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Manufacturing and characterizing AAV-based vectors for use in clinical studies

Gene Therapy volume 15, pages 840–848 (2008)Cite this article

Abstract

Recombinant adeno-associated virus (AAV)-based vectors expressing therapeutic gene products have shown great promise for human gene therapy. A major challenge for translation of promising research to clinical development is the manufacture and certification of AAV vectors for clinical use. This review summarizes relevant aspects of current Good Manufacturing Practice, focusing on considerations and challenges specific for recombinant AAV.

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References

  1. Carter BJ . Adeno-associated virus vectors in clinical trials. Hum Gene Ther 2005; 16: 541–550.

    Article  CAS  Google Scholar 

  2. Warrington KH, Herzog RW . Treatment of human disease by adeno-associated viral gene transfer. Hum Genet 2006; 119: 571–603.

    Article  CAS  Google Scholar 

  3. Fiandaca M, Forsayeth J, Bankiewicz K . Current status of gene therapy trials for Parkinson's disease. Exp Neurology 2008; 209: 51–57.

    Article  CAS  Google Scholar 

  4. 21CFR parts 120 and 211—current good manufacturing practice in manufacturing, processing, packing or holding of drugs general and current good manufacturing practice for finished pharmaceuticals. http://www.gpoaccess.gov/cfr/index.html.

  5. US Dept Health Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research. Guidance for FDA and sponsors: content and review of Chemistry, Manufacturing, and Control (CMC) information for human gene therapy Investigational New Drug applications (INDs). November, 2004. http://www.fda.gov/cber/guidelines.htm.

  6. US Dept Health Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research. Guidance for Industry: INDs—approaches to complying with CGMP during Phase 1. January 2006. http://www.fda.gov/cber/guidelines.htm.

  7. US Center for Drugs and Biologics, Center for Devices and Radiological Health, Food and Drug Administration. Guideline on general principles of process validation. May, 1987. http://www.fda.gov/cber/guidelines.htm.

  8. US Dept Health Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research. Guidance for industry: analytical procedures and methods validation. Chemistry, manufacturing and controls documentation. August 2000. http://www.fda.gov/cber/guidelines.htm.

  9. US Dept Health Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research, Center for Veterinary Medicine, Office of Regulatory Affairs. Guidance for industry: quality systems approach to pharmaceutical CGMP regulations. September 2006. http://www.fda.gov/cber/guidelines.htm.

  10. PDA. Points to consider for aseptic processing. J Pharm Sci Technol 2003; 57: 1–72.

    Google Scholar 

  11. US Dept Health Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research. Guidance for industry: Q8 pharmaceutical development. May 2006. http://www.fda.gov/cber/guidelines.htm.

  12. Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko J et al. Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response. Nat Med 2006; 12: 342–347.

    Article  CAS  Google Scholar 

  13. Mingozzi F, Maus MV, Hui DJ, Sabatino DE, Murphy SL, Rasko JE et al. CD8+ T-cell responses to adeno-associated virus capsid in humans. Nat Med 2007; 13: 419–422.

    Article  CAS  Google Scholar 

  14. US Dept Health Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research, Center for Veterinary Medicine. Guidance for industry: comparability protocols—protein drug products and biological products—Chemistry, Manufacturing, and Controls information. September 2003. http://www.fda.gov/cber/guidelines.htm.

  15. Grimm D, Kern A, Rittner K, Kleinschmidt JA . Novel tools for production and purification of recombinant adenoassociated virus vectors. Hum Gene Ther 1998; 9: 2745–2760.

    Article  CAS  Google Scholar 

  16. Matsushita T, Elliger S, Elliger C, Podsakoff G, Villarreal L, Kurtzman GJ et al. Adeno-associated virus vectors can be efficiently produced without helper virus. Gene Therapy 1998; 5: 938–945.

    Article  CAS  Google Scholar 

  17. Clark KR, Voulgaropoulou F, Fraley DM, Johnson PR . Cell lines for the production of recombinant adeno-associated virus. Hum Gene Ther 1995; 6: 1329–1341.

    Article  CAS  Google Scholar 

  18. Xiao X, Li J, Samulski RJ . Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 1998; 72: 2224–2232.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Clark KR, Liu X, McGrath JP, Johnson PR . Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type virus. Hum Gene Ther 1999; 10: 1031–1039.

    Article  CAS  Google Scholar 

  20. Conway JE, Rhys CMJ, Zolotukhin I, Zolotukhin S, Muzyczka N, Hayward GS et al. High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type 1 vector expressing AAV-2 Rep and Cap. Gene Therapy 1999; 6: 986–993.

    Article  CAS  Google Scholar 

  21. Urabe M, Ding C, Kotin RM . Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum Gene Ther 2002; 13: 1935–1943.

    Article  CAS  Google Scholar 

  22. Negrete A, Yang LC, Mendez AF, Levy JR, Kotin RM . Economized large-scale production of high yield of rAAV for gene therapy applications exploiting baculovirus expression system. J Gene Med 2007; 9: 938–948.

