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目的比较甘氨酸/Na Cl沉淀工艺(盐析工艺)和层析工艺制备的人凝血因子Ⅷ(FⅧ)。方法对盐析工艺和层析工艺制备的工艺过程样品和成品,采用双缩脲法检测蛋白含量,ACL7000血凝仪检测FⅧ凝固活性,并计算比活性,按《中国药典》三部(2010版)要求进行Tween-80残留量、TNBP残留量、外观、可见异物、水分、热原和异常毒性检测。结果层析工艺中以新鲜冰冻血浆为原料制备冷沉淀能较好地保证FⅧ的收率;聚乙二醇沉淀步骤能降低操作体积;层析处理能有效去除杂蛋白,使FⅧ比活至少提高25倍;S/D和80℃72 h病毒灭活处理对FⅧ质量无明显影响。两种工艺制备成品的外观、可见异物、异常毒性和热原检查均符合《中国药典》三部(2010版)相关规定,但层析工艺中FⅧ比活明显高于盐析工艺,并且对TNBP和Tween-80的去除效果更显著。结论层析工艺制备的FⅧ比活、Tween-80和TNBP残留量等关键性指标均优于甘氨酸/Na Cl沉淀工艺,该工艺更适于FⅧ的规模化生产。
Objective To compare the human factor Ⅷ (FⅧ) prepared by the glycine / NaCl precipitation process (salting out process) and the chromatographic process. Methods Samples and finished products prepared by salting-out and chromatography were determined by biuret method. The coagulation activity of F Ⅷ was detected by ACL7000 coagulation analyzer. The specific activity was calculated according to “Chinese Pharmacopoeia” (2010 edition ) Requires Tween-80 Residues, TNBP Residues, Appearance, Visible Foreign Body, Moisture, Pyrogen and Abnormal Toxicity Testing. Results The cryoprecipitate prepared by using fresh frozen plasma as raw material can better ensure the yield of FⅧ. The polyethylene glycol precipitation step can reduce the operating volume. The chromatographic treatment can effectively remove the impurity protein and increase the FⅧ specific activity 25 times; S / D and 72 h virus inactivation at 80 ℃ had no significant effect on the quality of FⅧ. The appearances of the finished products prepared by the two processes showed that foreign matter, abnormal toxicity and pyrogen test all met the requirements of the third part of the Chinese Pharmacopoeia (2010 edition), but the specific activity of FⅧ in the chromatography process was significantly higher than that of the salting-out process, And Tween-80 removal effect is more significant. Conclusion The key indexes of FⅧ specific activity, Tween-80 and TNBP residues prepared by chromatography are superior to those of glycine / NaCl precipitation, which is more suitable for the large-scale production of FⅧ.