Development of large-scale amplification process of rabies virus in 150 L bioreactor
Objective To develop a large-scale culture process for rabies virus(RABV)in 150 L bioreactor,and lay a foun-dation 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 x 107 cell/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 lgLD50/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).
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