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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.
RESUMEN
Objective To study the inlfuence factors on detection of activity of four coagulation factors in prothrombin complex concentrates (PCC) by several factors. Methods Using Pharmacopoeia of the People’s Republic of China (2010) as reference, the activity of four coagulation factors in PCC were investigated by choosing different pre-treatments, different diluents, different salt concentration, different standard human plasma and different company reagents. Results The activity of FII, FVII, FX were decreased and FIX was increased in the condition of adding protamine sulfate, and there were no differences of four coagulation factors whether warm bath in 37 for 15 min or not. However, the differences of four coagulation factors were significant by using deficient plasma, saline, distilled water and commercial dilution buffer(P<0.05). The activity of coagulation factor II, X in 1 mol/L salt concentration of PCC were significantly lower than in 0.25 mol/L(P<0.05), while coagulation factor VII, IX were not. The activity of FII, FVII, FIX, and FX were different by using different standard human plasma to make standard curve. The activity of four coagulation factors existed significant difference(P=0.00) by using SIEMENS company reagents and domestic reagents. Conclusion Choosing different pre-treatments, different dilution buffers, salt concentration, standard human plasma and commercial kits will inlfuence the detection result of coagulation factors.