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
Open Access
- 1 January 2022
- journal article
- research article
- Published by Scientific Research Publishing, Inc. in Journal of Biomedical Science and Engineering
- Vol. 15 (06), 157-178
- https://doi.org/10.4236/jbise.2022.156015
Abstract
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.Keywords
This publication has 24 references indexed in Scilit:
- Alternative methods and strategies to reduce, refine, and replace animal use for human vaccine post-licensing safety testing: state of the science and future directionsProcedia in Vaccinology, 2011
- A microcarrier cell culture process for propagating rabies virus in Vero cells grown in a stirred bioreactor under fully animal component free conditionsVaccine, 2007
- Optimization of virus yield as a strategy to improve rabies vaccine production by Vero cells in a bioreactorJournal of Biotechnology, 2006
- Vero-cell rabies vaccine produced using serum-free mediumVaccine, 2004
- Effect of shear stress on expression of a recombinant protein by Chinese hamster ovary cellsBiotechnology & Bioengineering, 2002
- A Simple Serum-free Freezing Medium for Serum-free Cultured CellsBiologicals, 1995
- Evaluation of the new serum-free medium (MDSS2) for the production of different biologicals: Use of various cell linesCytotechnology, 1994
- β-propiolactone treatment impairs the biological activity of residual DNA from BHK-21 cells infected with rabies virusVaccine, 1993
- Foaming and media surfactant effects on the cultivation of animal cells in stirred and sparged bioreactorsJournal of Biotechnology, 1992
- Molecular epidemiology of rabies virus in France: comparison with vaccine strainsJournal of General Virology, 1992