超大孔离子交换制备色谱分离纯化高活性凝血因子VIII

建立了一条从人血浆中分离高活性凝血因子VIII(FVIII)的纯化工艺。基于FVIII和介质孔径的尺度比及其对蛋白质活性影响的分析,设计了以超大孔离子交换制备色谱为核心步骤的新型分离纯化工艺。分别进行超大孔离子交换色谱与传统离子交换色谱的条件优化,并对优化工艺所得产品进行了活性检测(底物显色法)和纯度检测(高效凝胶过滤和凝胶电泳)。结果表明,超大孔介质结构不但可以有效地保护蛋白质大分子结构,而且能够大幅度地提高制备色谱的传质速率,从而得到具有高凝血活性的FVIII产品。FVIII在超大孔制备色谱过程中的回收率(85%)比传统离子交换制备色谱高4～5倍,产品比活高达154 IU/mg。此外,还研究了超大孔介质的再生程序,采用5个柱体积的1 mol/L NaOH低流速清洗色谱柱,保证了色谱工艺的稳定性。本纯化工艺步骤简单,重现性好,易于放大生产。

Separation of coagulation factor VIII with high activity using gigaporous anion exchange chromatography

A purification process to obtain coagulation factor Ⅷ(FⅧ) with high activity from human plasma was established.Based on the analysis of the size ratio between FⅧ and matrix porous medium and its effect on the protein activity,a novel purification process designed was superporous ion exchange chromatography(IEC).The operating conditions of gigaporous and traditional anion exchange chromatography were optimized separately.The chromogenic substrate,gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) were used to monitor the bioactivity and purity of the chromatographic products.The results showed that the superporous medium could not only protect structure of macro-protein but also enhance its mass transfer,finally giving FⅧ product with high activity.The yield of FⅧ in superporous chromatography was about five times of commercially agarose chromatography and the specific activity was up to 154 IU/mg protein.Furthermore,we studied the regeneration process of the superporous medium,washing the column with 5 column volumes of 1 mol/L NaOH at a low flow rate,to ensure the chromatographic stability.This purification process is simple,reproducible and suitable for large-scale production.