce unwanted and difficult to remove impurities, e.g., Triton™ X-100. A degradation product of Triton™ X-100 is 4-tert octylphenol, which mimics the hormone estradiol and leads to harmful effects to the endocrine system of aquatic organisms. In 2017, octylphenol ethoxylates (OPE) were added to the European Authorization list (Annex XIV) of REACH (Registration, Evaluation, Authorization and Restrictions of Chemicals). Annex XIV is a list of banned substances in the Ell. After January 2021 , OPE products, including Triton™ X-100 detergent, are prohibited in the Ell by the European Chemicals Agency (ECHA), unless authorization was granted by the authorities, or the intended use is exempt from authorization. Therefore, it would be beneficial to have an effective and sustainable reagent and method to lyse the cells and isolate and/or purify viruses like AAV from the cells in which they are produced. 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. Formula I with x = 7 to 15 and n + m = 8 to 12 Preferably x= 8 to 10, in a very preferred embodiment x = 9. 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. Typi