猪瘟疫苗微载体悬浮培养生产工艺试验

Process optimization for classical swine fever vaccine production based microcarrier suspension culture

为优化猪瘟病毒(CSFV)的BT细胞悬浮培养工艺以提高CSFV抗原含量,采用2 L生物反应器对BT细胞的最佳接种密度、CSFV的最佳接种剂量进行了摸索和优化,采用优化的工艺参数,进行了BT细胞5倍消化放大工艺验证,同时对比了BT细胞悬浮培养工艺与转瓶培养工艺增殖CSFV的差异。结果表明,在3 g/L微载体浓度下,采用1.5×10~5个/mg的细胞初始接种密度,培养72 h可获得最佳细胞密度;采用MOI(感染复数)为0.5的接种剂量可收获≥106.8FAID_(50)/mL的CSFV抗原;BT细胞从2 L到10 L生物反应器的5倍消化放大工艺验证试验,3批细胞培养96 h均能达到4.0×10~6个/mL以上;悬浮培养工艺增殖的CSFV抗原含量约是转瓶培养工艺的15倍。以上试验为猪瘟疫苗的生物反应器规模化生产奠定了基础。

In order to increase the antigen content of CSFV by optimizing the manufacture process for BT cell suspension culture,BT cells were cultured in a 2 L bioreactor,and the optimal inoculation density and dosage were explored and optimized. Five times digestion and amplification process of BT cells were confirmed using the optimized parameters. Meanwhile,the difference of CSFV propagation between suspension cultured in bioreactor and roller bottles was compared. Results showed that the optimum cell density peaked at 72 h by using the initial seeding density of 1. 5 × 10~5 cells/mg under the dosage of 3 g/L microcarrier. And the virus titer of harvested CSFV antigen reached more than 106. 8 FAID_（50）/mL at the MOI of 0. 5. According to the confirmation of 5 times amplification process of BT cells cultured in bioreactor from 2 L to 10 L,the density of three batches of cells reached more than4 × 10~6 cells/mL at 96 h. Thus the antigen content of CSFV suspension cultured in bioreactor was about 15 times higher than that cultured in roller bottle. All together,the study set a foundation for the large-scale production of CSF vaccine in bioreactor.