Continuous influenza virus production in a tubular bioreactor system provides stable titers and avoids the “von Magnus effect”


Autoři: Felipe Tapia aff001;  Daniel Wohlfarth aff001;  Volker Sandig aff002;  Ingo Jordan aff002;  Yvonne Genzel aff001;  Udo Reichl aff001
Působiště autorů: Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany aff001;  ProBioGen AG, Berlin, Germany aff002;  Chair for Bioprocess Engineering, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany aff003
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224317

Souhrn

Continuous cell culture-based influenza vaccine production could significantly reduce footprint and manufacturing costs compared to current batch processing. However, yields of influenza virus in continuous mode can be affected by oscillations in virus titers caused by periodic accumulation of defective interfering particles. The generation of such particles has also been observed previously in cascades of continuous stirred tank reactors (CSTRs) and is known as the “von Magnus effect”. To improve virus yields and to avoid these oscillations, we have developed a novel continuous tubular bioreactor system for influenza A virus production. It was built using a 500 mL CSTR for cell growth linked to a 105 m long tubular plug-flow bioreactor (PFBR). Virus propagation took place only in the PFBR with a nominal residence time of 20 h and a production capacity of 0.2 mL/min. The bioreactor was first tested with suspension MDCK cells at different multiplicities of infection (MOI), and then with suspension avian AGE1.CR.pIX cells at a fixed nominal MOI of 0.02. Maximum hemagglutinin (HA) titers of 2.4 and 1.6 log10(HA units/100 μL) for suspension MDCK cells and AGE1.CR.pIX cells, respectively, were obtained. Flow cytometric analysis demonstrated that 100% infected cells with batch-like HA titers can be obtained at a MOI of at least 0.1. Stable HA and TCID50 titers over 18 days of production were confirmed using the AGE1.CR.pIX cell line, and PCR analysis demonstrated stable production of full-length genome. The contamination level of segments with deletions (potentially defective interfering particles), already present in the virus seed, was low and did not increase. Control experiments using batch and semi-continuous cultures confirmed these findings. A comparison showed that influenza virus production can be achieved with the tubular bioreactor system in about half the time with a space-time-yield up to two times higher than for typical batch cultures. In summary, a novel continuous tubular bioreactor system for cell culture-based influenza virus production was developed. One main advantage, an essentially single-passage amplification of viruses, should enable efficient production of vaccines as well as vectors for gene and cancer therapy.

Klíčová slova:

Flow rate – Genetic oscillators – Influenza – Influenza A virus – Influenza viruses – Stainless steel – Viral replication – Batch culture


Zdroje

1. Bouvier NM, Palese P. The biology of influenza viruses. Vaccine. 2008;26(4): D49–53.

2. World Health Organization Note. Influenza virus infections in humans. 2014. Available from: https://www.who.int/influenza/vaccines/en/.

3. McLean KA, Goldin S, Nannei C, Sparrow E, Torelli G. The 2015 global production capacity of seasonal and pandemic influenza vaccine. Vaccine. 2016;34(45): 5410–13. doi: 10.1016/j.vaccine.2016.08.019 27531411

4. Perdue ML, Arnold F, Li S, Donabedian A, Cioce V, Warf T, et al. The future of cell culture-based influenza vaccine production. Expert Rev Vaccines. 2011;10(8): 1183–94. doi: 10.1586/erv.11.82 21854311

5. Buckland B, Boulanger R, Fino M, Srivastava I, Holtz K, Khramtsov N et al. Technology transfer and scale-up of the Flublok® recombinant hemagglutinin (HA) influenza vaccine manufacturing process. Vaccine. 2014;32(42): 5496–502. doi: 10.1016/j.vaccine.2014.07.074 25131727

6. Tapia F, Jordan I, Genzel Y, Reichl U. Efficient and stable production of Modified Vaccinia Ankara virus in two-stage semi-continuous and in continuous stirred tank cultivation systems. PLoS One. 2017;12(8): e0182553. doi: 10.1371/journal.pone.0182553 28837572

7. Konstantinov KB, Cooney CL. White paper on continuous bioprocessing May 20–21, 2014 continuous manufacturing symposium. J Pharm Sci. 2015;104(3): 813–20.

