摘要
目的应用7.5 L生物反应器,培养Vero细胞和乙型脑炎病毒,规模化生产乙型脑炎灭活疫苗。方法应用7.5 L生物反应器,分别以5、10、15、20、25 g/L的微载体密度加装,采用灌流方式培养Vero细胞,分别将P3株乙型脑炎病毒按0.005 00、0.002 50、0.001 60、0.001 25、0.001 00 MOI接种至生物反应器内,收获乙脑病毒液。经浓缩、灭活、纯化等工艺制备疫苗半成品,经疫苗灭活及纯化试验进行工艺验证,合格后分装为疫苗成品,按照《中国药典》三部(2010版)中《冻干乙型脑炎灭活疫苗(Vero细胞)》要求,进行疫苗全项检定。结果当微载体密度为20、25 g/L时,细胞密度可达1.2×107个/ml,病毒滴度平均达8.33～8.52 lgLD50/ml;当病毒接种量为0.001 25 MOI时,病毒最高滴度可达9.02 lgLD50/ml。经超滤浓缩后的病毒原液,使用1∶4 000β-丙内酯灭活72 h,可达到灭活效果;纯化后可去除90%以上杂蛋白。应用7.5 L生物反应器生产的6批乙型脑炎灭活疫苗,经检定各项指标均符合国家要求。结论应用7.5 L生物反应器培养Vero细胞和P3株乙型脑炎病毒,经连续灌流收获,可规模化生产Vero细胞乙型脑炎灭活疫苗。
Objective To culture Vero cells and Japanese encephalitis virus（JEV） and produce inactivated JE vaccine in a large scale by using 7.5 L bioreactor.Methods A 7.5 L bioreactor was loaded with microcarriers at densities of 5,10,15,20 and 25 g / L respectively,on which Vero cells were cultured by continuous perfusion and inoculated with JEV P3 strain at MOIs of 0.005 00,0.002 50,0.001 60,0.001 25 and 0.001 00 respectively.The virus liquid was harvested,concentrated,inactivated and purified to prepare the final bulk of JE vaccine,and the procedure was verified by vaccine inactivation and purification tests.The final bulk qualified in control tests were distributed to obtain final product which was subjected to overall control tests according to the requirements in monograph of Japanese Encephalitis Vaccine（Vero cell）,Inactivated,Freeze-dried in Chinese Pharmacopoeia（VolumeⅢ,2010 edition）.Results When the densities of microcarriers were 20 and 25 g / L,the density of Vero cells reached 1.2 × 107 cells / ml,while the mean virus titer was 8.33 ~ 8.52 lgLD50 / ml.However,when the MOI of virus was 0.001 25,the virus titer reached 9.02 lgLD50 / ml at most.The virus bulk concentrated by ultrafiltration was effectively inactivated after treatment with β-propionolactone at a dilution of 1 ∶ 4 000 for 72 h.Ninety percent of foreign protein was removed after purification.All the quality indexes of six batches of vaccine prepared by using 7.5 L bioreactor met the relevant national requirements.Conclusion Inactivated JE vaccine was produced in a large scale by inoculation of JEV P3 strain into Vero cells cultured in 7.5 L bioreactor by continuous perfusion.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第11期1414-1416,共3页
Chinese Journal of Biologicals
