Abstract
Fixed-bed bioreactors packed with macrocarriers show great potential to be used for vaccine process development and large-scale production due to distinguishing features of low shear force, high cell adhering surface area, and easy replacement of culture media in situ. As an initial step of utilizing this type of bioreactors for Pseudorabies virus production (PRV) by African green monkey kidney (Vero) cells, we developed a tube-fixed-bed bioreactor in the previous study, which represents a scale-down model for further process optimization. By using this scale-down model, here we evaluated impacts of two strategies (use of serum-free medium and low cell inoculum density) on PRV production, which have benefits of simplifying downstream process and reducing risk of contamination. We first compared Vero cell cultures with different media, bioreactors and inoculum densities, and conclude that cell growth with serum-free medium is comparable to that with serum-containing medium in tube-fixed-bed bioreactor, and low inoculum density supports cell growth only in this bioreactor. Next, we applied serum-free medium and low inoculum cell density for PRV production. By optimization of time of infection (TOI), multiplicity of infection (MOI) and the harvesting strategy, we obtained total amount of virus particles ~ 9 log10 TCID50 at 5 days post-infection (dpi) in the tube-fixed-bed bioreactor. This process was then scaled up by 25-fold to a Xcell 1-L fixed-bed bioreactor, which yields totally virus particles of 10.5 log10 TCID50, corresponding to ~ 3 × 105 doses of vaccine. The process studied in this work holds promise to be developed as a generic platform for the production of vaccines for animal and human health.
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References
Abdul-Hamid NA, Abas F, Maulidiani M, Ismail IS, Tham CL, Swarup S, Umashankar S (2019) NMR metabolomics for evaluating passage number and harvesting effects on mammalian cell metabolome. Anal Biochem 576:20–32. https://doi.org/10.1016/j.ab.2019.04.001
Barrett PN, Terpening SJ, Snow D, Cobb RR, Kistner O (2017) Vero cell technology for rapid development of inactivated whole virus vaccines for emerging viral diseases. Expert Rev Vaccines 16:883–894. https://doi.org/10.1080/14760584.2017.1357471
Bock A, Schulze-Horsel J, Schwarzer J, Rapp E, Genzel Y, Reichl U (2011) High-density microcarrier cell cultures for influenza virus production. Biotechnol Prog 27:241–250. https://doi.org/10.1002/btpr.539
Butler M (2015) Serum and protein free media. In: Butler M (ed) Animal cell culture. Cell engineering. Springer, Cham, pp 223–236
Cherry RS, Papoutsakis ET (1986) Hydrodynamic effects on cells in agitated tissue culture reactors. Bioprocess Eng 1:29–41. https://doi.org/10.1007/bf00369462
Dong B, Zarlenga DS, Ren X (2014) An overview of live attenuated recombinant pseudorabies viruses for use as novel vaccines. J Immunol Res 2014:824630. https://doi.org/10.1155/2014/824630
Gallo-Ramirez LE, Nikolay A, Genzel Y, Reichl U (2015) Bioreactor concepts for cell culture-based viral vaccine production. Expert Rev Vaccines 14:1181–1195. https://doi.org/10.1586/14760584.2015.1067144
Genzel Y, Reichl U (2009) Continuous cell lines as a production system for influenza vaccines. Expert Rev Vaccines 8:1681–1692. https://doi.org/10.1586/Erv.09.128
Green JA (1986) Unusual monocyte-lymphocyte interactions determine the specificity of the immune-specific interferon response induced by newcastle disease and herpes simplex viruses. J Med Virol 20:381–389. https://doi.org/10.1002/jmv.1890200411
