不同吸附体系及因素水平对PCC中4类抗凝蛋白的影响研究

Effect of different adsorption system on the coagulation inhibitors in prothrombin complex concentrate

目的探讨不同吸附体系及其柠檬酸钠、氯化钠、pH、电导4因素对凝血酶原复合物(PCC)中蛋白C、蛋白S、蛋白Z、抗凝血酶收率及比活的影响。方法选择柠檬酸钠、氯化钠、pH3因素3水平设计正交试验,配制9种PCC平衡吸附体系;凝胶平衡后,吸附血浆,洗涤、洗脱,制备PCC浓缩物;测定浓缩物中蛋白C和S、抗凝血酶活性、蛋白Z含量、总体积及蛋白浓度,获得四类抗凝蛋白收率及比活,比较不同吸附体系收率、比活总趋势;利用正交试验直观分析法分析柠檬酸钠、氯化钠、pH对蛋白C、S和Z收率及比活的影响;测定吸附体系电导率,分析电导率与4类抗凝蛋白收率及比活的关系。结果不同吸附体系4类抗凝蛋白收率及比活有较大差异,抗凝血酶二者均极低。柠檬酸钠对蛋白C收率及比活、蛋白S收率影响较大;pH对蛋白S比活、蛋白Z收率及比活影响极差分别为:0.120、6.847、0.716,高于其他因素;氯化钠对其影响均最小。柠檬酸钠浓度越低,蛋白C收率及比活越高,蛋白S收率则越低,0.015 mol/L水平蛋白Z收率为49.11%,高于其他水平,而蛋白S和Z比活最低;pH越低,蛋白Z收率则越高,pH7.0水平蛋白C和S收率及比活、蛋白Z比活最高;氯化钠浓度0.075 mol/L水平,蛋白C收率及比活、蛋白S和Z比活分别为:29.37%、0.533 IU/mg、0.401 IU/mg、2.362μg/mg,均高于其他水平,而蛋白S、Z收率最低。不同电导率条件,蛋白C收率及蛋白Z比活变化较大,其中蛋白Z与S收率、蛋白Z与S比活、蛋白C收率与比活变化趋势基本一致;(10.00±0.20)ms/cm条件,蛋白C、S和Z比活较高。结论不同吸附体系抗凝血酶收率及比活均极低,蛋白C、S和Z收率及比活间无规律性。氯化钠对蛋白C、S和Z收率及比活影响较小,柠檬酸钠、氯化钠、pH不同水平对蛋白C、S和Z收率影响效应无共性,0.01 mol/L柠檬酸钠,0.075 mol/L氯化钠、pH7.0水平蛋白C、S和Z比活最佳。电导率与四类抗凝蛋白收率及比活间无线性关系。

Objective To study the effect of different adsorption systems for preparing prothrombin complex concen- trates （PCC） and their four variables （sodium citrate, NaC1, pH, and conductivity） on the yield and activity of proteins C （PC） ,S （PS） ,Z （PZ）, and antithrombin （AT） during PCC production. Methods 9 adsorption conditions for purifying PCC were prepared according to the orthogonal table designed by three variables （sodium citrate, NaC1, and pH）, and each in three levels. The gel was equilibrated by the corresponding adsorption condition, and then mixed with cryo-poor plasma to adsorb PCC. After the gel was washed, PCC was eluted. The PC, PS, and AT activity, PZ content, total volume and protein concentration of PCC were measured to obtain the yield and purity of the above four inhibitors, and then their yields and pu- rity in different adsorption conditions were compared. The effects of sodium citrate,NaC1 and pH on the yield and purity of PC, PS, and PZ were analyzed by heuristics analysis method. In addition, the conductivity was determined and its impact on the yield and purity of the four coagulation inhibitors was analyzed. Results Under different adsorption conditions, the yield and purity of the four inhibitors were quite different. Both the yield and the purity of AT were very low. Of the three varia- bles, sodium citrate had greater impact on PC yield and purity, as well as PS yield, and pH showed greater impact on PZ yield and purity, and PS purity. For three levels, the higher the sodium citrate content, the lower the yield and purity of PC,and the higher the yield of PS. When sodium citrate was 0. 015 mol/L,the yield of PZ was highest,but the purities of PS and PZ were the lowest. The lower pH, and the higher PZ yield. At pH7.0,the yield and purity of PC and PS,and the purity of PZ was the highest. When sodium chloride was 0. 075 tool/L, the PC yield and purity, and PS and PZ purity were the highest, but PS and PZ yields were the lowest. With the variation of conductivity, the PC yield and PZ purity showed greater fluctua- tion,furthermore the change tendency of PZ and S yield, PZ and S purity, and PC yield and purity, was similar. Conclusion Of all the adsorption systems, the yield and purity of AT are very low, and there is no association between the yield and purity of PC, PS, and PZ. Sodium chloride has less effect on the yield and purity. The effect of different levels of sodium cit- rate,NaCl,pH on their yield shows no uniform character,however their parity are the maximum at sodium citrate O. O l mol/ L, NaC1 0. 075 mol / L, and pH 7.0. There is no obvious linear relationship between the conductivity and the yield, as well as the purity of the four inhibitors.