Preparation technology of new gel-purified prothrombin complexes concentrates
10.3969/j.issn.1005-1678.2016.11.058
- VernacularTitle:新型凝胶纯化凝血酶原复合物的工艺探讨
- Author:
Min ZHOU
;
Renyong ZENG
;
Yuanyuan LIU
;
Changqing LI
;
Haijun CAO
;
Zechao HE
- Keywords:
prothrombin complex concentrate;
Capto DEAE-gel;
ion-exchange column chromatography
- From:
Chinese Journal of Biochemical Pharmaceutics
2016;36(11):192-196
- CountryChina
- Language:Chinese
-
Abstract:
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 FⅦwere very low.When the three variables ( sodium citrate, NaCl and pH) 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/mL.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 2.17-3.31IU/mg.Therefore, these studies will lay the groundwork for exploring novel preparation process of producing PCC.