Ho L, Greene CL, Schmidt AW, Huang LH (2004) Cultivation of HEK 293 cell line and production of a member of the superfamily of G-protein coupled receptors for drug discovery applications using a highly efficient novel bioreactor. Cytotechnology 45:117–123. https://doi.org/10.1007/s10616-004-6402-8
Hu AY et al (2008) Microcarrier-based MDCK cell culture system for the production of influenza H5N1 vaccines. Vaccine 26:5736–5740. https://doi.org/10.1016/j.vaccine.2008.08.015
Huang D et al (2015) Serum-free suspension culture of MDCK cells for production of influenza H1N1 vaccines. PLoS ONE 10:e0141686. https://doi.org/10.1371/journal.pone.0141686
Kaufman JB, Wang G, Zhang W, Valle MA, Shiloach J (2000) Continuous production and recovery of recombinant Ca2+ binding receptor from HEK 293 cells using perfusion through a packed bed bioreactor. Cytotechnology 33:3–11. https://doi.org/10.1023/A:1008143132056
Krell PJ (1996) Passage effect of virus infection in insect cells. Cytotechnology 20:125–137. https://doi.org/10.1007/BF00350393
Le Ru A, Jacob D, Transfiguracion J, Ansorge S, Henry O, Kamen AA (2010) Scalable production of influenza virus in HEK-293 cells for efficient vaccine manufacturing. Vaccine 28:3661–3671. https://doi.org/10.1016/j.vaccine.2010.03.029
Liu CC et al (2011) Purification and characterization of enterovirus 71 viral particles produced from vero cells grown in a serum-free microcarrier bioreactor system. PLoS ONE 6:e20005. https://doi.org/10.1371/journal.pone.0020005
Mattos DA, Silva MV, Gaspar LP, Castilho LR (2015) Increasing Vero viable cell densities for yellow fever virus production in stirred-tank bioreactors using serum-free medium. Vaccine 33:4288–4291. https://doi.org/10.1016/j.vaccine.2015.04.050
McFerran JB, Dow C (1975) Studies on immunisation of pigs with the Bartha strain of Aujeszky’s disease virus. Res Vet Sci 19:17–22. https://doi.org/10.1016/s0034-5288(18)33548-3
Meuwly F, Ruffieux PA, Kadouri A, von Stockar U (2007) Packed-bed bioreactors for mammalian cell culture: bioprocess and biomedical applications. Biotechnol Adv 25:45–56. https://doi.org/10.1016/j.biotechadv.2006.08.004
Muench LJRH (1937) A simple method of estimating fifty per cent endpoints. Am J Epidemiol 27:493–497
Nauwynck H, Glorieux S, Favoreel H, Pensaert M (2007) Cell biological and molecular characteristics of pseudorabies virus infections in cell cultures and in pigs with emphasis on the respiratory tract. Vet Res 38:229–241. https://doi.org/10.1051/vetres:200661
Nie J et al (2019) Production process development of pseudorabies virus vaccine by using novel scale-down model of fixed-bed bioreactor. J Pharm Sci. https://doi.org/10.1016/j.xphs.2019.10.002
Onyekaba C, Bueon L, King P, Fahrmann J, Goyal SM (1987) Susceptibility of various ceil culture systems to pseudorabies virus. Comp Immunol Microbiol Infect Dis 10:163–166. https://doi.org/10.1016/0147-9571(87)90027-0
Organization WH (1987) Requirements for continuous cell lines used for biological substances. Tech Rep Ser 745(1):99–115
Ozturk SS, Palsson BO (1991) Examination of serum and bovine serum albumin as shear protective agents in agitated cultures of hybridoma cells. J Biotechnol 18:13–28. https://doi.org/10.1016/0168-1656(91)90232-k
Portner R, Platas OB, Fassnacht D, Nehring D, Czermak P, Markl H (2007) Fixed bed reactors for the cultivation of mammalian cells: design, performance and scale-up. Open Biotechnol J 1:41–46. https://doi.org/10.2174/1874070700701010041
Rourou S, van der Ark A, van der Velden T, Kallel H (2007) A microcarrier cell culture process for propagating rabies virus in Vero cells grown in a stirred bioreactor under fully animal component free conditions. Vaccine 25:3879–3889. https://doi.org/10.1016/j.vaccine.2007.01.086
