超滤截留狂犬疫苗残留DNA的工艺优化和机制探讨

Mechanism and process optimization of ultrafiltration of residual DNA in rabies vaccine

目的通过优化生产工艺来控制以VERO细胞为载体的狂犬疫苗生产中的残留DNA。方法采用双抗体夹心ELISA法检测评估抗原回收率,通过DNA探针杂交法检测残留DNA。采用检测抗原回收率和DNA残留率对狂犬疫苗生产中的核心环节-超滤的3个关键因素:超滤膜包的选择、超滤压力以及浓缩倍数进行评估,并评价鱼精蛋白预处理超滤液对于上述两指标的影响。结果与结论综合抗原回收率、DNA去除率以及生产层面的因素,获得了超滤工艺的最优条件,即以截留相对分子质量为7.5×105的超滤膜包、20倍浓缩和15 psi的超滤压力作为超滤工艺的核心参数。通过鱼精蛋白的预处理,初步探明超滤截留是通过分子筛的模式截留DNA,而鱼精蛋白的加入可以结合碎片化的DNA,形成较大的分子片段,从而更利于超滤截留。

Objective To control residual DNA by optimizing methodology during the production of rabies vaccine using Vero cells as a vector.Methods The antigen recovery rate was assessed by linked immunosorbent assay-sandwich technique while the residual DNA was detected by DNA probe hybridization method.Antigen recovery and removal of DNA were the main indexes for evaluateing ultrafiltration,the vital part of rabies vaccine production.Three key factors in ultrafiltration were assessed：selection of membrane packages,ultrafiltration pressure and the concentration ratio.Then protamine was used to pretreat ultrafiltrates.Based on the two indicators mentioned above,the effect of protamine pretreatment on the ultrafiltrate was evaluated.Results and Conclusion The optimum condition of ultrafiltration was obtained on the basis of the general antigen recovery rate,DNA removal rate and actual production.The primary parameters of ultrafiltration were as follows：7.5 × 10^5 ultrafiltration membrane packages,20 times concentrated,15 psi ultrafiltration pressure.After pretreatment with protamine,ultrafiltration has proved to be a molecular sieve in intercepting DNA,while protamine can tangle the fragmented DNA and form a larger molecular segment,which is believed to be more conducive to ultrafiltration interception.