用细胞工厂培养增殖病毒制备冻干甲型肝炎减毒活疫苗

Using Cell-Stack Apparatus to Culture Virus and to Produce Hepatitis A Attenuated Live Vaccine (Freeze-dried)

目的解决冻干甲型肝炎(甲肝)减毒活疫苗(Hepatitis A Attenuated Live Vaccine,Freeze-dried;HepA-L-Fd)原液生产的"瓶颈"问题,提高疫苗的产量。方法以转瓶培养为对照,采用细胞工厂培养人胚肺二倍体细胞,来增殖甲肝病毒,制备成试验疫苗,观察细胞的生长、病毒收获物中的病毒滴度、试验疫苗的质量,并对细胞工厂的可重复使用性进行初步研究。结果三批细胞工厂的平均细胞计数为1.16×105/cm2,其细胞产量明显高于转瓶培养(8.71×104/cm2);细胞工厂培养所得的收获物,病毒滴度的均值[8.56LgCCID50/ml(Median Infective Dose of CellCulture,50%细胞感染剂量)/毫升]比转瓶培养的病毒滴度均值(8.28LgCCID50/ml)有所提高;以此病毒收获物分别制备成的三批试验疫苗,各项检测指标均符合HepA-L-Fd质量标准。加速稳定性试验显示,疫苗在37℃存放14d后,病毒滴度仍保持稳定;未发现因重复使用细胞工厂而导致病毒滴度的下降。结论细胞工厂培养有节省培养空间、减少污染风险、缩短操作时间等优点,可替代转瓶,成为新一代用于疫苗规模化生产的细胞培养技术。

Objective To solve the bottle neck problem of bulk production of hepatitis A attenuated live vaccine （freeze-dried） and to increase the yield of vaccine. Methods To take rolling bottle as comparison, and to produce virus and vaccine for experiment using human diploid cells cultured on cell-stack apparatus （cell factory）. The growth of cell, virus titer of the harvest and quality of tested vaccine are observed The feasibility of re-use of cell factory is studied. Results The average quantity of cell cultured in cell factory is 1.16 × 10^5 / cm^2, which is apparently more than those cultured in rolling bottle （8.71 × 10^4/cmU）. The virus mean titer （8.56 LgCCID50/ml） cultured by cell factory is higher than that by rolling bottle （8.28LgCCID50/ml）. Three batches of vaccine made from the harvests are all qualified and comply with the standard of hepatitis A attenuated live vaccine （freeze-dried）, the accelerating stability test shows that the virus titer of vaccine remain steady after 14 day＇ s storing at 37℃ . The decrease of virus titer caused by-use of cell factory is not observed. Conclusion The cell factory has the advantages of saving space, reducing the risk of pollution and shortening the operation time. It could replace rolling bottle to be the new generation of cell culture technique tbr vaccine standardized production.