8. Gori GB. Continuous cultivation of virus in cell suspensions by use of the lysostat. Appl Microbiol. 1965;13(6): 909–17. 4286249

9. Roth F, Ullrich MW, Elizondo Herrera A, Seifert HS. Kontinuierliche Producktion von MKS-Virus in einem zweistufigen. Berl Munch Tierarztl Wochenschr. 1994;107(4): 123–7. 7993350

10. Frensing T, Heldt FS, Pflugmacher A, Behrendt I, Jordan I, Flockerzi D, et al. Continuous influenza virus production in cell culture shows a periodic accumulation of defective interfering particles. PLoS One. 2013 8(9): e72288. doi: 10.1371/journal.pone.0072288 24039749

11. Giri L, Feiss MG, Bonning BC, Murhammer DW. Production of baculovirus defective interfering particles during serial passage is delayed by removing transposon target sites in fp25k. J Gen Virol. 2012;93(Pt 2): 389–99. doi: 10.1099/vir.0.036566-0 21994323

12. Frensing T, Pflugmacher A, Bachmann M, Peschel B, Reichl U. Impact of defective interfering particles on virus replication and antiviral host response in cell culture-based influenza vaccine production. Appl Microbiol Biotechnol. 2014;98(21): 8999–9008. doi: 10.1007/s00253-014-5933-y 25132064

13. Von Magnus P. Incomplete forms of influenza virus. Adv Virus Res. 1954;2: 59–79. 13228257

14. Šantek B, Ivančić M, Horvat P, Novak S, Marić V. Horizontal Tubular Bioreactors in Biotechnology. Chem. Biochem. Eng. Q. 2006;20(4): 389–99.

15. Ersu CB, Ong SK. Treatment of wastewater containing phenol using a tubular ceramic membrane bioreactor. Environ Technol. 2008;29(2): 225–34. doi: 10.1080/09593330802029012 18613621

16. Moser A. Tubular bioreactors: case study of bioreactor performance for industrial production and scientific research. Biotechnol Bioeng. 1991;37(11): 1054–65. doi: 10.1002/bit.260371111 18597337

17. Molina E, Fernández J, Acién FG, Chisti Y. Tubular photobioreactor design for algal cultures. J Biotechnol. 2001;92(2): 113–31. doi: 10.1016/s0168-1656(01)00353-4 11640983

18. Levenspiel O. Chemical reaction engineering. 1st ed. John Wiley & Sons; 1962.

19. Tapia F, Genzel Y, Reichl U. Plug flow tubular bioreactor, system containing the same and method for production of virus. Patent application WO2017190790A1.

20. Lohr V. Characterization of the avian designer cells AGE1.CR and AGE1.CR.pIX considering growth, metabolism and production of influenza virus and Modified Vaccinia Virus Ankara (MVA). PhD. Thesis, Otto-von-Guericke University Magdeburg. 2014.

21. Lohr V, Genzel Y, Behrendt I, Scharfenberg K, Reichl U. A new MDCK suspension line cultivated in a fully defined medium in stirred-tank and wave bioreactor.Vaccine. 2010;28(38):6256–64. doi: 10.1016/j.vaccine.2010.07.004 20638458

22. Kalbfuss B, Knöchlein A, Kröber T, Reichl U. Monitoring influenza virus content in vaccine production: precise assays for the quantitation of hemagglutination and neuraminidase activity. Biologicals. 2008;36(3): 145–61. 18561375

23. Genzel Y, Reichl U. Vaccine production—state of the art and future needs in upstream processing. In: Poertner R, editor. Animal cell biotechnology: methods and protocols. Humana Press Inc.; 2007. pp. 457–73.

24. Frensing T, Kupke SY, Bachmann M, Fritzsche S, Gallo-Ramirez LE, Reichl U. Influenza virus intracellular replication dynamics, release kinetics, and particle morphology during propagation in MDCK cells. Appl Microbiol Biotechnol. 2016;100: 7181–92. doi: 10.1007/s00253-016-7542-4 27129532

25. Peschel B, Frentzel S, Laske T, Genzel Y, Reichl U. Comparison of influenza virus yields and apoptosis-induction in an adherent and a suspension MDCK cell line. Vaccine. 2013;31(48): 5693–9. doi: 10.1016/j.vaccine.2013.09.051 24113260

26. Genzel Y, Vogel T, Buck J, Behrendt I, Ramirez DV, Schiedner G, et al. High cell density cultivations by alternating tangential flow (ATF) perfusion for influenza A virus production using suspension cells. Vaccine. 2014;32(24): 2770–81. doi: 10.1016/j.vaccine.2014.02.016 24583003

27. Stettler M, Zhang X, Hacker DL, De Jesus M, Wurm FM. Novel orbital shake bioreactors for transient production of CHO derived IgGs. Biotechnol Prog. 2007;23(6): 1340–6. doi: 10.1021/bp070219i 17914862

28. Ingham J, Dunn IJ, Heinzle E, Prenosil JE, Snape JB. Chemical Engineering Dynamics, An Introduction to Modelling and Computer Simulation. 3rd completely revised ed. Wiley Germany; 2007.


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2019 Číslo 11