Pilot-Scale Production of Lyophilized Inactivated Rabies Vaccine Candidate in Vero Cells under Fully Animal Component-Free Conditions Using Microcarrier Technology and Laboratory Animal Trials
The upstream process was carried out in an animal component-free medium on Cytodex 1 microcarriers. Recombinant trypsin is a non-animal derived protease used as an alternative to animal-derived trypsin. To inactivate recombinant trypsin, a soybean trypsin inhibitor (STI) should be added to the medium. A protocol was first tested in T-flasks and then passaged to 500 mL and 3 L spinner flasks. Cell detachment was completed in 10 - 12 min, and 0.4 g/L STI was added to a 3L spinner, and cells were transferred into a 30 L stirred tank bioreactor. On day 5, the cell density had reached its maximum (around 1.8 × 106 cells/mL). At an MOI of 0.3 with serum-free medium conditions, cell infection yielded a maximal rabies virus titer of 1.82 × 107 FFU/mL at 5 days. All cell culture conditions and virus growth kinetics in serum-free media were investigated. In conclusion, Vero cells were grown on Cytodex 1 with serum-free media and a high amount of rabies virus was obtained. A mouse challenge was used to determine the immune response to an inactivated rabies virus vaccine candidate. Also, we evaluated inactive rabies vaccine candidate safety, and immunogenicity in mice, sheep, horses, and cattle. We found that no horses, sheep, or cattle who were given vaccine IM at 3.2 IU/dose exhibited any clinical sign of disease and all developed high VNA titers (up to 10.03 IU/mL) by 3 - 4 WPI. After the accelerated stability studies, the lyophilized inactivated rabies vaccine candidate showed enough antigenic potency (2.6 IU/mL) in the mouse challenge test. Also, 18-month long-term stability studies showed enough immune response (1.93 IU/mL) on day 14. The activity of the vaccine candidate showed a good immune response and safety criteria that meet WHO requirements. This is the first pilot-scale mammalian cell-based viral rabies vaccine production study in Türkiye that used microcarriers.
References
[1]
Kayser, V., Françon, A., Pinton, H., Saluzzo, J.F. and Trout, B.L. (2017) Rational Design of Rabies Vaccine Formulation for Enhanced Stability. Turkish Journal of Medical Sciences, 47, 987-995. https://doi.org/10.3906/sag-1610-82
[2]
Hosseini, et al. (2015) Evaluation of a Novel Adjuvant in Rabies Vaccine Formulation. International Conference of Social Science, Medicine and Nursing (SSMN-2015), Istanbul, 5-6 June 2015, 133-134.
[3]
Sacramento, D., Badrane, H., Bourhy, H. and Tordo, N. (1992) Molecular Epidemiology of Rabies Virus in France: Comparison with Vaccine Strains. Journal of General Virology, 73, 1149-1158. https://doi.org/10.1099/0022-1317-73-5-1149
[4]
Trabelsi, K., Rourou, S., Loukil, H., Majoul, S. and Kallel, H. (2005) Comparison of Various Culture Modes for the Production of Rabies Virus by Vero Cells Grown on Microcarriers in a 2-l Bioreactor. Enzyme and Microbial Biotechnology, 36, 514-519. https://doi.org/10.1016/j.enzmictec.2004.11.008
[5]
WHO (1993) World Survey of Rabies 27 (for Year 1991). WHO: Rabies: 93.209. WHO, Geneva.
[6]
World Health Organization (2010) Recommendations for the Evaluation of Animal Cell Cultures as Substrates for the Manufacture of Biological Medicinal Products and for the Characterization of Cell Banks. Proposed Replacement of TRS 878, Annex 1; 61st Meeting of WHO Expert Committee on Biological Standardization. WHO, Geneva.
[7]
Wiktor, T.J., Fanget, B.J., Fournier, P. and Montagnon, B.J. (1987) Process for the Large-Scale Production of Rabies Vaccine. US Patent No. 4,664,912.
[8]
Montagnon, B.J., Fanget, B. and Nicolas, A.J. (1981) The Large-Scale Cultivation of VERO Cells in Micro-Carrier Culture for Virus Vaccine Production. Preliminary Results for Killed Poliovirus Vaccine. Developments in Biological Standardization, 47, 55-64.
