优化辛酸沉淀工艺参数后结合两步层析法分离纯化IgG效果

Effect of separation and purification of IgG by two-step chromatography after optimization of caprylic acid precipitation process

目的优化辛酸沉淀工艺参数,并结合两步层析纯化,评价10%静注人免疫球蛋白(intravenous immunoglobulin,IVIG)中杂蛋白去除效果。方法对溶解倍数、反应液终pH值、辛酸钠浓度3个因素进行优化,设计3因素3水平的正交试验方案,优选辛酸沉淀工艺参数;取100 g组分Ⅱ+Ⅲ沉淀,按最佳工艺参数进行辛酸沉淀,过滤收集上清经Q、QXP两步层析纯化后,进行IgG、IgA、IgM、Alb含量及纯度检测,评价杂蛋白去除效果。结果辛酸沉淀法最佳工艺参数为溶解倍数13倍,反应液终pH值5.1,反应液辛酸钠终浓度20 mmol/L;按最佳工艺参数获得的辛酸沉淀上清经两步层析纯化后纯度达99.99%,杂蛋白含量降至1 mg/L以下。结论辛酸沉淀法可最大限度提升IgG收率,给合后续两步层析纯化,可获得高纯度IgG。该工艺可用于临床安全的人免疫球蛋白产品开发。

Objective To optimize process parameters of caprylic acid precipitation and evaluate the removal efficiency of foreign protein in 10%intravenous immunoglobulin(IVIG)by the optimized process in combination with two-step chromatography.Methods An orthogonal test including three factors,dissolution ratio,final pH value of reaction solution and sodium caprylate concentration,at three levels were performed to optimize the process parameters of caprylic acid precipitation.A portion of 100 g fractionsⅡ+Ⅲwere subjected to caprylic acid precipitation by the optimized process,of which the supernatant was collected,purified by Q and QXP two-step chromatography,and determien d for IgG,IgA,IgM and Alb contents as well as purity to evaluate the removal effect of foreign protein.Results The optimal dissolution ratio for caprylic acid precipitation was 13,while the optimal final pH value of reaction solution was 5.1,and the optimal sodium caprylate concentration was 20 mmol/L.The purity of supernatant of caprylic acid precipitation was 99.99%after purification by two-step chromatography,and the foreign protein content decreased to less than 1 mg/L.Conclusion Caprylic acid precipitation maximized the yield of IgG,by which highly purified IgG was obtained in combination with two-step chromatography.The process may be used for the development of clinically safe human immunoglobulin products.