Optimization of virus yield as a strategy to improve rabies vaccine production by Vero cells in a bioreactor

To improve rabies vaccine production by Vero cells, we have developed a strategy based on high cell density culture and optimization of virus yield. We have first optimized cell growth in spinner flask using a Taouchi's L8 experimental design. We analyzed the effects of the following factors: initial glucose and glutamine concentrations, Cytodex 1 concentration and the regulation of glucose level at I g l(-1). We have also investigated the effect of the following factor interactions: Cytodex I concentration/glutamine concentration, Cytodex 1 concentration/glucose concentration and glucose concentration/glutamine concentration. Statistical analysis of the collected data pointed to the initial glucose concentration, the regulation of glucose level at I a l(-1) and the interactions between Cytodex 1 concentration/initial glucose concentration and Cytodex 1 concentration/initial glutamine concentration as the parameters that affected cell growth. Using the optimal conditions determined earlier, we have studied Vero cell growth in a 7-1 bioreactor and in batch culture, and obtained a cell density level equal to 3.6 +/- 0.2 x 10(6) cells ml(-1). Cell infection with rabies virus (LP 2061[Vero strain) at a multiplicity of infection (MOI) of 0.3 using M 199 medium supplemented with 0.2% bovine serum albumin (BSA), yielded a maximal virus titer equal to 8 +/- 1.6 x 10(7) Fluorescent Focus Units (FFU) ml(-1). We have also studied Vero cell growth in a 7-1 bioreactor using recirculation as a per-fusion culture mode during cell proliferation step and perfusion for virus multiplication phase. In comparison to batch culture, we reached a higher cell density level that was equal to 10.1 +/- 0.5 x 10(6) cells ml(-1). Cell infection under the conditions previously indicated, yielded 141 of virus harvest that had a virus titer equal to 2.6 +/- 0.5 x 10(7) FFU ml(-1). The activity of the inactivated virus harvest showed a protective activity that meets WHO requirements. (c) 2005 Elsevier B.V. All rights reserved.