凝血酶原复合物制备过程吸附条件的研究

Studies of adsorption conditions in prothrombin complex concehtrates production

目的探讨凝血酶原复合物制备过程凝血因子Ⅱ、Ⅶ、Ⅸ、Ⅹ的吸附条件,提高活性收率。方法选择DEAE-SephadexA-50凝胶平衡体系中柠檬酸钠、氯化钠、pH 3种可变因素,每因素取3水平,根据L9(33)正交表设计试验,配制平衡液,分别对DEAE-SephadexA-50凝胶进行平衡处理后,按1.67g/L加入等量健康人混合去冷沉淀血浆,4℃搅拌,吸附凝血因子;吸附后凝胶洗涤、洗脱,检测洗脱液中凝血因子Ⅱ、Ⅶ、Ⅸ、Ⅹ活性并进行活性回收率正交试验直观分析,获得较优吸附条件并进行验证。此外,分析吸附平衡体系电导率与4种因子活性收率间关系。结果柠檬酸钠、氯化钠、pH对凝血因子Ⅱ、Ⅶ、Ⅸ、Ⅹ吸附影响不同;在所考察范围内,pH对4种凝血因子吸附影响效应趋势一致,pH越低,吸附效果越佳;柠檬酸钠及氯化钠浓度对4种凝血因子吸附影响效应趋势不一致,柠檬酸钠浓度越高,凝血因子Ⅱ、Ⅶ、Ⅹ吸附越佳,而凝血因子Ⅸ在(0.012～0.02)mol/L水平较佳;氯化钠浓度越低,凝血因子Ⅱ、Ⅶ、Ⅸ吸附越佳,而凝血因子Ⅹ在(0.1～0.14)mol/L水平较佳;平衡体系电导率与4种因子活性收率间不存在线性关系。结论吸附条件中pH及柠檬酸钠浓度对凝血因子Ⅱ、Ⅶ、Ⅸ、Ⅹ吸附效果影响较大。柠檬酸钠(0.02～0.028)mol/L,氯化钠(0.02～0.06)mol/L,pH6.6～6.9条件,4种凝血因子吸附效果相对较佳。电导率对4种因子吸附效果影响不明显。

Objective To study adsorption conditions of coagulation factor Ⅱ、Ⅶ、Ⅸ、Ⅹ in prothromhin complex con- centrates （PCC） production and improve activity recovery of PCC. Methods Orthogonal experiment was designed for the e- quilibration system of DEAE-SephadexA-50 gel. Three variable factors, sodium citrate, sodium chloride and pH, and their re- spective three levels were selected, and then equilibration buffer were prepared based on L9（33 ）table. 9 equal parts of the gel were equilibrated respectively by equilibration buffer, and then different equilibrated DEAE-SephadexA-50 gel was mixed with cryoprecipitate supcrnatant in the proportion of 1.67g： 1L for adsorption of coagulation factor Ⅱ、Ⅶ、Ⅸ、Ⅹ, at 4℃. After that, the gel was washed, and then the coagulation factor Ⅱ、Ⅶ、Ⅸ、Ⅹ were eluted. The activity and their recovery of the coagulation factors in the eluate was measured and analyzed on the basis of orthogonal design-direct method. According to analysis results, the better adsorption conditions were obtained, and then were verified. Furthermore, the relationship between conductivity of equilibration system and activity recovery of four kinds coagnlation factors was analyzed. Results Sodium citrate,sodium chloride and pH have diverse impacts on activity recovery of four coagulation factors. The effect of pH on four factors shows same tendency,the lower pH the higher recovery,on the contrary,sodium citrate and sodium chloride have distinct effects on four factors. The higher sodium citrate concentration the higher activity recovery of factors II , g~, X, however the recovery of factor IX is higher when sodium citrate is in （0. 012 - 0. 02）mol/L. The lower sodium chloride concentration the higher recovery of factors Ⅱ、Ⅶ、Ⅸ ,however the recovery offactor X is higher when sodium chloride is in （0. 1 -0. 14） mol/L. It was not line-relation that between conductivity of equilibration system and activity recovery of four kinds coagulation factors. Conclusion The pH and concentration of sodium citrate have greater impact than sodium chloride on the activity recovery of coagulation factors Ⅱ、Ⅶ、Ⅸ、Ⅹ. The recovery of four kinds coagulation factors is relatively higher when sodium citrate （0. 02 - 0. 028 ） tool/L, sodium chloride （0. 02 - 0. 06） mol / L, and pH 6. 6 - 6. 9. The effect of conductivity of equilibration system on activity recovery of four kinds coagulation factors is not obvious.