Standardization of microcarrier based bioreactor culture in parallel with roller bottle for rabies virus (PV-11) propagation in Vero cell using MEM eagle’s and RPMI 1640 medium

Abstract

The Vero cell is the continuous cell line used as a cell substrate for many viral vaccines manufacturing including Rabies vaccine. The Pitman and PV-11 strains of rabies virus are commonly used for production of rabies vaccines. In our study the PV - 11 strain is used as seed virus for the production of Vero cell derived inactivated Anti Rabies Vaccines (Lyophilized). The working cell bank and working virus seed lot were prepared and tested for its sterility and mycoplasma contamination. In this study the roller bottle method and micro carrier (cytodex-1) based bioreactor culturing method were standardized with MEM Eagles and RPMI 1640 medium. The outcome of the two methods were compared ,to find out the medium and culturing method which can give higher concentration of Vero cells to get higher viral titre in mass production. The quantities of Vero cells obtained from passage 144 to 148 were assessed using a haemocytometer cell count procedure. The quantity of the Vero cell obtained per roller bottle ranged from 222.82x106 cells to 236.67x106 cells in MEM Eagles medium and 227.80x106 cells to 230.66x106 cells in RPMI 1640 medium. The average quantity of Vero cells in roller bottles were 1.12 x 106 cells per ml of culture. In the bioreactor culturing method the Vero cells obtained in the concentration of 1.69 to 1.75 million cells / ml of culture by MEM Eagles medium and 1.67 to 1.70 million cells / ml by RPMI 1640 medium. In the bioreactor the average yield of vero cells ranged 1.70 x106 cells per ml of culture. It is estimated that about 35% increase in Vero cell quantity in bioreactor system than the roller bottle culturing method. 0.3 MOI (Multiplicity of Infection) of rabies virus was kept as constant for infection of confluent monolayered Vero cells in both roller bottle and bioreactor culture. Four viral harvests were collected at three day intervals and replenished with fresh culturing medium each time. The viral titres in each viral harvest were quantified using in-house standardized RT-PCR method; the roller bottle method yielded viral titre log 6.044 to 7.106 and the bioreactor culture yielded log 6.559 to 7.216 rabies viral particles. It is observed that the bioreactor culture yielded more viral titre than the roller bottle culture using both the medium.

Comparative Analysis of MEM, RPMI 1640 Media on Rabies Virus Propagation in Vero Cells and Virus Quantification by FAT

“…The P-155 batch of MEM media gave a yield of 10.05 x 10 6 cells whereas newly adapted P-155 batch of RPMI-1640 media gave a yield of 7.56 x 10 6 cells and there is ~25% of reduction in RPMI 1640 during initial stage of adaptation. The cell line is passaged further 3 times from passage 156 to 158 for complete adaptation and the cell enumeration results shows that the RPMI 1640 cell count as equal to the MEM media [14]. The cell count in RPMI 1640 media was 10.35 x 10 6 and in MEM it was 10.28 x 10 6 cells per 25 cm 2 tissue culture flasks at passage level 158 in respective media.…”