Silva AC, Delgado I, Sousa MF, Carrondo MJ, Alves PM (2008) Scalable culture systems using different cell lines for the production of Peste des Petits ruminants vaccine. Vaccine 26:3305–3311. https://doi.org/10.1016/j.vaccine.2008.03.077
Slivac I, Srček VG, RadoŠević K, Kmetič I, Kniewald Z (2006) Aujeszky’s disease virus production in disposable bioreactor. J Biosci 31:363–368. https://doi.org/10.1007/bf02704109
Srcek VG, Cajavec S, Sladic D, Kniewald Z (2004) BHK 21 C13 cells for Aujeszky's disease virus production using the multiple harvest process. Cytotechnology 45:101–106. https://doi.org/10.1007/s10616-004-2551-z
Tapia F, Vogel T, Genzel Y, Behrendt I, Hirschel M, Gangemi JD, Reichl U (2014) Production of high-titer human influenza A virus with adherent and suspension MDCK cells cultured in a single-use hollow fiber bioreactor. Vaccine 32:1003–1011. https://doi.org/10.1016/j.vaccine.2013.11.044
Toriniwa H, Komiya T (2007) Japanese encephalitis virus production in Vero cells with serum-free medium using a novel oscillating bioreactor. Biologicals 35:221–226. https://doi.org/10.1016/j.biologicals.2007.02.002
Trabelsi K, Rourou S, Loukil H, Majoul S, Kallel H (2006) Optimization of virus yield as a strategy to improve rabies vaccine production by Vero cells in a bioreactor. J Biotechnol 121:261–271. https://doi.org/10.1016/j.jbiotec.2005.07.018
Varani J, Inman DR, Fligiel SE, Hillegas WJ (1993) Use of recombinant and synthetic peptides as attachment factors for cells on microcarriers. Cytotechnology 13:89–98. https://doi.org/10.1007/bf00749935
Wang S-J, Zhong J-J, Chen Y-L, Yu J-T (1995) Characterization and modeling of oxygen transfer in a 20-l modified cell-lift bioreactor with a double-screen cage. J Ferment Bioeng 80:71–77. https://doi.org/10.1016/0922-338x(95)98179-o
Wen Y, Yang ST (2011) Microfibrous carriers for cell culture: a comparative study. Biotechnol Prog 27:1126–1136. https://doi.org/10.1002/btpr.625
Wittmann G (1991) Spread and control of aujeszky's disease (AD). Comp Immunol Microbiol Infect Dis 14:165–173. https://doi.org/10.1016/0147-9571(91)90129-2
Wu S-C (1999) Influence of hydrodynamic shear stress on microcarrier-attached cell growth: cell line dependency and surfactant protection. Bioprocess Eng 21:201. https://doi.org/10.1007/s004490050663
Yuk IH et al (2006) A serum-free Vero production platform for a chimeric virus vaccine candidate. Cytotechnology 51:183–192. https://doi.org/10.1007/s10616-006-9030-7
Zhang S, Handa-Corrigan A, Spier RE (1992) Foaming and media surfactant effects on the cultivation of animal cells in stirred and sparged bioreactors. J Biotechnol 25:289–306. https://doi.org/10.1016/0168-1656(92)90162-3
Zhou J, Li S, Wang X, Zou M, Gao S (2017) Bartha-k61 vaccine protects growing pigs against challenge with an emerging variant pseudorabies virus. Vaccine 35:1161–1166. https://doi.org/10.1016/j.vaccine.2017.01.003
Acknowledgements
This work was supported by the National Key Research and Development Program of China (No.2018YFA0900804); the National Natural Science Foundation of China (No. 21878124 and No. 31570034), the Collaborative Innovation Center of Jiangsu Modern Industrial Fermentation, the 111 Project (No. 111-2-06), and TaiShan Industrial Experts Programme (tscy20160307).
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Nie, J., Sun, Y., Peng, F. et al. Pseudorabies virus production using a serum-free medium in fixed-bed bioreactors with low cell inoculum density. Biotechnol Lett 42, 2551–2560 (2020). https://doi.org/10.1007/s10529-020-02987-x
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DOI: https://doi.org/10.1007/s10529-020-02987-x