多层细胞工厂在水痘-带状疱疹减毒毒种(OKa株)传代中的应用

Application of multilayer cell factory in subculture of attenuated varicella-zoster virus OKa strain

目的改进水痘-带状疱疹减毒活疫苗毒种(OKa株)培养方法,提高毒种的均一性及稳定性,延长保存时间。方法分别采用原培养方式(2 L方瓶培养法)和改进的培养方式(40层细胞工厂)制备水痘-带状疱疹病毒OKa株工作种子批,检测病毒滴度及-65℃以下保存1、3、6、12个月的稳定性,并用该种子批分别制备3批水痘减毒活疫苗后,进行疫苗成品的全面检定。结果 2 L方瓶培养法和40层细胞工厂制备的水痘-带状疱疹病毒工作种子批OKa-2015[1]C和OKa-2015[2]C的病毒滴度分别为6.1和6.0 lg PFU/ml;无菌试验、支原体检查、鉴别试验、外源因子检查结果均符合《中国药典》三部(2015版)相关要求;-65℃以下保存12个月,滴度均无明显下降。两种培养方式各制备的3批疫苗成品的各项检定结果均符合《中国药典》三部(2015版)相关要求,各批疫苗的病毒滴度及37℃热稳定性差别不明显。结论 40层细胞工厂制备水痘-带状疱疹病毒毒种(OKa株),-65℃以下长期保存具有稳定性,该培养方式可提高毒种的均一性,降低微生物污染风险。

Objective To improve the method for culture of OKa strain of live attenuated varicella-zoster vaccine so as to improve the homogeneity and stability and prolong the time for storage of the strain. Methods The working seeds of OKa strain was cultured in 2 L flask and 40-layer cell factory respectively, of which the titer was determined, and the stability was evaluated after storage at-65 ℃ for 1, 3, 6 and 12 months. Three batches of live attenuated vaccine were prepared with the seeds and subjected to overall control tests. Results The working seeds OKa-2015[1]C cultured in2 L flask and Oka-2015[2]C cultured in 40-layer cell factory reached titers of 6. 1 and 6. 0 lg PFU/ml respectively,of which the results of sterility test, mycoplasma test, identity test and adventitious agent test met the requirements in Chinese Pharmacopoeia（Volume Ⅲ, 2015 edition）. The titers showed no significant decrease after storage at-65 ℃ for12 months. All the quality indexes of vaccines prepared with the virus seeds cultured by two methods met the requirements in Chinese Pharmacopoeia（Volume Ⅲ, 2015 edition）, while the virus titers and stabilities at 37 ℃ of various batches showed no significant difference. Conclusion The Oka strain cultured in 40-layer cell factory showed high stability after long-term storage at-65 ℃, of which the homogeneity was improved and the risk of contamination with microorganisms decreased.