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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 the residual DNA in rabies vaccine production with VERO cells by optimizing the production process. Methods Antibody recovery was evaluated by double antibody sandwich ELISA and residual DNA was detected by DNA probe hybridization. The key factors of ultrafiltration, including the selection of ultrafiltration membrane, the ultrafiltration pressure and the concentration ratio, were evaluated by the detection of antigen recovery rate and DNA residual rate. The effects of protamine pretreatment The effect of the filtrate on these two indicators. RESULTS AND CONCLUSIONS The optimum conditions of ultrafiltration were obtained by comprehensively recovering antigen, DNA removal rate and production factors. The optimal conditions for ultrafiltration were as follows: entrapment of ultrafiltration membrane with relative molecular mass of 7.5 × 10 5, 20-fold enrichment and 15 psi UF pressure as the core parameters of ultrafiltration process. Through the pretreatment of protamine, it is initially proved that the ultrafiltration retentate intercepts the DNA through the molecular sieve mode, and the addition of protamine can bind the fragmented DNA to form a larger molecular fragment, thereby facilitating the ultrafiltration retention.