Desalination effect on FⅧ components: a compartive study among 5 desalination methods

Abstract

Objective To compare the desalination effects of five desalination methods and their effects on the components for human coagulation factor Ⅷ(FⅧ), and provide reference for selection of protein desalination methods. Methods Sephadex G-25 Medium gel, Fractogel EMD BioSEC gel, ultrafiltration, room temperature dialysis and 4℃ dialysis were used to desalt human FⅧ. The desalination effect was evaluated by the removal rate of Na +, citrate ion and glycine. FⅧ protein recovery, FⅧ activity (FⅧ∶C), VWF antigen (VWF∶Ag), VWF activity(VWF∶Ac), VWF polymers and SDS-PAGE analysis before and after desalination were compared to evaluate the effect of desalination on FⅧ components. Results In terms of desalination effect, the removal rate of Na+ was the lowest in ultrafiltration desalination, while that of Fractogel EMD BioSEC gel was the highest [(97.90±0.06) % vs (99.82±0.07) %]. Except that there was no statistical significance between Sephadex G-25 Medium gel desalination and Fractogel EMD BioSEC gel desalination (P=0.90), the removal rates of the other four methods were statistically significant. The removal rate of glycine was the lowest in ultrafiltration desalination, wihle that of Fractogel EMD BioSEC gel desalination was the highest [(95.78±0.42) % vs (99.81±0.08) %]. Significant difference in glycine removal was noticed in ultrafiltration desalination, but not among the other four desalination methods. There was no significant difference in the removal rate of citrate ions among the five methods (P=0.85). For the effect of FⅧ components, FⅧ∶C, VWF∶Ag, VWF∶Ac and protein recovery rates of ultrafiltration desalination were the highest, with (18.34±1.99) IU/mL, (11.81±0.33) IU/mL, (12.26±0.58) IU/mL and (97.13±1.37) %, respectively. There was no significant change in VWF∶Ac/VWF∶Ag before and after desalination by the five methods. SDS-PAGE and VWF polymer analysis showed that different desalination methods had no significant impact on protein composition. Conclusion Although different desalination methods had no significant effect on the composition of FⅧ protein, the desalination effect was different. Moreover, different desalination methods had significant effects on protein recovery, FⅧ∶C, VWF∶Ag and VWF∶Ac. The selection of desalination methods should be more considered during protein processing,

Keywords

• human coagulation factor ⅷ(fⅷ)
• desalination
• gel chromatography
• ultrafiltration
• dialysis