Process intensification for an enhanced replication of a newly adapted RM-65 sheep pox virus strain in Vero cells grown in stirred bioreactor

International audienceSheep pox virus initially adapted to replicate in primary lamb kidney cells was adapted to Vero cells by serial passages in monolayer cultures. After nine passages the virus was able to correctly replicate in Vero cells, virus titer achieved was 10(5.875) TCID50 (median tissue culture infective dose) ml(-1). To optimize the production process, the effects of MO! (multiplicity of infection), TOI (time of infection) and the culture medium were investigated. Cell infection at a MO! of 0.005 concurrently with cell seeding showed the best results in terms of specific virus productivity. The effect of MEM enrichment with several components was investigated using the experimental design approach. 67 experiments were performed in 6-well plates to select the best combination. The highest titer was achieved when MEM was supplemented with 5 mM glucose, 5 mM fructose and 25 mM sucrose. Spinner culture confirms these data; virus titer was 10(7.375) TCID50 ml(-1). In addition Vero cells were cultivated in a 7-1 bioreactor in batch mode on 3 gl(-1) Cytodex1, and infected at cell seeding at a MO! of 0.005. Maximal virus titer was 10(7.275) TCID50 ml(-1). This corresponds to 44-fold factor enhancement compared to spinner cultures conducted in MEM + 2% FCS. (C) 2014 Elsevier B.V. All rights reserved

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

International audienceTo 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 Taguchi'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 1 g l(-1). We have also investigated the effect of the following factor interactions: Cytodex 1 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 1 g 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-l 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 M199 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-l bioreactor using recirculation as a perfusion 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 14l 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

Comparison of various culture modes for the production of rabies virus by Vero cells grown on microcarriers in a 2-l bioreactor

International audienceWe studied Vero cell growth on Cytodex 1 (3 g/l) and MEM supplemented with foetal calf serum (FCS) in a 2-l bioreactor using continuous (recirculation and perfusion) and batch culture modes. We achieved a cell density level equal to 4.35 × 106 cells/ml using the recirculation culture mode while perfused cultures yielded a higher cell density level equal to 4.73 × 106 cells/ml. However, as compared to the recirculation culture mode, the specific medium consumption rate was two-fold higher. Batch culture of Vero cells resulted in 2.2 × 106 cells/ml with a specific medium consumption rate similar to recirculation culture.Rabies virus production (LP 2061/Vero strain) by Vero cells was also investigated. We analysed in spinner flasks, the effects on virus multiplication of multiplicity of infection (MOI), regulation of glucose level to 1 g/l and type of medium. The highest virus titer was reached when cells were infected at an MOI of 0.3 in M199 medium supplemented with 0.2% of bovine serum albumin (BSA) and without regulating glucose level. In these conditions, virus titer was equal to 1.5 × 106 fluorescent focus units (FFU)/ml.We then evaluated rabies virus production by Vero cells grown on 3 g/l of Cytodex 1 using recirculation culture mode during the growth phase. Cells were infected at the optimal conditions previously determined. The maximal virus titer was equal to 2 × 107 FFU/ml. The activity of the experimental vaccine prepared showed a protective activity that meets WHO requirements