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@#Objective To investigate the effect of amplification culture of micro-carrier Vero cells from 30 L bioreactor to 300 L bioreactor after extra-tank trypsinization on the virus-producing ability of rabies virus(RABV)CTN-1Ⅴ strain.Methods The 140-passage of Vero cells were cultured at 37 ℃ for 72-120 h,then amplified by passaging at a cell density ratio of 1∶4 into the 10 × cell factory. After incubation at 37 ℃ for 72-120 h,the monolayer cells were detached and inoculated into the 30 L bioreactor with micro-carriers 7-10 g/L,culture temperature 37 ℃,pH 7. 0-7. 4,dissolved oxygen 30%-80%,stirring speed 10-50 r/min,and continuous perfusion culture 72-120 h. Total three batches of microcarrier Vero cells were cultured,which were amplified to the 300 L bioreactor after extra-tank trypsinization,with microcarrier5-8 g/L,culture temperature 37 ℃,pH 7. 0-7. 4,dissolved oxygen 30%-80%,stirring speed 30-80 r/min,and perfusion culture 72-120 h. RABV CTN-1Ⅴ was inoculated at the MOI of 0. 05,and the virus solution was harvested every 24 h and detected for the virus titer and antigen content. Results The cell density was about 1 × 10~7 cells/mL after culture for 96 h in the 30 L bioreactor,and was about 7. 4 × 10~6 cells/mL after culture for 96 h in the 300 L bioreactor. At 96 h after virus inoculation,the virus harvest solution reached the peak potency,with the average virus titer of 6. 8 lgLD_(50)/mL and the average antigen content of 2. 58 IU/mL. Conclusion The scale-up culture process of micro-carrier Vero cells after extra-tank trypsinization from 30 L bioreactor to 300 L bioreactor is stable and feasible,with no significant effect on the virus-producing ability of RABV CTN-1Ⅴ strain,which provides a reference for the large-scale production of inactivated RABV vaccine
RESUMEN
@#Objective To develop a large-scale culture process for rabies virus(RABV)in 150 L bioreactor,and lay a foundation for the further development of a larger-scale and high-density microcarrier reactor process.Methods Vero cells and RABV strain CTN-1V were cultured in 30 L(model:C30-2)and 150 L(model:VESSEL FERMENTER 300L)bioreactors by perfused culture with 20 g/L Cytodex-1 microcarrier and DO 20%-60%,at culture temperature 36-38 ℃ and pH 7.0-7.4.During the culture process,the cell density and virus titer were measured.The virus culture media was harvested for consecutive 13 d and detected for the sterility,mycoplasma,and the residues of antigen,host cell protein(HCP),bovine serum albumin(BSA)and DNA.Results The density of cultured cells in 30 L and 150 L bioreactors all reached above 1.2 ×10~7cell/mL.There was no significant difference in cell density at different time points during the culture(t = 0.225-2.173,P = 0.096-0.833).The highest virus titer(8.5 lgLD_(50)/mL)was found in the both bioreactors 6 d after infection with no significant difference(t = 1.000,P = 0.374).The residues of antigen,HCP,BSA and DNA in the virus suspension from the two bioreactors were basically the same.Conclusion 150 L bioreactor can be used for the large-scale culture of RABV,and the harvested virus conformed to the relevant standards in Chinese Pharmacopoeia(Volume Ⅲ,2020 edition).