离子交换层析法制备高纯度人凝血因子Ⅷ的研究

Preparation of high purity human coagulation factor Ⅷ using ion exchange chromatography

目的：从血浆冷沉淀中采用离子交换层析法纯化人凝血因子Ⅷ。方法：冰冻血浆在2~8℃环境下融化,离心法收获冷沉淀。用注射用水溶解冷沉淀,探索了5种冷沉淀溶解液预纯化的方法。冷沉淀溶解液经S/D病毒灭活后采用离子交换层析纯化人凝血因子Ⅷ,经超滤、配制、除菌分装、冻干、干热病毒灭活,获得人凝血因子Ⅷ成品,分别检测流穿液、洗涤液和洗脱液中人凝血因子Ⅷ效价和蛋白含量。观察了冻干和不同干热条件前后凝血因子Ⅷ活性的变化。观察人凝血因子Ⅷ冻干品的外观和复溶时间。结果：冷沉淀中人凝血因子Ⅷ的回收率为40%~70%。5种冷沉淀溶解液预纯化方法中,用盐酸调节p H至6.6、6.3的方法人凝血因子Ⅷ回收率较高。离子交换层析流穿液中FⅧ损失率小于5%,而洗涤液中FⅧ损失率较高,最高损失率超过20%。人凝血因子Ⅷ中蛋白质含量较低,均小于0.65 mg·m L-1,比活性为50~100IU·m L-1。吐温-80和TNBP的残留量均符合《中国药典》的要求。冻干过程中人凝血因子Ⅷ效价下降大约1%~5%。100℃,30 min干热处理对FⅧ效价的破坏小于80℃,72 h。人凝血因子Ⅷ冻干品外观和复溶时间均达到中国药典标准。结论：采用离子交换层析法从血浆冷沉淀中分离出高纯度人凝血因子Ⅷ,可以进行工艺放大研究。

Objective： To purify coagulation factor Ⅷ from human plasma using ion exchange chromatography. Methods： The cryoprecipitate was harvested by using centrifugation after thawing of fresh frozen plasma under2 ~ 8 ℃. The cryoprecipitate was dissolved with water for injection,and five prepurification methods of dissolved cryoprecipitate liquid were explored. The coagulation factor Ⅷ was purified by using ion exchange chromatography after inactivation of virus followed by ultrafiltration,preparation,aseptic packaging,lyophilization,and dry heat inactivation of virus to obtain the final products. The activity and protein content of coagulation factor Ⅷ in the flowing through liquid,washing liquid,and elution liquid were determined. The activities of coagulation factor Ⅷ products undering lyophilizatio and dry heat inactivation of virus were compared. The appearance and dissolving time of coagulation factor Ⅷ were observed. Results： The recovery of coagulation factor Ⅷ activity was 40% ~ 70% from frozen plasma after centrifugating. The recoveries at p H 6. 6 and 6. 3 were higher among the five methods. The loss ratio of flow though was less than 5%,and the top loss ratio of washing reached 20%. The protein content of coagulation factor Ⅷ was less than 0. 65 mg·m L- 1,and the specific activity was 50 ~ 100 IU·m L- 1. The residue amount of Teween-80 and tributyl phosphate conformed to the standard of Chinese Pharmacopoeia. The activity of coagulation factor Ⅷ decreased by about 1% ~ 5% after lyophilizatio,and the activity was decreased in a smaller magnitude by dry heat under the condition of 100 ℃ 30 min than that under 80 ℃ 72 h. The appearance and dissol-ving time of coagulation factor Ⅷ purified by ion exchange chromatography met the standards. Conclusion： High purity coagulation factor Ⅷ can be obtained by using ion exchange chromatography and this process can be scaled-up.