血浆特定蛋白检测在IgG层析法中试纯化工艺中的应用  

Application of plasma specific protein determination to pilot purification of IgG by chromatography

作　　者：李策生[1] 周志军[1] 胡勇[1] 李陶敬[1] 林连珍[1] 彭焱[1] 高少阳[1] 

机构地区：[1]武汉生物制品研究所有限责任公司血液制剂研究室,湖北武汉430060

出　　处：《中国生物制品学杂志》2013年第11期1663-1667,1671,共6页Chinese Journal of Biologicals

摘　　要：目的通过血浆特定蛋白检测，对层析法纯化IgG的中试工艺进行质量控制。方法采用免疫散射比浊法及BNP特定蛋白分析仪及配套试剂，全面检测生产用和中试纯化工艺用原料血浆的组成，建立血浆中白蛋白（albumin，ALB）、IgG、IgA、IgM、补体3（compliment3，C3）、c4、转铁蛋白（transferin，TRF）、α1-抗胰蛋白酶（α1-antitrypsin，AAT）、d2巨球蛋白（alpha2-macroglobin，A2M）、触珠蛋白（haptoglobin，HPT）、α1酸性糖蛋白（alphal-acid glycoprotein，AAG）、铜蓝蛋白（ceruloplasmin，CER）、纤维蛋白原（fibfinogen，FIB）、抗凝血酶Ⅲ（antithrombinⅢ，ATⅢ）、IgG1、IgG2、IgG3、IgG4 18项特定蛋白分布的数据库；对3批中试纯化工艺各步骤中ALB、IgG、IgA、IgM、FIB、AAT的去除情况及IgG回收率进行监测，验证工艺的稳定性；将3批中试纯化工艺制备的原液及成品中IgG含量的检测结果与凯氏定氮法检测结果进行比较，验证免疫散射比浊法的准确度及不同人员重复检测的中间精密度；对中试纯化工艺及低温乙醇法制备的IgG原液的杂质含量及亚类进行比较分析。结果中试纯化工艺用原料混浆中仅TRF、A2M、ATⅢ含量与生产用原料混浆差异有统计学意义（P〈0．05）；中试纯化工艺用原料血浆经A2P亲和层析去除了大部分的ALB、AAT，辛酸盐沉淀去除了剩余的ALB、FIB和IgM，DEAE离子交换纯化去除了IgA和ALB，IgG总回收率高于70％，表明该工艺稳定性好，提高了目标蛋白的得率。中试纯化工艺制备的IgG原液及成品的检测CV值均〈10％，回收率在90％-110％之间，表明该方法准确度及精密度良好；与低温乙醇法制备的IgG原液相比，中试纯化工艺制备的原液杂质含量更低，IgG的亚类分布与所用的原料血浆相似。结论血浆特定蛋白检测在血浆蛋白纯化工艺的监控和优化中具有应用价值。Objective To control the quality of pilot purification procedure of IgG by chromatography through determina-tion of plasma specific protein. Methods The compositions of material plasma used for production and pilot purification were analyzed by immune scatter turhidimetry using Dade Behring BN ProSpec analyzer （BNP） and ancillary reagents, based on which a data bank of distributions of albumin （ALB）, IgG, IgA, IgM, complement 3 （C3）, C4, trnasferin （TRF）, α 1-antitrypsin （AAT）, α2-macroglobin （ A2M ）, haptoglobin （HPT）, α 1 -acid glycoprotein （ AAG ）, ceruloplasmin （CER）, fibrinogen（FIB）, antithrombin Ⅲ （ATⅢ）, IgG1, IgG2, IgG3 and IgG4 was established. The removals of ALB, IgG, IgA, IgM, FIB and AAT as well as recovery rate of IgG at various steps of pilot purification procedure of three batches of plasma were monitored to verify the stability of the procedure. The determination results of IgG contents in three batches of bulks and final products prepared by the pilot purification procedure were compared with those by Kjel-dahal method to verify the accuracy and precision in intermediate assay by various staff. The foreign matter contents and subclasses of bulks of IgG prepared by pilot purification procedure and cold ethanol fractionation were analyzed and com-pared. Results Only the TRF, A2M and AT m contents in pooled material plasmid used for pilot purification procedure showed significant difference with those in the plasma for production （P 〈 0. 05）. Most of ALB and AAT were removed from the plasma used for pilot purification by A2P affinity chromatography, while the residual ALB, FIB and IgM by capry-late precipitation, and IgA and ALB by DEAE ion exchange chromatography. The total recovery rate of IgG was more than 70%, indicating high stability of the procedure. The CVs of determination results of IgG contents in bulk and final product prepared by pilot purification procedure were less than 10%, while the recovery rates were 90% - 110%, indicating

关 键 词：血浆蛋白 免疫比浊法 纯化 工艺 IGG 

分 类 号：R392-33[医药卫生—免疫学]