生物反应器培养Vero细胞制备Ⅱ型肾综合征出血热灭活疫苗

Preparation of inactivated type Ⅱ hemorrhagic fever with renal syndrome vaccines by culture of Vero cells in bioreactor

目的在生物反应器中用微载体连续灌流培养Vero细胞,制备Ⅱ型肾综合征出血热灭活疫苗。方法在50 L体积的生物反应器中,加入含10 g/L微载体的DMEM培养基,接种Vero细胞,当细胞密度约达5×10~6个/ml时,感染肾综合征出血热(hemorrhagic fever with renal syndrome,HFRS)毒种,接毒后72 h开始收获,连续收获17 d,收获的病毒原液经浓缩、灭活、Sepharose 4FF凝胶层析纯化后制备疫苗,检测病毒滴度、抗原含量及残余牛血清白蛋白、DNA、宿主蛋白含量,并接种新西兰家兔,检测疫苗效力。结果确定最佳Vero细胞接种浓度为1×10~5个/ml,病毒最佳MOI为0.015。利用优化后的培养条件收获的病毒滴度在6.5~8.0 log CCID_(50)/ml,且各项检测指标均符合《中国药典》三部(2010版)相关规定。结论用生物反应器微载体灌流培养制备Ⅱ型肾综合征出血热灭活疫苗的小试工艺可行。

Objective To establish a technology for preparation of inactivated type Ⅱ hemorrhagic fever with renal syndrome（HFRS）vaccine by continuous perfusion culture of Vero cells on microcarriers in bioreactor. Methods A 50 L bioreactor was filled with DMEM containing 10 g/L of microcarriers, on which Vero cells were inoculated. After the cell density reached 5 × 10^6cells/ml, HFRS virus was inoculated at a MOI of 0. 015, and the virus liquid was harvested 72 h later for 17 consecutive days, then concentrated, inactivated, and purified by Sepharose 4FF gel chromatography. The prepared vaccine was determined for virus titer, antigen content as well as residual bovine serum albumin, DNA and host cell protein contents, and inoculated to rabbits to determine the potency. Results The optimal Vero cell concentration for inoculation was 1 × 10^5cells/ml, while the optimal MOI of virus inoculation was 0. 015. The titer of harvested virus cultured under the optimized condition was 6. 5 ~ 8. 0 log CCID_（50）/ml, while the quality indexes met the requirements in Chinese Pharmacopoeia（Volume Ⅲ, 2010 edition）. Conclusion The pilot procedure for preparation of inactivated typeⅡHFRS vaccine by continuous perfusion culture of Vero cells on microcarriers in bioreactor was feasible.