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目的探讨篮式生物反应器制备森林脑炎灭活疫苗(Vero细胞)。方法应用14 L篮式生物反应器,以Vero细胞作为森林脑炎病毒增殖的细胞基质,采用片状载体对Vero细胞进行高密度培养,探讨不同细胞接种浓度对生物反应器内细胞密度及病毒产量的影响。连续收获的森林脑炎病毒液经超滤浓缩、1∶4 000(v/v)β-丙内酯灭活、柱层析纯化后,获得疫苗原液,按照《中国药典》三部(2010版)进行相关检测。结果用(5~7.5)×105个/ml细胞浓度接种Vero细胞培养森林脑炎病毒可连续收获30 d以上,平均病毒滴度为8.4 lg LD50/ml;1∶4 000(v/v)β-丙内酯灭活24 h可完全灭活病毒,3批病毒浓缩液灭活验证试验均未检出残余活病毒;柱层析纯化后杂蛋白去除率可达89.56%;疫苗原液经无菌检查合格,其他各项质量指标均符合《中国药典》三部(2010版)规定。结论应用篮式生物反应器培养Vero细胞制备了合格的森林脑炎灭活疫苗,该工艺适用于森林脑炎灭活疫苗的规模化生产。
Objective To investigate the preparation of rabies encephalitis inactivated vaccine (Vero cells) by a basket bioreactor. Methods Using 14 L basket bioreactor, Vero cells were used as the cell matrix for the proliferation of forest encephalitis virus. The Vero cells were cultured in high density using the sheet carrier. The cell density and the virus production in the bioreactor Impact. Successive harvest of forest encephalitis virus solution was concentrated by ultrafiltration, 1: 4000 (v / v) β-propiolactone inactivated, purified by column chromatography to obtain the vaccine stock solution, according to “Chinese Pharmacopoeia” three ) For the relevant testing. Results Vero cells were inoculated with Vero cells at a concentration of (5 ~ 7.5) × 105 cells / ml for continuous harvesting for more than 30 days with an average virus titer of 8.4 lg LD50 / ml and 1: 4000 (v / v) β - inactivation of propiolactone for 24 h completely inactivated virus, three batches of virus concentrate inactivation validation test were not detected residual virus; Purification by column chromatography after removal of contaminated proteins up to 89.56%; Vaccine stock solution by sterile Inspection qualified, the other quality indicators are in line with “Chinese Pharmacopoeia” three (2010 version) requirements. Conclusions The qualified inactivated forest encephalitis vaccine was prepared by using the borage bioreactor to culture Vero cells. This process is suitable for the large-scale production of forest encephalitis inactivated vaccine.