人凝血因子Ⅷ原液生产工艺放大可行性研究

The feasibility study of mass production of human coagulation factor Ⅷ in manufacturing

目的对比3批小试（50g冷沉淀）、3批小规模放大（400g冷沉淀）和3批中试级（5-10 kg冷沉淀）试验结果探讨人凝血因子Ⅷ原液生产工艺放大的可行性。方法 Tris溶液复溶冷沉淀,经聚乙二醇沉淀后,0.3%磷酸三丁酯和1%Tween 80处理6h以灭活脂包膜病毒,然后经DEAE 650M层析纯化。绝大部分杂蛋白和磷酸三丁酯及Tween80随流穿液直接流穿,洗脱峰样品即为人凝血因子Ⅷ原液。结果 3批小试人凝血因子Ⅷ活性回收率分别为30.50%、24.40%和25.60%,平均值26.83%,回收率变异系数8.52%;3批小规模放大回收率分别为19.40%、21.70%和22.00%,平均值21.04%,回收率变异系数5.54%;3批中试级回收率分别为14.50%、16.50%、17.80%,平均值16.27%,回收率变异系数8.34%。结论对比级放大试验各级回收率及变异系数发现,工艺重复性好,放大可行。

Objective To study the feasibility of mass production of human coagulation factor Ⅷ（ FⅧ） in accordance with the results from three small-scale tests（ 50 g cryoprecipitates）,three large-scale tests（ 400 g cryoprecipitates） and three pilot-level ed productions（ 5 ~ 10 kg cryoprecipitates）,. Methods Cryoprecipitates centrifuged from human fresh frozen plasma were dissolved in Tris buffer. The dissolved solution was again centrifuged,followed by precipitation by PEG4000. The resulting supernatant was treated with solvent / detergent（ S / D,1% tween 80 and 0. 3% Tn BP） by the means of virus inactivation method for six hours. The supernatant was then subjected to purification through weak anion exchanger（ Toyopearl DEAE 650M） in ion-exchange chromatography. Impure proteins and solvent / detergent were first eluted with EQ and washing buffer. The final eluate was identified as the pure FⅧ（ human）. Results The recovery rates of FⅧ in three small-scale tests were 30. 50%,24. 40% and 25. 60%,with an average of 26. 83% and a coefficient of variation（ CV） of 8. 52%. The recovery rates in three large-scale tests were 19. 40%,21. 70% and 22. 00%,with an average of 21. 04% and a CV of 5.54%. The rates of three pilot-leveled productions were 14. 50%,16. 50% and 17. 80%,with an average of 16. 27% and a CV of 8. 34%. Conclusion Comparing the recovery rates and CV of FⅧ at different scales,the mass production of FⅧ was determined to be stable and the manufacturing process was deemed feasible.