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目的采用正交试验法优化凝血酶原复合物(Prothrombin complex concentrate,PCC)的制备工艺。方法取DEAE-Sephadex A-50凝胶平衡体系中的柠檬酸钠、氯化钠、pH值3因素,每因素取3水平设计正交试验;称取9等份DEAE-Sephadex A-50凝胶干粉,分别采用试验设计的平衡体系进行平衡;平衡后的凝胶分别与等量健康人去冷沉淀混合血浆混合,制备凝血酶原复合物浓缩物;测定浓缩物蛋白浓度及凝血因子Ⅱ、Ⅶ、Ⅸ、Ⅹ活性,获得4种凝血因子比活;通过正交试验直观分析及直接对比法确定较优及较差比活制备条件,对确定的条件进行验证;测定平衡体系电导率,分析正交试验中平衡体系电导率与4种凝血因子比活的相关性。结果正交试验直观分析法与直接对比法确定的凝血因子Ⅱ、Ⅶ、Ⅸ、Ⅹ较优比活凝胶平衡条件一致:柠檬酸钠0.005~0.012 mol/L,氯化钠0.06~0.10 mol/L,pH 6.9~7.2;两种方法确定的较差平衡条件不同,分别为:柠檬酸钠0.012~0.02 mol/L、氯化钠0.1~0.14 mol/L,pH 7.2~7.5及柠檬酸钠0.005~0.012 mol/L,氯化钠0.1~0.14 mol/L,pH 7.2~7.5,经实验验证,两种方法确定的较优条件比活均优于较差条件,但两种较差条件中,直接对比法确定的条件更符合实际。平衡液电导率与4种凝血因子比活之间不存在线性关系,但在不同电导率条件下,4种凝血因子比活有较大波动,电导率9.67~10.34 ms/cm,4种凝血因子比活均较高;电导率12.16~12.90 ms/cm,4种凝血因子比活均较低。结论正交试验可用于凝血酶原复合物制备工艺优化,但不同分析方法结果有一定差异。平衡体系电导率对凝血酶原复合物比活有一定影响,在工艺优化时应予注意。
Objective To optimize the preparation of Prothrombin complex concentrate (PCC) by orthogonal test. Methods Take DEAE-Sephadex A-50 gel balance system of sodium citrate, sodium chloride, pH value of 3 factors, each factor to take the level of 3 orthogonal design; weighed 9 equal parts DEAE-Sephadex A-50 gel The dry powders were respectively equilibrated with the balance system designed by the experiment. The equilibrated gels were respectively mixed with equal volume of healthy people to precipitate the mixed plasma to prepare prothrombin complex concentrates. The concentrations of the concentrated protein and coagulation factors II and VII , Ⅸ and Ⅹ activities. Four kinds of clotting factor specific activity were obtained. The optimal and poor specific preparation conditions were determined by orthogonal test and direct contrast method, and the conditions were verified. The conductivity of the equilibrium system was determined, The correlation between the conductivity of the equilibrium system and the specific activity of four clotting factors during the cross test. Results The results of orthogonal test showed that the coagulation factors Ⅱ, Ⅶ, Ⅸ and Ⅹ, which were determined by the direct comparison method, were better than the equilibrium conditions of the live gels: sodium citrate 0.005 ~ 0.012 mol / L, sodium chloride 0.06 ~ 0.10 mol / L, pH 6.9 ~ 7.2. The equilibrium conditions determined by the two methods were different, which were as follows: sodium citrate 0.012-0.02 mol / L, sodium chloride 0.1-0.14 mol / L, pH 7.2-7.5 and sodium citrate 0.005 ~ 0.012 mol / L NaCl 0.1 ~ 0.14 mol / L, pH 7.2 ~ 7.5. The experimental results show that the optimal conditions determined by the two methods are better than the worse conditions than the poor conditions. However, Direct contrast method to determine the conditions more realistic. There was no linear relationship between the BLC and the specific activity of the four clotting factors, but under different conductivity conditions, the specific activity of the four clotting factors fluctuated greatly with the conductivity of 9.67 ~ 10.34 ms / cm and the four coagulation factors Specific activity were higher; electrical conductivity 12.16 ~ 12.90 ms / cm, four kinds of coagulation factors than living low. Conclusion The orthogonal test can be used to optimize the preparation process of prothrombin complex, but the results of different analytical methods are different. The conductivity of the equilibrium system has a certain impact on the specific activity of prothrombin complex, should be noted when optimizing the process.