人血浆载脂蛋白AⅠ冻干品干热法灭活病毒的效果验证  

Verification of virus inactivation efficacy in freeze-dried human plasma-derived apolipoprotein A Ⅰ by dry heat method

作　　者：卞炯[1] 何毅明[1] 吴玮[1] 刁武平[1] 朱威[1] 

机构地区：[1]上海生物制品研究所有限责任公司血液制剂室,上海200052

出　　处：《中国生物制品学杂志》2014年第4期559-563,共5页Chinese Journal of Biologicals

摘　　要：目的采用干热法对纯化的人血浆载脂蛋白AⅠ(apolipoprotein AⅠ,ApoAⅠ)冻干品进行病毒灭活处理,并对灭活效果进行验证。方法将脂包膜病毒伪狂犬病病毒(pseudorabies virus,PRV)、辛德毕斯(Sindbis)病毒及非脂包膜病毒脑心肌炎(encephalomyocarditis,EMC)病毒按1∶9(v∶v)的比例分别加至3批ApoAⅠ样品中,冻干后于水浴中(100±1)℃加热30 min灭活,分别于加热后5、10、20、30 min取样,检测病毒滴度,绘制病毒灭活动力学曲线,并检测干热法灭活病毒对目的蛋白理化性质及生物活性的影响。结果干热法对ApoAⅠ样品中的脂包膜和非脂包膜病毒均可有效灭活,灭活20 min后,3种指示病毒的滴度均迅速下降;干热法灭活后,冻干品的外观、复溶时间、复溶后澄明度、目的蛋白的平均回收率(95.44%)均符合要求,样品的SDS-PAGE、Western blot图谱、免疫电泳行为、二级结构和生物活性均无显著变化。结论干热法能有效灭活脂包膜和非脂包膜病毒;在ApoAⅠ纯化工艺中采用S/D法和干热法两步病毒灭活步骤,可保证制品的安全性。Objective To inactivate the virus in freeze-dried apolipoprotein A Ⅰ （ApoA Ⅰ ）, prepared from purified human plasma, by dry heat method, and verify the inactivation efficacy. Methods Three batches of samples of ApoA Ⅰ were added with lipid-enveloped pseudorabies virus （PRV） and Sindbis virus as well as non-lipid enveloped encephalomyocarditis （EMC） virus, each at a ratio of 1 ： 9 （v ： v）, respectively, then lyophilized and incubated in （100±1）℃ water bath for 30 min. Samples were taken 5, 10, 20 and 30 rain after incubation and determined for virus titer, based on which the dynamic curve of virus inactivation was plotted, and the effects of virus inactivation by dry heat on physiochemical properties and biological activity of target protein were evaluated. Results Both lipid-enveloped and non-lipid enveloped viruses in ApoA I were effectively inactivated by dry heat method. The titers of three viruses decreased significantly after inactivation for 20 min. The appearance, time for reconstitution, clarity after reconstitution and average recovery rate （95. 44%） of target protein after inactivation met the relevant requirements, while the SDS-PAGE profile, Western blot profile, immunoelee- trophoretic behavior, secondary structure and biological activity of samples showed no obvious change. Conclusion Dry heat method was effective for inactivation of lipid-enveloped and non-lipid enveloped viruses. Two step virus inactivation by S / D and dry heat method in purification of ApoA Ⅰ may ensure the safety of product.

关 键 词：载脂蛋白A Ⅰ血浆 冻干品 灭活 干热法 生物活性 

分 类 号：R392.33[医药卫生—免疫学]