人凝血因子Ⅷ制备工艺在放大过程中的稳定性研究

Study on the stability of human coagulation factor Ⅷ process during development

目的评价人凝血因子Ⅷ制备工艺在放大过程的稳定性。方法人凝血因子Ⅷ工艺放大依次经过BPG50小试阶段、BPG 100中试阶段和BPG 300规模化制备阶段。按《中国药典》2010版三部要求,对工艺放大过程中制品关键质量指标,如蛋白质含量、人凝血因子Ⅷ效价、吐温80残留量、磷酸三丁酯残留量、外观、可见异物、水分、热原和异常毒性等指标进行分析。结果冷沉淀溶解效果稳定,FⅧ回收率依次为(46.8±8.8)%、(46.4±8.2)%和(43.7±5.4)%(P>0.05);S/D处理对FⅧ无显著性影响,FⅧ回收率依次为(95±22)%、(111±20)%和(102±8)%(P>0.05);层析过程对杂蛋白去除效果明显,比活提高倍数分别为(52.4±23.2)、(45.8±17.2)和(55.3±5.6)倍(P>0.05),对吐温80和磷酸三丁酯去除效果稳定,Tween-80残留量分别为(10±1.8)、(8.7±3.5)和(11.3±1.5)μg·m L-1(P>0.05),磷酸三丁酯残留量分别为(0.27±0.31)、(1.2±2.6)和0μg·m L-1(P>0.05);干热处理对FⅧ活性、外观和可见异物指标无显著性影响,FⅧ回收率分别为(90±6)%、(92±4)%和(94±3)%(P>0.05)。3种规模制备的人凝血因子Ⅷ成品关键质量指标稳定。结论该工艺放大过程稳定可靠,适合于人凝血因子Ⅷ规模化制备。

Objective To evaluate the stability of human coagulation factor Ⅷ process during its development. Methods The development process of human coagulation factor Ⅷ included BPG 50 laboratory scale, BPG 100 pilot scale and BPG300 industry scale. The critical test items included protein content,FⅧ activity,Tween 80 content,tri（ n-butyl） phosphate（ TNBP） content,appearance,visible foreign matter,moisture,pyrogen test and abnormal toxicity test,which were determined according to the requirements in Chinese Pharmacopeia 2010 edition（ Volume Ⅲ）. Results During scale-up process,cryoprecipitate could be dissolved stably. The recovery rates of FⅧ were 46. 8 ± 8. 8%,46. 4 ± 8. 2% and 43. 7 ± 5. 4%（ P 〉0. 05）,respectively. The S / D treatment had no significant influence on FⅧ activity. The recovery rates of FⅧ were 95 ± 22%,111 ± 20% and 102 ± 8%（ P 〉0. 05）,respectively. The chromatographic process could remove hybrid protein,Tween 80 and TNBP obviously. The ratio of the increase in specific activity were 52. 4 ± 23. 2,45. 8 ± 17. 2 and 55. 3 ± 5. 6（ P 〉0. 05）,respectively. The residual amounts of Tween-80 were 10 ± 1. 8,8. 7 ± 3. 5 and 11. 3 ± 1. 5μg·m L^- 1（ P〉 0. 05）,respectively. The residual amounts of phosphate were 0. 27 ± 0. 31 and 1. 2 ± 2. 6 and 0μg·m L^- 1（ P〉 0. 05）,respectively. The dry-heat treatment had no significant influence on FⅧ activity,appearance nor visible foreign matter. The recovery rates of FⅧ were 90 ± 6%,92 ± 4% and 94± 3%（ P〉 0. 05）,respectively. The critical test items of human coagulation factor Ⅷ remained stable during scale-up process. Conclusion The scale-up process is stable and reliable,and is suitable for FⅧ preparation.