新型凝胶纯化凝血酶原复合物的工艺探讨

Preparation technology of new gel-purified prothrombin complexes concentrates

目的探索新型凝胶Capto DEAE离子交换柱层析纯化凝血酶原复合物的工艺条件。方法健康人混血浆经离心去除冷沉淀后,采用DEAE-Sephadex A-50凝胶进行吸附,经洗涤、洗脱得到洗脱液;将得到的洗脱液超滤脱盐后,再通过Capto DEAE凝胶柱纯化,制备凝血酶原复合物（prothrombin complex concentrates,PCC）。测定PCC中凝血因子Ⅱ、Ⅶ、Ⅸ、Ⅹ及抗凝蛋白C的活性并计算收率;采用Bradford法测定PCC蛋白浓度,再根据测定的4种凝血因子和蛋白C的活性,计算有效成分的比活;以此分析所选工艺条件下Capto凝胶对PCC的纯化效果。结果不同实验方案下,所得制品中FⅡ、FⅨ、FⅩ收率和纯度均较高,且该3种凝血因子均衡度较好;但FⅦ收率和纯度均较低;其中A方案下PCC收率和纯度较好,即Capto DEAE凝胶体系平衡液、洗涤液、洗脱液中柠檬酸钠、Na Cl、p H分别为0.020-0.028 mol/L、0.10-0.15 mol/L、6.9-7.2;0.012-0.020 mol/L、0.10-0.15 mol/L、6.9-7.2;0.005-0.012 mol/L、2.4 mol/L、7.2-7.5时,FⅨ收率为（74.40±10.89）%,比活为（3.31±0.31）IU/mg,抗凝蛋白C的收率和比活均较高。结论采用DEAE-Sephadex A-50批式吸附及Capto DEAE柱层析相结合的新方法制备PCC,在所选择条件下,PCC制品的纯度为2.17-3.31 IU/mg,远优于传统工艺制得的产品,此为高品质PCC的制备提供了新的方法。

Objective To study the process conditions for new gel Capto DEAE ion exchange chromatography to purify prothrombin complexes concentrates. Methods After removal of cryoprecipitate by centrifugation,healthy human plasma was mixed with DEAE-Sephadex A-50 gel. After that,the gel were washed and eluted to obtain eluate; then,the eluate,after being ultrafiltered,was loaded on a column packed with Capto DEAE-gel for chromatography to prepare PCC which was later determined for activities of coagulation factors Ⅱ,Ⅶ,Ⅸ,Ⅹ and anticoagulation protein C,with their yield calculated. Besides,the protein concentration of PCC was determined using the Bradford method,based on which the specific activity of the four coagulation factors and protein C were calculated. According to the results,purification effect of Capto DEAE-gel on the PCC was analyzed.Results Under different experimental conditions,the yield and purity of the coagulation factors FⅡ,FⅨ and FⅩ were high,and the equilibrum degree of the three factors was good; however,the yield and purity of coagulation factor FⅦ were very low. When the three variables（ sodium citrate,Na Cl and p H） in balanced solution,washing solution and elution were 0. 020-0. 028 mol/L,0. 10-0. 15 mol/L and 6. 9-7. 2; 0. 012-0. 020 mol/L,0. 10-0. 15 mol/L and 6. 9-7. 2; 0. 005-0. 012 mol/L,2. 4 mol/L and 7. 2-7. 5,respectively,the yield and purity of PCC prepared from Capto DEAE-gel were good.Under this condition,yield of factor Ⅸ was（ 74. 40 ± 10. 89） % and purity of factor Ⅸ was（ 3. 31 ± 0. 31） IU/m L. Under different experimental conditions,yield and specific activity of anticoagulant protein C were higher. Conclusion The purity of four coagulation factors and anticoagulation protein C of PCC prepared by the new method that combined the batch adsorption with DEAE-sephadex A-50 was combined with column chromatography packed with Capto DEAE-gel are higher than those prepared by the routine procedure. Furthermore,the PCC are better than these products obtained by traditional process,whose purity are 2. 17-3. 31 IU/mg. Therefore,these studies will lay the groundwork for exploring novel preparation process of producing PCC.