带状疱疹疫苗生产中2BS细胞细胞工厂培养工艺的建立

Development of culture procedure for 2BS cell factory during production of herpes zoster vaccine

目的建立带状疱疹疫苗生产中2BS细胞在细胞工厂中的培养工艺。方法将2BS细胞复苏(28代)后,连续传代至35、36代,再分别按1∶4、1∶2比例传至10层细胞工厂中,采用不同的加液量(200、300、400 ml/层)进行培养,显微镜下观察细胞形态,并进行细胞计数。取细胞工厂,按MOI 0.008接种水痘-带状疱疹病毒,制备成带状疱疹减毒活疫苗,检测疫苗原液及成品的滴度;疫苗成品进行37℃稳定性试验;采用酶联免疫法检测抗生素残留量和牛血清白蛋白残留量;凝胶法检测内毒素含量;水分容量滴定法检测水分含量。结果 2BS细胞按1∶2比例传代,细胞工厂单层加液量为300 ml,可制备单层致密、优质的细胞,细胞工厂收获时细胞病变达80%以上,且病变细胞圆润、典型、广泛,细胞形态保持较好;传代比例为1∶4的细胞制备的疫苗原液及成品的滴度明显低于传代比例为1∶2的细胞制备的疫苗,且1∶4传代的细胞制备的疫苗成品37℃稳定性滴度不符合带状疱疹疫苗放置1周病毒滴度不小于4.6 lg PFU/ml的质量标准;其他各项质量指标均符合要求。结论建立了带状疱疹疫苗生产中2BS细胞在细胞工厂中的培养工艺,利用细胞工厂可制备出质量均一、致密优质的2BS细胞。

Objective To develop a culture procedure for 2BS cell factory during production of herpes zoster vaccine.Methods The 2BS cells were resuscitated（passage 28）and subcultured to passages 35 and 36,then to the ten layer cell factory at mass ratios of 1 ∶ 4 and 1 ∶ 2 respectively. The cells on each layer were added with various volumes（200,300 and400 ml） of medium,observed for morphology by microscopy and counted. The cell factory was inoculated with varicellazoster virus at a MOI of 0. 008 and prepared into live attenuated vaccine. The bulk and final product of vaccine were determined for titers. The final product was subjected to stability test at 37 ℃. The residual antibiotics and bovine albumin contents were determined by ELISA,while the endotoxin content by gel method,and the moisture content by volume titration method. Results Dense 2BS cell monolayer with high quality was prepared by subculture at a mass ratio of 1 ∶ 2 and addition of 300 ml of medium,of which the CPE reached more than 80% at time of harvest. The cells with CPE were round,most of which were in typical shape. The titers of bulk and final product of vaccine prepared with cells subcultured at a mass ratio of 1 ∶ 4 were significantly lower than those at 1 ∶ 2. However,the titer in stability test at 37 ℃ of final product of vaccine prepared with the cells subcultured at a mass ratio of 1 ∶ 4 failed to met the requirements of not less than 4. 6 lg PFU / ml after storage for one week. Other indexes met the relevant requirements. Conclusion A culture procedure for 2BS cell factory during production of herpes zoster vaccine was developed,by which dense 2BS cells with even and high quality were prepared.