超大孔离子交换制备色谱分离纯化高活性凝血因子Ⅷ

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

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。

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 sub- strate, 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 chromatogra- phy and the specific activity was up to 154 IU/mg protein. Furthermore, we studied the regen- eration process of the superporous medium, washing the column with 5 colunm volumes of 1mol/L NaOH at a low flow rate, to ensure the chromatographic stability. This purification process is simple, reproducible and suitable for large-scale production.