去除无细胞百日咳疫苗丝状血凝素纯化液中内毒素TritonX-114液相分离法的建立及其效果评价

Establishment and effect evaluation of TritonX⁃114 liquid phase separation method for removal of endotoxin in purified filamentous hemagglutinin of acellular pertussis vaccine

目的建立去除无细胞百日咳疫苗丝状血凝素(filamentous hemagglutinin,FHA)纯化液中内毒素的TritonX-114液相分离法,并评价其效果。方法以影响TritonX-114液相分离法效果的TritonX-114加入量、4℃混匀时间、37℃分层时间、25℃16743×g离心时间为试验因素,每个因素设计3个水平,通过正交试验设计12组试验。每组试验获得的样品均检测蛋白回收率,以蛋白回收率最高的条件为最佳。采用最佳条件去除3批无细胞百日咳疫苗FHA纯化液内毒素,检测FHA蛋白含量、内毒素含量及TritonX-114残留量。结果TritonX-114液相分离法最佳条件:TritonX-114加入量为1%,4℃混匀时间为30 min,37℃分层时间为60 min,25℃16743×g离心时间为10 min。最佳条件下去除3批无细胞百日咳疫苗FHA纯化液内毒素后,内毒素含量均<0.25 EU/mL,TritonX-114残留量均值为0.183%,蛋白回收率均值为91.04%。结论TritonX-114液相分离法能够有效去除无细胞百日咳疫苗FHA纯化液中的内毒素,且方法耗时短,操作简便。

Objective To develop the TritonX-114 liquid phase separation method for removal of endotoxin in purified filamentous hemagglutinin(FHA)of acellular pertussis vaccine and evaluate its effect.Methods Twelve groups of test were designed by orthogonal experimental design on the influencing factors of TritonX-114 liquid phase separation,including dosage of TritonX-114,time for mixing up at 4℃,time for separation at 37℃and time for centrifugation at 16743×g and 25℃,three levels for each.The samples obtained from each group were determined for recovery rate of protein,and the condition under which the recovery rate was the highest was served as optimal one.After removal of endotoxin under the optimal condition,three batches of purified FHA of acellular pertussis vaccine were determined for FHA content,endotoxin content and residual Triton X-114 content.Results The optimal dosage of TritonX-114 for removal of endotoxin in purified FHA of acellular pertussis vaccine was 1%,while the optimal time for mixing up at 4℃was 30 min,the optimal time for separation at 37℃was 60 min,and the optimal time for centrifugation at 16743×g and 25℃was 10 min.After removal of endotoxin under the optimal condition,all the endotoxin contents in three batches of purified FHA of acellular pertussis vaccine were less than 0.25 EU/mL,while the mean residual TritonX-114 content was 0.183%,and the mean protein recovery rate was 91.04%.Conclusion TritonX-114 liquid phase separation method is effective in removal of bacterial endotoxin from purified FHA of acellular pertussis vaccine,which is time-saving and simple.