Vaccine Production: Upstream Processing with Adherent or Suspension Cell Lines

  • Yvonne Genzel
  • Jana Rödig
  • Erdmann Rapp
  • Udo Reichl
Part of the Methods in Molecular Biology book series (MIMB, volume 1104)


The production of viral vaccines in cell culture can be accomplished with primary, diploid, or continuous (transformed) cell lines. Each cell line, each virus type, and each vaccine preparation require the specific design of upstream and downstream processing. Media have to be selected as well as production vessels, cultivation conditions, and modes of operation. Many viruses only replicate to high titers in adherently growing cells, but similar to processes established for recombinant protein production, an increasing number of suspension cell lines is being evaluated for future use. Here, we describe key issues to be considered for the establishment of large-scale virus production in bioreactors. As an example upstream processing of cell culture-derived influenza virus production is described in more detail for adherently growing and for suspension cells. In particular, use of serum-containing, serum-free, and chemically defined media as well as choice of cultivation vessel are considered.

Key words

Vaccine Virus Influenza Large-scale production Microcarrier MDCK AGE1.CR Adherent cells Suspension cells Virus harvest Virus quantification Wave bioreactor Stirred tank bioreactor 



The authors thank N. Wynserski, C. Best, S. König, and I. Behrendt for their excellent technical assistance. The authors would like to thank I. Jordan (ProBioGen AG) for the fruitful discussions on the AGE1.CR cell line and for allowing us to use this cell line. Equally, the authors thank B. Hundt (IDT Biologika GmbH) for the provision of the egg-derived influenza virus.


