两种凝胶过滤介质纯化Sabin株脊髓灰质炎病毒效果的比较

Comparison of effectiveness of two kinds of gel filtration medium in purification of polio virus Sabin strain

目的比较两种不同的凝胶过滤介质对Sabin株脊髓灰质炎病毒的分离纯化效果。方法将Ⅰ、Ⅱ、Ⅲ型Sabin株脊髓灰质炎病毒收获液各3批经超滤浓缩后,分别采用Sepharose CL-6B和Sepharose 6FF填料进行凝胶过滤纯化,分段收集各纯化峰,检测D抗原及蛋白含量,计算比活性、蛋白去除率及抗原回收率,确定病毒峰收集范围。取抗原含量最高的病毒收获液,利用DEAE Sepharose FF离子交换填料进行纯化后,检测Vero细胞DNA残留量、Vero细胞蛋白残留量、牛血清白蛋白残留量、抗生素残留量。结果 Sepharose 6FF填料按6%柱体积上样进行凝胶纯化,纯化的Ⅰ、Ⅱ、Ⅲ型病毒收获液抗原回收率均值分别为78.69%、78.03%和78.37%,与Sepharose CL-6B填料按3%柱体积上样(77.29%、78.60%和77.36%)相比,差异无统计学意义(P>0.05)。两种填料纯化的各型收获液再经离子交换纯化后,蛋白去除率、比活性、Vero细胞DNA残留量、Vero细胞蛋白残留量、牛血清白蛋白残留量、抗生素残留量检测结果均符合《中国药典》三部(2015版)相关要求,两种填料纯化的各型收获液比活性差异无统计学意义(P>0.05)。结论使用Sepharose 6FF纯化的各型病毒纯化液抗原回收率和比活性与Sepharose CL-6B无明显差异,但可以减少纯化的上样次数,大幅提高纯化效率,可用Sepharose 6FF替换Sepharose CL-6B。

Objective To compare the effectiveness of two kinds of gel filtration medium in purification of polio virus Sabin strain. Methods Three batches of harvested liquids of polio virus Sabin strain of types Ⅰ, Ⅱ and Ⅲ were concentrated by ultrafilitration, and purified by Sepharose CL-6B sepharose 6FF gel filtration chromatography separately.The purification peaks were collected and determined for D antigen and protein contents to calculate the specific activity,protein removal rate and antigen recovery rate, based on which the collection range of virus peaks was confirmed. The harvested virus liquid with the highest antigen content was purified by DEAE Sepharose FF ion exchange chromatography,and determined for residual Vero cell DNA, residual Vero cell protein, residual bovine serum albumin and residual antibiotics contents. Results In Sepharose 6FF gel filtration chromatography, samples were loaded at 6% of column volume, while the mean recovery rates of virus of types Ⅰ, Ⅱand Ⅲ were 78. 69%, 78. 03% and 78. 37% respectively.However, in Sepharose CL-6B chromatography, the samples were loaded at 3% of the column volume, and the mean recovery rates were 77. 29%, 78. 60% and 77. 36% respectively, which showed no significant difference with those by Sepharose 6FF chromatography（P〉0. 05）. After further purification by ion exchange chromatography, the protein removal rate, specific activity, residual Vero cell DNA content, residual Vero cell protein content, residual bovine serum albumin content and residual antibiotics content of harvest liquids of various types met the requirements in Chinese Pharmacopoeia（Volume Ⅲ, 2015 edition）, which showed no significant difference in those previously purified by Sepharose 6FF and Sepharo se CL-6B chromatography. Conclusion The recovery rate and specific activity of virus antigen of various types purified by Sepharose 6FF chromatography showed no significant difference with those by Sepharose CL-6B chromatography. However, the times of sample loading decreased, and the purification efficacy increased remarkably, indicating that Sepharose CL-6B might be substituted with Sepharose 6FF.