    Article  CAS  Google Scholar 

  23. US Dept Health Human Services, Food and Drug Administration. International conference on harmonisation; guidance on viral safety evaluation of biotechnology products derived from cell lines of human or animal origin. Fed Regist 1998; 63: 51074–51084. http://www.fda.gov/cber/gdlns/virsafe.pdf.

    Google Scholar 

  24. Muzyczka N . Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Top Microbiol Immunol 1992; 158: 97–129.

    CAS  PubMed  Google Scholar 

  25. Allen JM, Debelak DJ, Reynolds TC, Miller AD . Identification and elimination of replication-competent adeno-associated virus (AAV) that can arise by non-homologous recombination during AAV vector production. J Virol 1997; 71: 6816–6822.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Samulski RJ, Shang L-S, Shenk T . Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol 1989; 61: 3096–3101.

    Google Scholar 

  27. Smith PH, Wright JF, Qu G, Patarroyo-White S, Parker A, Sommer JM . Packaging of host cell and plasmid DNA into recombinant adeno-associated virus particles produced by triple transfection. Mol Ther 2003; 7: S348.

    Google Scholar 

  28. Chadeuf G, Ciron C, Moullier P, Salvetti A . Evidence for encapsidation of prokaryotic sequences during recombinant adeno-associated virus production and their in vivo persistence after vector delivery. Mol Ther 2005; 12: 744–753.

    Article  CAS  Google Scholar 

  29. European Medicines Agency. Report from the CHMP Gene Therapy Expert Group meeting. European Medicines Agency 2005; EMEA/CHMP/183989/2004. http://www.emea.europa.eu/pdfs/human/genetherapy/18398904en.pdf.

  30. Hauck B, Murphy S, Liu X, Zelenaia O, High KA, Wright JF . Trace capsid DNA impurities in AAV2 vectors are not transcribed and are unlikely to contribute to capsid peptide-MHC class I complexes. Blood 2007; 110: 090A.

    Google Scholar 

  31. World Health Organization. Requirements for the use of animal cells as in vitro substrates for the production of biologicals (Requirement for Biological substance No. 50). In: WHO Expert Committee on Biological Standardization. Geneva, 1998, Annex 1 (WHO Technical Report Series, No. 878).

  32. Sommer JM, Smith PH, Parthasarathy S, Isaacs J, Vijay S, Kieran J et al. Quantification of adeno-associated virus particles and empty capsids by optical density measurement. Mol Ther 2003; 7: 122–128.

    Article  CAS  Google Scholar 

  33. Schultz RB, Gregorevic P, Finn E, Doremus C, Meuse L, Haraguchi M et al. Intravenous injection cofactors of adeno-associated virus produce varying tissue-specific transduction levels in adult mice. Mol Ther 2007; 15: S276.

    Google Scholar 

  34. Parker A, Nagy D, Vargas J, Anand V, Qu G, Sommer J et al. In vivo performance of AAV2 vectors purified by CsCl gradient centrifugation or column chromatography. Mol Ther 2003; 7: S390.

    Google Scholar 

  35. Pien GC, Hasbrouck NC, Maus MV, Mingozzi F, High KA . Quantifying capsid peptide: MHC I complexes following adeno-associated virus (AAV) transduction. Blood 2007; 110: 1090A.

    Article  Google Scholar 

  36. Sadana A (ed) Bioseparation of Proteins. Academic Press: New York, 1998.

    Google Scholar 

  37. Belter PA, Cussler CL, Hu WS . Bioseparations. Wiley: New York, 1988.

    Google Scholar 

  38. Zhen Z, Espinoza Y, Bleu T, Sommer JM, Wright JF . Infectious titer assay for adeno-associated virus vectors with sensitivity sufficient to detect single infectious events. Hum Gene Ther 2004; 15: 709–715.

    Article  CAS  Google Scholar 

  39. Sommerford C, Samulski RJ . Membrane-associated heparin sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J Virol 1998; 72: 1438–1445.

    Google Scholar 

  40. Gao G, Qu G, Burnham MS, Huang J, Chirmule N, Joshi B et al. Purification of recombinant adeno-associated virus vectors by column chromatography and its performance in vivo. Hum Gene Ther 2000; 11: 2079–2091.

    Article  CAS  Google Scholar 

  41. Snyder RO, Flotte TR . Production of clinical-grade recombinant adeno-associated virus vectors. Curr Opin Biotechnol 2002; 13: 418–423.

    Article  CAS  Google Scholar 

  42. Chiorini JA, Kim F, Yang L, Kotin RM . Cloning and characterization of adeno-associated virus type 5. J Virol 1999; 73: 1309–1319.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Rutledge EA, Halbert CL, Russell DW . Infectious clones and vectors derived from adeno-associated virus (AAV) other than AAV type 2. J Virol 1998; 72: 309–319.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Walters RW, Yi SMP, Keshavjee S, Brown KE, Welsh MJ, Chiorini JA et al. Binding of adeno-associated virus type 5 to 2,3-linked sialic acid is required for gene transfer. J Biol Chem 2001; 276: 20610–20616.