  1. 1.
    Kaufmann SHE (2004) Novel vaccination strategies. Wiley-VCH, WeinheimCrossRefGoogle Scholar
  2. 2.
    Huang DB, Wu JJ, Tyring SK (2004) A review of licensed viral vaccines, some of their safety concerns, and the advances in the development of investigational viral vaccines. J Infect 49(3):179–209CrossRefGoogle Scholar
  3. 3.
    Aunins JG (2000) Viral vaccine production in cell culture. Encyclopedia of cell technology. Wiley, New YorkGoogle Scholar
  4. 4.
    Feng SZ, Jiao PR, Qi WB et al (2011) Development and strategies of cell-culture technology for influenza vaccine. Appl Microbiol Biotechnol 89(4):893–902CrossRefGoogle Scholar
  5. 5.
    Tree JA, Richardson C, Fooks AR et al (2001) Comparison of large-scale mammalian cell culture systems with egg culture for the production of influenza virus A vaccine strains. Vaccine 19(25–26):3444–3450CrossRefGoogle Scholar
  6. 6.
    Robertson JS, Cook P, Attwell AM et al (1995) Replicative advantage in tissue culture of egg-adapted influenza virus over tissue-culture derived virus: implications for vaccine manufacture. Vaccine 13(16):1583–1588CrossRefGoogle Scholar
  7. 7.
    Govorkova EA, Kodihalli S, Alymova IV et al (1999) Growth and immunogenicity of influenza viruses cultivated in Vero or MDCK cells and in embryonated chicken eggs. Dev Biol Stand 98:39–51, discussion 73–4Google Scholar
  8. 8.
    Perdue ML, Arnold F, Li S, Donabedian A et al (2011) The future of cell culture-based influenza vaccine production. Expert Rev Vaccines 10(8):1183–1194CrossRefGoogle Scholar
  9. 9.
    Shaw A (2012) New technologies for new influenza vaccines. Vaccine 30(33): 4927–4933CrossRefGoogle Scholar
  10. 10.
    Genzel Y, Reichl U (2009) Continuous cell lines as a production system for influenza vaccines. Expert Rev Vaccines 8(12):1681–1692CrossRefGoogle Scholar
  11. 11.
    Barrett PN, Portsmouth D, Ehrlich HJ (2010) Developing cell culture-derived pandemic vaccines. Curr Opin Mol Ther 12(1):21–30Google Scholar
  12. 12.
    Jordan I, Northoff S, Thiele M et al (2011) A chemically defined production process for highly attenuated poxviruses. Biologicals 39(1):508CrossRefGoogle Scholar
  13. 13.
    Lohr V, Genzel Y, Jordan I et al (2012) Live attenuated influenza viruses produced in a suspension process with avian AGE1.CR.pIX cells. BMC Biotechnol 12:79CrossRefGoogle Scholar
  14. 14.
    Lohr V, Genzel Y, Behrendt I et al (2010) A new MDCK suspension line cultivated in a fully defined medium in stirred-tank and wave bioreactor. Vaccine 28(38):6256–6264CrossRefGoogle Scholar
  15. 15.
    Schwarzer J, Rapp E, Hennig R et al (2009) Glycan analysis in cell culture-based influenza vaccine production: influence of host cell line and virus strain on the glycosylation pattern of viral hemagglutinin. Vaccine 27(32): 4325–4336CrossRefGoogle Scholar
  16. 16.
    Genzel Y, Behrendt I, Rodig J et al (2013) CAP, a new human suspension cell line for influenza virus production. Appl Microbiol Biotechnol 97(1):111–122CrossRefGoogle Scholar
  17. 17.
    Roedig JV, Rapp E, Bohne J et al (2013) Impact of cultivation conditions on N-glycosylation of influenza virus A hemagglutinin produced in MDCK cell culture. Biotechnol Bioeng. doi: 10.1002/bit.24834 Google Scholar
  18. 18.
    Hutter J, Rodig JV, Hoper D et al (2013) Toward animal cell culture-based influenza vaccine design: viral hemagglutinin n-glycosylation markedly impacts immunogenicity. J Immunol 190(1):220–230CrossRefGoogle Scholar
  19. 19.
    Bahnemann HG (1990) Inactivation of viral antigens for vaccine preparation with particular reference to the application of binary ethylenimine. Vaccine 8(4):299–303CrossRefGoogle Scholar
  20. 20.
    Budowsky EI, Zalesskaya MA (1991) Principles of selective inactivation of viral genome. V. Rational selection of conditions for inactivation of the viral suspension infectivity to a given extent by the action of beta-propiolactone. Vaccine 9(5):319–325CrossRefGoogle Scholar
  21. 21.
    Budowsky EI, Friedman EA, Zheleznova NV et al (1991) Principles of selective inactivation of viral genome. VI. Inactivation of the infectivity of the influenza virus by the action of beta-propiolactone. Vaccine 9(6):398–402CrossRefGoogle Scholar
  22. 22.
    Mahy BWJ, Kangro HO (1996) Virology methods manual. Academic, LondonGoogle Scholar
  23. 23.
    Kalbfuss B, Knochlein A, Krober T et al (2008) Monitoring influenza virus content in vaccine production: precise assays for the quantitation of hemagglutination and neuraminidase activity. Biologicals 36(3):145–161CrossRefGoogle Scholar
  24. 24.
    Schwarzer J, Rapp E, Reichl U (2008) N-glycan analysis by CGE-LIF: profiling influenza A virus hemagglutinin N-glycosylation during vaccine production. Electrophoresis 29(20):4203–4214CrossRefGoogle Scholar
  25. 25.
    Roedig JV, Rapp E, Hoper D et al (2011) Impact of host cell line adaptation on quasispecies composition and glycosylation of influenza A virus hemagglutinin. PLoS One 6(12):e27989CrossRefGoogle Scholar
  26. 26.
    Smith PK, Krohn RI, Hermanson GT et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150(1):76–85CrossRefGoogle Scholar
  27. 27.
    Rödig JV, Rapp E, Hennig R et al (2009) Optimized CGE-LIF-based glycan analysis for high-throughput applications. In: Jenkins N, Barron N, Alves PM (eds) 21st annual meeting of the European society for animal cell culture technology (ESACT). Springer Science+Business Media BV, Dublin, Ireland, pp 599–603Google Scholar
  28. 28.
    MHRA (2007) Rules and guidance for pharmaceutical manufacturers and distributors 2007—the ‘Orange Guide’. Pharmaceutical Press, London, UK. ISBN 9-78-085369719-0Google Scholar
  29. 29.
    European Pharmacopoeia (EP): Maisonneuve S.A., France; continuously updatedGoogle Scholar
  30. 30.
    United States Pharmacopoeia (USP). Rockville, MD, USA: US Pharmacopoeial Convention; continuously updatedGoogle Scholar
  31. 31.
    Points to consider in the characterization of cell lines used to produce biologicals: Department of Health and Human Services, Food and Drug Administration; 1993. Report No.: Docket No. 84N-0154Google Scholar
  32. 32.
    Guidance on viral safety evaluation of biotechnology products derived from cell lines and animal origin: Department of Health and Human Services, Food and Drug Administration; 1998. Report No.: Docket No. 96D-0058Google Scholar
  33. 33.
    Gregersen J-P (1994) Research and development of vaccines and pharmaceuticals from biotechnology. Wiley-VCH, Weinheim, GermanyCrossRefGoogle Scholar
  34. 34.
    Mohler L, Flockerzi D, Sann H et al (2005) Mathematical model of influenza A virus production in large-scale microcarrier culture. Biotechnol Bioeng 90(1):46–58CrossRefGoogle Scholar
  35. 35.
    Genzel Y, Reichl U (2007) Vaccine production: state of the art and future needs. In: Pörtner R (ed) Animal cell biotechnology—methods and protocols, 2nd edn. Humana Press, New York, US, pp 457–474Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Yvonne Genzel
    • 1
  • Jana Rödig
    • 1
  • Erdmann Rapp
    • 1
    • 2
  • Udo Reichl
    • 1
    • 3
  1. 1.Max Planck Institute for Dynamics of Complex Technical SystemsMagdeburgGermany
  2. 2.glyXera GmbHMagdeburgGermany
  3. 3.Lehrstuhl für BioprozesstechnikOtto-von-Guericke-Universität MagdeburgMagdeburgGermany

Personalised recommendations