Wu Suh-Chin, Liu Chia-Chyi, Lian Wei-Cheng
Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan.
Vaccine. 2004 Sep 28;22(29-30):3858-64. doi: 10.1016/j.vaccine.2004.05.037.
Enterovirus 71 (EV71) is an enterovirus that could lead to severe neurological disorders and fatalities. The inactivated vaccine is an appropriate EV71 vaccine format for meeting current needs. Large-scale preparation of the inactivated EV71vaccine depends on a scalable cell culture system for industrial mass production. In this paper, Vero cells were found to produce higher titers of EV71 than did MRC-5 and WI-38 cells. High-density microcarrier Vero cell cultures were established using 5g/L Cytodex 1 microcarriers and found to promote the release of EV71s from infected Vero cells. For the large-scale production of the inactivated vaccine antigen, the extracellular virus titers produced in the 2L bioreactor were found to be 10 times lower than the spinner flask culture but improved by 30-folds using glucose/glutamine feedings during infection. A serum-free Vero cell microcarrier culture was also established in the bioreactor, yielding a high-titer of 5.8 x 10(7) TCID50/mL for EV71 production. The immunogenicity of the inactivated virions produced in serum-free culture elicited a slightly higher level of neutralizing antibody response in immunized mice. These results constitute valuable information on the development of a large-scale microcarrier cell culture process for producing inactivated EV71 vaccine.
肠道病毒71型（EV71）是一种可导致严重神经紊乱和死亡的肠道病毒。灭活疫苗是满足当前需求的一种合适的EV71疫苗形式。大规模制备灭活EV71疫苗依赖于用于工业大规模生产的可扩展细胞培养系统。在本文中，发现Vero细胞产生的EV71滴度高于MRC-5和WI-38细胞。使用5g/L Cytodex 1微载体建立了高密度Vero细胞微载体培养物，发现其可促进感染的Vero细胞释放EV71。对于大规模生产灭活疫苗抗原，发现2L生物反应器中产生的细胞外病毒滴度比转瓶培养低10倍，但在感染期间通过葡萄糖/谷氨酰胺补料提高了30倍。还在生物反应器中建立了无血清Vero细胞微载体培养物用于生产EV71，其滴度高达5.8×10⁷ TCID₅₀/mL。无血清培养产生的灭活病毒颗粒的免疫原性在免疫小鼠中引发了略高水平的中和抗体反应。这些结果为开发用于生产灭活EV71疫苗的大规模微载体细胞培养工艺提供了有价值的信息。