    Article  CAS  Google Scholar 

  45. Brument N, Morenweiser R, Blouin V, Toublance E, Raimbaud I, Cherel Y et al. A versatile and scalable two-step ion-exchange chromatography process for the purification of recombinant adeno-associated virus serotypes-2 and -5. Mol Ther 2002; 6: 678–686.

    Article  CAS  Google Scholar 

  46. Kaludov N, Handelman B, Chiorini JA . Scalable purification of adeno-associated virus type 2, 4, or 5 using ion-exchange chromatography. Hum Gene Ther 2002; 13: 1235–1243.

    Article  CAS  Google Scholar 

  47. Zolotukhin S, Byrne BJ, Mason E, Zolotukhin I, Potter M, Chestnut K et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Therapy 1999; 6: 973–985.

    Article  CAS  Google Scholar 

  48. Potter M, Chestnut K, Muzyczka N, Flotte T, Zolotukhin S . Stream-lined large-scale production of recombinant adeno-associated virus (rAAV) vectors. Methods Enzymol 2002; 346: 413–430.

    Article  CAS  Google Scholar 

  49. Qu G, Bahr-Davidson J, Prado J, Tai A, Cataniag F, McDonnell J et al. Separation of adeno-associated virus type 2 empty particles from genome containing vector by anion-exchange column chromatography. J Virol Meth 2007; 140: 183–192.

    Article  CAS  Google Scholar 

  50. Smith RH, Ding C, Kotin RM . Serum-free production and column purification of adeno-associated virus type 5. J Virol Meth 2003; 114: 115–124.

    Article  CAS  Google Scholar 

  51. Davidoff AM, Ng CY, Sleep S, Gray J, Azam S, Zhao Y et al. Purification of recombinant adeno-associated virus type 8 vectors by ion exchange chromatography generates clinical grade vector stocks. J Virol Meth 2004; 121: 209–215.

    Article  CAS  Google Scholar 

  52. Offit PA . The Cutter Incident—How America's First Polio Vaccine Led to the Growing Vaccine Crisis. Yale University Press: New Haven and London, 2005.

    Google Scholar 

  53. US Dept Health Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research. Guidance for Industry: Nonclinical studies for the safety evaluation of pharmaceutical excipients. May 2005. http://www.fda.gov/cber/guidelines.htm.

  54. Wright JF, Qu G, Tang C, Sommer JM . Recombinant adeno-associated virus: formulation challenges and strategies for a gene therapy vector. Curr Op Drug Disc Dev 2003; 6: 174–178.

    CAS  Google Scholar 

  55. Wright JF, Le T, Prado J, Bahr-Davidson J, Smith PH, Zhen Z et al. Identification of factors that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during vector purification and formulation. Mol Ther 2004; 12: 171–178.

    Article  Google Scholar 

  56. Maecker HT, Ghanekar SA, Suni MA, He XS, Picker LJ, Maino VC . Factors affecting the efficiency of CD8+ T cell cross-priming with exogenous antigens. J Immunol 2001; 166: 7268–7275.

    Article  CAS  Google Scholar 

  57. Bennicelli J, Wright JF, Komaromy A, Jacobs JB, Hauck B, Zelenaia O et al. Reversal of visual deficits in animal models of Leber congenital amaurosis within weeks after treatment using optimized AAV2-mediated gene transfer. Mol Ther 2008; 16: 458–465.

    Article  CAS  Google Scholar 

  58. US Dept Health Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research, Office of Regulatory Affairs. Guidance for industry: sterile drug products produced by aseptic processing—current good manufacturing practice. September 2004. http://www.fda.gov/cber/guidelines.htm.

  59. US Dept Health Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research. Guidance for industry: container closure systems for packaging human drugs and biologics. May 1999. http://www.fda.gov/cber/guidelines.htm.

  60. Gombold J, Peden K, Gavin D, Wei Z, Baradaran K, Mire-Sluis A et al. Lot release and characterization testing of live-virus-based vaccines and gene therapy products, Part 1: factors influencing assay choices. BioProcess Int 2006; 4: 46–56.

    CAS  Google Scholar 

  61. Mohiuddin I, Loiler S, Zolotukhin I, Byrne BJ, Flotte TR, Snyder RO . Herpesvirus-based infectious titering of recombinant adeno-associated viral vectors. Mol Ther 2005; 11: 320–326.

    Article  CAS  Google Scholar 

  62. US Dept Health Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research. Guidance for industry: Q1A (R2) stability testing of new drug substances and products. November 2003. http://www.fda.gov/cber/guidelines.htm.

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Acknowledgements

We thank Drs Guang Qu, Olga Zelenaia and Bernd Hauck for helpful discussions and review of the manuscript.

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  1. Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA

    J F Wright

  2. Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA

    J F Wright

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Correspondence to J F Wright.

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Wright, J. Manufacturing and characterizing AAV-based vectors for use in clinical studies. Gene Ther 15, 840–848 (2008). https://doi.org/10.1038/gt.2008.65

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