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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(compliment 3,C3)、C4、转铁蛋白(transferin,TRF)、α1-抗胰蛋白酶(α1-antitrypsin,AAT)、α2巨球蛋白(alpha2-macroglobin,A2M)、触珠蛋白(haptoglobin,HPT)、α1酸性糖蛋白(alpha1-acid glycoprotein,AAG)、铜蓝蛋白(ceruloplasmin,CER)、纤维蛋白原(fibrinogen,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 test the quality of the pilot-scale process for the purification of IgG by chromatography. Methods Immunofluorescence nephelometry and BNP-specific protein analyzer and reagents were used to detect the composition of plasma in the production and pilot-scale purification processes. The levels of albumin (ALB), IgG, IgA, IgM, complement 3, C3, C4, transferin (TRF), α1-antitrypsin (AAT), alpha2-macroglobin (A2M), haptoglobin HPT, alpha1-acid glycoprotein (AAG), ceruloplasmin (CER), fibrinogen (FIB), antithrombinⅢ (ATⅢ), IgG1, IgG2 and IgG3 , IgG4 18 specific protein distribution database; the removal of ALB, IgG, IgA, IgM, FIB, AAT and IgG recovery rate in each of three batches of the pilot purification process were monitored to verify the stability of the process; three batches Pilot test purification solution prepared from the finished product and the IgG content of the test results and Kjeldahl method test results were compared to verify the accuracy of immune nephelometry and repeated testing of different personnel of the intermediate precision; And low temperature ethanol prepared IgG original Comparative analysis of impurity content and subclasses. Results There were significant differences in the contents of TRF, A2M and ATⅢ between the raw materials and the raw materials during the pilot-scale purification process (P <0.05). The plasma from the pilot-scale purification process was removed by A2P affinity chromatography Part of the ALB, AAT, caprylate precipitation to remove the remaining ALB, FIB and IgM, DEAE ion exchange purification to remove IgA and ALB, IgG recovery was higher than 70%, indicating that the stability of the process and improve the target protein The yield. The CV and CV values of both the IgG stock solution and the finished product prepared by the pilot-scale purification process were both less than 10% and the recoveries were between 90% and 110%, indicating that the method was accurate and accurate. Compared with the IgG solution prepared by the low- Compared with the pilot-scale purification process, the original liquid impurity content is lower, and the subclass of IgG is similar to the raw material plasma used. Conclusion The detection of plasma specific proteins has application value in the monitoring and optimization of plasma protein purification process.