Purification of Plasma-Derived Coagulation Factor VIII by Immobilized-Zn2+ and -Co2+ Affinity Chromatography
- 222 Downloads
Coagulation factor VIII (FVIII) is a glycoprotein that plays a crucial role in the clotting cascade. Replacement therapies with recombinant and plasma-derived concentrates of FVIII are used for treatment of hemophilia A. We have previously purified the human plasma FVIII by immobilized metal affinity chromatography (IMAC) using Cu2+ as the metal ligand. In this work we report the purification of FVIII using Zn2+ and Co2+, two metal ions that bind proteins more weakly. Human plasma was directly applied to the anion-exchange ANX Sepharose FF column and the eluate was used as starting material for the studies in IMAC columns. Using imidazole as desorbing agent, FVIII was recovered with 65% activity in the IMAC-Zn2+ column and with 74% activity in the IMAC-Co2+ column. Purification factors were 4 and 9, respectively. Using a pH gradient, FVIII was eluted at pH 5.0 with 17% activity in the IMAC-Zn2+ and 77% activity in the IMAC-Co2+. Vitamin K-dependent proteins, a family of proteins that includes Prothrombin and coagulation factor IX, coeluted with FVIII in the ANX Sepharose FF column and were recovered with the unbound proteins on both IMAC columns. Therefore, Co2+ and Zn2+ columns were as effective as the Cu2+ column in separating FVIII from vitamin K-dependent proteins. Finally, we have shown that FVIII remained complexed with the von Willebrand factor.
KeywordsAnion-exchange chromatography Coagulation factor VIII Immobilized metal affinity chromatography Plasma protein purification Vitamin K-dependent proteins
The authors thank Hemocentro de Ribeirão Preto, SP, Brazil, for the plasma donation, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for CIV’s fellowship and Programa de Aprimoramento Profissional (SES-FUNDAP) for RC’s fellowship. This work was supported by Fundação Butantan.
Compliance with Ethical Standards
Conflict of interest
All authors declare that they have no conflicts of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- 12.Franchini M, Frattini F, Crestani S, Sissa C, Bonfanti C (2013) Treatment of hemophilia B: focus on recombinant factor IX. Biologics 7:33–38Google Scholar
- 13.Minford A, Behnisch W, Brons P, David M, Gomez Gomez N, Hertfelder HJ, Kruempel A, Kurnik K, Mathias M, Molines Honrubia A, Monagle P, Morgan M, Nowak-Gottl U, Olivieri M (2014) Subcutaneous protein C concentrate in the management of severe protein C deficiency—experience from 12 centres. Br J Haematol 164:414–421CrossRefGoogle Scholar