全层析工艺制备10%静脉注射用水痘-带状疱疹人免疫球蛋白的质量控制  

Quality control of 10%human varicella-zoster immunoglobulin for intravenous injection prepared by full chromatography

作　　者：周志军 岳胜兰 彭焱 陈克金 陈曦 李娟 汪菲菲 李策生 胡勇 张金 ZHOU Zhijun;YUE Shenglan;PENG Yan;CHEN Kejin;CHEN Xi;LI Juan;WANG Feifei;LI Cesheng;HU Yong;ZHANG Jin(Scientific Research and Development,Sinopharm Wuhan Biopharmaceuticals Co.,Ltd.,Wuhan 430207,Hubei Province,China)

机构地区：[1]国药集团武汉生物制药有限公司科研开发部,湖北武汉430207

出　　处：《微生物学免疫学进展》2024年第5期48-55,共8页Progress In Microbiology and Immunology

基　　金：湖北省中国科学院科技合作专项。

摘　　要：目的用全层析工艺制备10%静脉注射人水痘-带状疱疹免疫球蛋白[human varicella zoster immune globulin for intravenous administration,VZIG(IV)],对其纯化过程的关键步骤进行质量控制(包括IgG相对纯度、抗原回收率、杂质去除情况及半成品检定)。方法取100袋含高效价水痘-带状疱疹病毒(varicella-zoster virus,VZV-IgG),ELISA效价>1.500 IU/mL的原料血浆融化后制备约50 L合并血浆,按照全层析工艺进行10%VZIG(IV)的制备,对其关键步骤样品用免疫比浊法进行特定血浆蛋白的蛋白含量检测,计算目标组分中各种蛋白的占比、IgG步骤回收率和工艺总回收率;用BCA法进行总蛋白含量检测,计算IgG相对纯度;对合并血浆和半成品按照《中华人民共和国药典》2020版(三部)(简称《中国药典》)要求进行关键项目(蛋白含量、分子大小分布、纯度、抗A抗B血凝素、激肽释放酶原激活剂)检测,同时增加半成品的辛酸钠、IgG亚类、IgA、IgM、ALB的蛋白含量检测,合并血浆及半成品的VZV-IgG的ELISA效价检测。结果全层析工艺中A50吸附主要去除的是CER、FNC、C1I,去除率分别是39.321%、46.407%、74.132%;IgG步骤回收率为97.44%,相对纯度为15.17%。亲和层析主要去除了血浆中大部分杂蛋白,其洗脱液中主要成分为IgG和FIB(占比分别为69.891%和17.994%),其他蛋白占比均<5%,IgG的步骤回收率为69.47%,相对纯度为63.86%。辛酸盐沉淀主要去除了上一步残留的大部分杂质蛋白,仅有微量IgM(占比0.607%)和ALB(占比0.229%)残留,IgG的步骤回收率为81.00%,相对纯度为98.98%。DEAE离子交换层析主要去除的是IgM,IgG的步骤回收率为95.69%,相对纯度为95.01%。半成品的相对纯度为98.79%,整个工艺IgG的总回收率达到46.26%。全层析工艺将原料血浆的VZV-IgG的ELISA效价提高了18.33倍,抗原浓度浓缩了10.8倍,比活性从0.047 IU/mg提高到0.533 IU/mg。半成品各项关键指标检测合格,辛酸钠残留量<Objective To prepare 10%human varicella-zoster immunoglobulin for intravenous administration[VZIG(IV)]by full chromatography.Quality control of key steps in the purification process(including IgG relative purity,antigen recovery,impurity removal and final bulk testing)was performed.Methods 100 bags of plasma with high potency varicella-zoster virus antibody(VZV-IgG)(ELISA potency>1.500 IU/mL)were melted to prepare about 50 liters pool plasma,and 10%VZIG(IV)was prepared by full chromatography process.Specific plasma protein antigen content was detected by immunoturbidimetry in key step samples,and total protein content was detected by BCA method.The proportion of various proteins in target components,relative purity of IgG,step recovery and total process recovery of IgG antigen were calculated.According to the requirements of Pharmacopoeia of the People's Republic of China(2020 Edition,PartⅢ)(referred to as Chinese Pharmacopoeia),part of key items(protein content,molecular size distribution,purity,anti-A and anti-B hemagglutinin,kallikrein-zymogen activator)were detected for final bulk,at the same time,the content detection of Sodium octanoate,IgG subclasses,IgA,IgM and ALB in final bulk were added,and the potency of VZV-IgG in pool plasma and final bulk were detected.Results The A50 adsorption in the full chromatography process mainly removed CER,FNC and C1I,the removal rates were 39.321%,46.407%and 74.132%respectively,the step recovery of IgG was 97.44%and the relative purity was 15.17%.The affinity chromatography mainly removed most of the impurity proteins in plasma.The main components in the eluent were IgG and FIB(accounting for 69.891%and 17.994%respectively),and the other proteins accounted for less than 5%.The step recovery of IgG was 69.47%and the relative purity was 63.86%.Most of the impurity proteins remaining in the previous step were removed by caprylate precipitation,and only trace amount of IgM(0.607%)and ALB(0.229%)remained.The step recovery of IgG was 81.00%,and the relative purity was 98.98%;

关 键 词：10%静脉注射用水痘-带状疱疹人免疫球蛋白 全层析工艺 特定蛋白检测 BCA试验 杂质去除 回收率 比活性 

分 类 号：R392-33[医药卫生—免疫学]