[9]
Sugawara, K., Nishiyama, K., Ishikawa, Y., Abe, M., Sonoda, K., Komatsu, K., et al. (2002) Development of Vero Cell-Derived Inactivated Japanese Encephalitis Vaccine. Biologicals, 30, 303-314. https://doi.org/10.1006/biol.2002.0345
[10]
Rourou, S., van der Ark, A., van der Velden, T. and 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
[11]
WHO Expert Committee on Rabies (1984) Seventh Report. Technical Report Series 709. World Health Organization, Geneva, 21.
[12]
WHO Expert Committee on Rabies (1992) Eighth Report. Technical Report Series 824. World Health Organization, Geneva, 16.
[13]
Merten, O.W., Kierulff, J.V., Castignolles, N. and Perrin, P. (1994) Evaluation of the New Serum-Free Medium (MDSS2) for the Production of Different Biologicals: Use of Various Cell Lines. Cytotechnology, 14, 47-59. https://doi.org/10.1007/BF00772195
[14]
Merten, O.W., Kallel, H., Manuguerra, J.C., Tardy-Panit, M., Crainic, R., Delpeyroux, F., et al. (1999) The New Medium MDSS2N, Free of Any Animal Protein Supports Cell Growth and Production of Various Viruses. Cytotechnology, 30, 191-201. https://doi.org/10.1023/A:1008021317639
[15]
Frazatti-Gallina, N.M., Paoli, R.L., Moura-Fuches, R.M., Jorge, S.C. and Pereira, C.A. (2001) Higher Production of Rabies Virus in Serum-Free Medium Cell Cultures on Microcarriers. Journal of Biotechnology, 92, 67-72. https://doi.org/10.1016/S0168-1656(01)00362-5
[16]
Frazatti-Gallina, N.M., Mourao-fuches, R.M., Paoli, R.L., Silva, M.L.N., Miyaki, C., Valentini, E.J.G., et al. (2004) Vero-Cell Rabies Vaccine Produced Using Serum-Free Medium. Vaccine, 23, 511-517. https://doi.org/10.1016/j.vaccine.2004.06.014
[17]
Trabelsi, K., Rourou, S., Loukil, H., Majoul, S. and Kallel, H. (2006) Optimization of Virus Yield as a Strategy to Improve Rabies Vaccine Production by Vero Cells in a Bioreactor. Journal of Biotechnology, 121, 261-271. https://doi.org/10.1016/j.jbiotec.2005.07.018
[18]
Croughan, M.S., Hamel, J.F. and Wand, D.I.C. (2000) Hydrodynamic Effects on Animal Cells Grown in Microcarriers Cultures. Biotechnology and Bioengineering, 67, 541-582. https://doi.org/10.1002/(SICI)1097-0290(20000320)67:6<841::AID-BIT19>3.0.CO;2-K
[19]
Keanne, J.T., Ryan, D. and Gray, P.P. (2003) Effect of Shear Stress on Expression of a Recombinant Protein by Chinese Hamster Ovary Cells. Biotechnology and Bioengineering, 81, 211-220. https://doi.org/10.1002/bit.10472
[20]
Van der Pol, L. and Tramper, J. (1998) Shear Sensitivity of Animal Cells from a Culture-Medium Perspective. Tibtech, 16, 323-328. https://doi.org/10.1016/S0167-7799(98)01209-8
[21]
Zhang, S., Handa-Corrigan, A. and Spier, R.E. (1992) Foaming and Media Surfactant Effects on the Cultivation of Animal Cells in Stirred and Sparged Bioreactors. Journal of Biotechnology, 25, 289-306. https://doi.org/10.1016/0168-1656(92)90162-3
[22]
Morgeaux, S., Tordo, N., Gontier, C. and Perrin, P. (1993) β Propiolactone Treatment Impairs the Biological Activity of Residual DNA from BHK-21 Cells Infected with Rabies Virus. Vaccine, 11, 82-90. https://doi.org/10.1016/0264-410X(93)90343-V
[23]
Trabelsi, K., Zakour, M. and Kallel, H. (2019) Purification of Rabies Virus Produced in Vero Cells Grown in Serum-Free Medium. Vaccine, 37, 7052-7060. https://doi.org/10.1016/j.vaccine.2019.06.072
[24]
Severo, M.G., Zeferino, A.S. and Soccol, C.R. (2017) Development of a Rabies Vaccine in Cell Culture for Veterinary Use in the Lyophilized Form. In: Thomaz-Soccol, V., Pandey, A. and Resende, R.R., Eds., Current Developments in Biotechnology and Bioengineering, Elsevier, Amsterdam, Vol. 21, 523-560. https://doi.org/10.1016/B978-0-444-63660-7.00021-8
[25]
World Health Organization (2005) Recommendations for Inactivated Rabies Vaccine for Human Use Produced in Cell Substrates and Embryonated Eggs. Annexe 2, 56th Meeting of WHO Expert Committee on Biological Standardization. WHO, Geneva.
