目的研究在不同工艺参数下,使用超滤膜包和中空纤维柱超滤浓缩基于MDCK细胞培养的B型流感病毒液的效果。
方法制备6批次基于MDCK细胞培养的B型流感病毒澄清液，采用超滤膜包和中空纤维柱对B型流感病毒澄清液2倍浓缩后洗滤。取鸡红细胞悬液检测病毒浓缩液的血凝滴度，使用Lowry法2测定蛋白含量并计算抗原回收率和杂蛋白去除率，以考察不同洗滤次数和超滤膜类型对以上指标的影响；对洗滤10次的病毒洗滤液进行超滤浓缩，分析使用2种超滤膜超滤浓缩后不同膜清洗次数下的病毒残留情况；评价病毒浓缩液在不同保存温度下的稳定性。
结果超滤膜包和中空纤维柱洗滤后，病毒洗滤液的血凝滴度保持稳定，洗滤次数为5次时，杂蛋白去除率较高，达到90%以上。中空纤维柱处理后的病毒浓缩液抗原回收率（61.95%）略高于超滤膜包（49.91%），2种超滤膜处理后收获的病毒浓缩液杂蛋白去除率均在96%左右。冲洗超滤浓缩后的超滤膜包和中空纤维柱1次，超滤系统中含有残留的病毒抗原，占病毒澄清液抗原含量的10%以上。2种超滤膜处理后的病毒浓缩液在2～8 ℃保存1周血凝滴度稳定，在-70 ℃保存1周后血凝滴度大幅度下降。
结论超滤膜包和中空纤维柱洗滤病毒澄清液的次数在5次及以上时，可获得较高病毒含量、低杂蛋白含量的病毒液。
ObjectiveTo study the effect of ultrafiltration concentration of influenza B virus liquid based on MDCK cell culture using ultrafiltration membrane package and hollow fiber column under different process parameters.
MethodsSix batches of influenza B virus clarified liquid based on MDCK cell culture were prepared. Ultrafiltration membrane packages and hollow fiber columns were used to make 2× concentrated influenza B virus clarified liquid, which was then washed and filtered. Hemagglutination titers of the virus concentrates were measured using chicken erythrocyte suspensions, protein content was determined by Lowry method 2 for the caculation of antigen recovery and miscellaneous protein removal rate, in order to investigate the effects of different washing times and types of ultrafiltration membranes on the above indices. Ultrafiltration concentration was carried out on the virus filtration solution that was washed and filtered 10 times to analyze the virus residue under different membrane cleaning times after ultrafiltration concentration using two types of ultrafiltration membranes. The stability of the virus concentrates stored at various temperatures was evaluated.
ResultsAfter filtration through the ultrafiltration membrane package and hollow fiber column, the hemagglutination titer of the virus filtrate remained stable, and the miscellaneous protein removal rate was relatively high at 5 times of filtration, reaching more than 90%. The antigen recovery rate of virus concentrates treated by hollow fiber column (61.95%) was slightly higher than that of ultrafiltration membrane package (49.91%), while the overall protein removal rates of the virus concentrates using both types of membranes were around 96%. After the ultrafiltration membrane package and hollow fiber column were washed once following ultrafiltration concentration, residual virus antigen in the ultrafiltration system accounted for over 10% of the antigen content of the virus clarification liquid. The virus concentrates processed with two types of membranes showed high stability after being stored at 2-8 ℃ for 1 week, while the hemagglutination titer dropped dramatically after being stored at -70 ℃ for 1 week.
ConclusionWhen the virus clarification liquid is washed and filtered using ultrafiltration membrane packages and hollow fiber columns 5 times and above, the virus solution obtained has high viral content and low heterogeneous protein content.
吴熠潇,刘海东,朱绍荣. 基于MDCK细胞培养的B型流感病毒液的超滤浓缩工艺研究[J]. 国际生物制品学杂志,2025,48(01)：26-31.
DOI:10.3760/cma.j.cn311962-20240730-00052版权归中华医学会所有。
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吴熠潇、刘海东：实验设计、研究实施、数据采集、数据分析、统计分析、论文撰写；朱绍荣：研究指导、论文修改、经费获取
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