重组人IL-12病毒去除/灭活工艺的建立及验证  

Development and verification of procedure for virus inactivation / removal in recombinant human interleukin-12

作　　者：赵峰[1] 张宜俊 冉艳红[1] 王翠玲 李平 李弘剑[1] 

机构地区：[1]暨南大学生命科学技术学院,广东广州510632 [2]广州市恺泰生物科技有限公司,广东广州510663

出　　处：《中国生物制品学杂志》2014年第7期950-953,958,共5页Chinese Journal of Biologicals

基　　金：广东省科技攻关计划基金(00728291120224013)

摘　　要：目的建立重组人白细胞介素-12(recombinant human IL-12,rhIL-12)的病毒去除/灭活工艺,并进行验证。方法采用预过滤器(Viresolve Shield,纳米滤膜3.1 cm2)和除细小病毒过滤器(Viresolve Pro,纳米滤膜3.1 cm2)串联过滤,样品体积为90 ml,过滤时间为2 h,安全系数为1.88,压力为30 psi,建立rhIL-12病毒去除工艺,分析该工艺对产品的比活性及回收率的影响,并以猪细小病毒(porcine parvovirus,PPV)作为指示病毒,对该工艺进行病毒去除效果验证;采用低pH孵放法(用1 mol/L HCl调pH值至3.8±0.1,室温放置180 min后,用1 mol/L NaOH调pH值至7.4)建立rhIL-12病毒灭活工艺,分析该工艺对产品的蛋白含量及比活性的影响,并分别以水疱性口炎病毒(vesicular stomatitis virus,VSV)和伪狂犬病病毒(pseudorabies virus,PRV)作为指示病毒,对该工艺进行病毒灭活效果验证。结果纳米膜串联过滤病毒去除工艺中,蛋白回收率为95.14%,比活性为过滤前的95.12%,对rhIL-12活性基本无影响;当滤出液达90 ml时,PPV下降滴度均大于4 log10,符合《生物组织提取制品和真核细胞表达制品的病毒安全性评价技术审评一般原则》的要求。低pH孵放法病毒灭活工艺中,rhIL-12的蛋白含量在病毒灭活前后分别为359和338μg/ml,生物学活性分别为3.392×106和3.356×106 IU,均无明显变化;室温处理180 min后,指示病毒PRV、VSV的下降滴度均>4 log10,符合《生物组织提取制品和真核细胞表达制品的病毒安全性评价技术审评一般原则》的要求。结论建立的rhIL-12膜过滤病毒去除工艺和低pH孵放法病毒灭活工艺均可有效地去除/灭活病毒,保证了产品的质量及安全性。Objective To develop and verify the procedure for virus inactivation / removal in recombinant human interleukin-12（rhIL-12）. Methods A procedure for virus removal in rhIL-12 was developed by using pre-filter（Viresolve Shield,nano film 3. 1 cm2）combined with the filter for parvovirus（Viresolve Pro,nano film 3. 1 cm2）. The volume of sample was 90 ml,while the time for filtration was 2 h,the safety coefficient was 1. 88 and the pressure was 30 psi. The procedure was analyzed for effects on specific activity and recovery of product,and verified for efficacy of virus removal using porcine parvovirus（PPV）as an indicator. A procedure for virus inactivation in rhIL-12 was developed by low pH incubation,in which the pH value was adjusted to 3. 8 ± 0. 1 with 1 mol / L hydrochloric acid and,after incubation at room temperature for 180 min,to 7. 4 with 1 mol / L sodium hydroxide. The procedure was analyzed for effect on content and specific activity of product,and verified for the efficacy of virus inactivation using vesicular stomatitis virus（VSV）as an indicator. Results The recovery rate of protein in the developed virus removal procedure was 95. 14%,while the specific activity was 95. 12% of that before filtration. The procedure showed little influence on rhIL-12 activity. When the volume of filtrate reached 90 ml,the titer of PPV decreased by more than 4 log10,which met the General Requirements for Technology of Evaluation on Safety of Virus in Tissue Extracts and Expressed Products in Eukaryotic Cells. However,in the virus inactivation procedure,the protein contents before and after virus inactivation were 359 and 338 μg / ml,while the biological activities were 3. 392 × 106and 3. 356 × 106IU,respectively,which showed no significant change.After treatment at room temperature for 180 min,both the titers of PRV and VSV decreased by 4 log10,which met the Requirements for Technology of Evaluation on Safety of Virus in Tissue Extracts and Expressed Products in Eukaryotic Cells.Conclusion The developed

关 键 词：白细胞介素-12 过滤 低pH孵放 病毒灭活 病毒去除 

分 类 号：Q93-334[生物学—微生物学] R392-33[医药卫生—免疫学]