[26]
Chabaud-Riou, M., Moreno, N., Guinchard, F., Nicolai, M.C., Niogret-Siohan, E., Sève, N., Manin, C., Guinet-Morlot, F. and Riou, P. (2017) G-Protein Based ELISA as a Potency Test for Rabies Vaccines. Biologicals, 46, 124-129. https://doi.org/10.1016/j.biologicals.2017.02.002
[27]
Isbrucker, R., Levis, R., Casey, W., McFarland, R., Schmitt, M., Arciniega, J., Descamps, J., Finn, T., Hendriksen, C., Horiuchi, Y., Keller, J., Kojima, H., Sesardic, D., Stickings, P., Johnson, N.W. and Allen, D. (2011) Alternative Methods and Strategies to Reduce, Refine, and Replace Animal Use for Human Vaccine Post-Licensing Safety Testing: State of the Science and Future Directions. Procedia in Vaccinology, 5, 47-59. https://doi.org/10.1016/j.provac.2011.10.004
[28]
OIE Office International des Epizooties (2000) Manual of Standards for Diagnostic Tests and Vaccines. 276-291.
[29]
Koprowski, H. (1996) The Mouse Inoculation Test. In: Meslin, F.X., Kaplan, M.M. and Koprowski, H., Eds., Laboratory Techniques in Rabies, 4th Edition, WHO, Geneva, 80-87.
[30]
Reed, L.J. and Muench, H.A. (1938) Simple Method for Estimating 50% Endpoints. The American Journal of Hygiene, 27, 493-497. https://doi.org/10.1093/oxfordjournals.aje.a118408
[31]
Wilbur, L.A. and Aubert, M.F.A. (1996) The NIH Test for Potency. In: Meslin, F.X., Kaplan, M.M. and Koprowsky, H., Eds., Laboratory Techniques in Rabies, WHO, Geneva, 360-368.
[32]
Rourou, S., Riahi, N., Majoul, S., Trabelsi, K. and Kallel, H. (2013) Development of an in Situ Detachment Protocol of Vero Cells Grown on Cytodex1 Microcarriers under Animal Component-Free Conditions in Stirred Bioreactor. Applied Biochemistry and Biotechnology, 170, 1724-1737. https://doi.org/10.1007/s12010-013-0307-y
[33]
Merten, O.W., Petres, S. and Couve, E. (1995) A Simple Serum-Free Freezing Medium for Serum-Free Cultured Cells. Biologicals, 23, 185-189. https://doi.org/10.1006/biol.1995.0030
[34]
Hesse, F., Scharfenberg, K. and Wagner, R. (1999) Cryopreservation under Protein-Free Medium Conditions: A Reliable Way to Safe Cell Banking. In: Bernard, A., Griffits, B., Noe, W. and Wurm, F., Eds., Animal Cell Technology: Products from Cells, Cells as Products, Kluwer Academic Publishers, Dordrecht, 501-503. https://doi.org/10.1007/0-306-46875-1_106
[35]
Gamoh, K., Shimazaki, Y., Senda, M., Makie, H., Itoh, O., Muramatsu, M., Hirayama, N. and Hatakeyama, H. (2003) Establishment of a Potency Test by ELISA for a Rabies Vaccine for Animal Use in Japan. The Journal of Veterinary Medical Science, 65, 685-688. https://doi.org/10.1292/jvms.65.685
