, Volume 80, Issue 9, pp 1319–1327 | Cite as

Purification of Plasma-Derived Coagulation Factor VIII by Immobilized-Zn2+ and -Co2+ Affinity Chromatography

  • Claudia Iwashita Verinaud
  • Gabriel Pinna Feliciano
  • Roberta Rodrigues de Carvalho
  • Alexandre Paulo Yague Lopes
  • Isaías Raw
  • Elizabeth Angélica Leme Martins
  • Elisabeth Cheng


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.


Anion-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.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. 1.
    Sulkowski E (1985) Purification of proteins by IMAC. Trends Biotechnol 3:1–7CrossRefGoogle Scholar
  2. 2.
    Honda RT, Araújo RM, Horta BB, Val AL, Demasi M (2005) One-step purification of metallothionein extracted from two different sources. J Chromatogr B Analyt Technol Biomed Life Sci 820:205–210. doi: 10.1016/j.jchromb.2005.03.017 CrossRefGoogle Scholar
  3. 3.
    Andersson L, Sulkowski E (1992) Evaluation of the interaction of protein alpha-amino groups with M(II) by immobilized metal ion affinity chromatography. J Chromatogr 604:13–17CrossRefGoogle Scholar
  4. 4.
    Kagedal L (2011) Immobilized metal ion affinity chromatography. In: Janson J-C (ed) Protein purification: principles, high resolution methods, and applications. Wiley, New Jersey, pp 183–201CrossRefGoogle Scholar
  5. 5.
    Hemdan ES, Zhao YJ, Sulkowski E, Porath J (1989) Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography. Proc Natl Acad Sci USA 86:1811–1815CrossRefGoogle Scholar
  6. 6.
    Fay PJ (2004) Activation of factor VIII and mechanisms of cofactor action. Blood Rev 18:1–15CrossRefGoogle Scholar
  7. 7.
    Burnouf T, Caron C, Burkhardt T, Goudemand J (2004) Content and functional activity of von Willebrand factor in apheresis plasma. Vox Sang 87:27–33CrossRefGoogle Scholar
  8. 8.
    Cheng E, Jinzenji D, Lorthiois AP, de Carvalho RR, Tanaka-Azevedo AM, Raw I, Martins EA (2010) Purification of coagulation factor VIII using chromatographic methods. Direct chromatography of plasma in anion exchange resins. Biotechnol Lett 32:1207–1214CrossRefGoogle Scholar
  9. 9.
    Rodrigues ES, Verinaud CI, Oliveira DS, Raw I, Lopes AP, Martins EA, Cheng E (2015) Purification of coagulation factor VIII by immobilized metal affinity chromatography. Biotechnol Appl Biochem 62:343–348CrossRefGoogle Scholar
  10. 10.
    Sarode R (2014) Four-factor prothrombin complex concentrate versus plasma for urgent vitamin K antagonist reversal: new evidence. Clin Lab Med 34:613–621CrossRefGoogle Scholar
  11. 11.
    Teitel JM, Sholzberg M (2013) Current status and future prospects for the prophylactic management of hemophilia patients with inhibitor antibodies. Blood Rev 27:103–109CrossRefGoogle Scholar
  12. 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. 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
  14. 14.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  15. 15.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  16. 16.
    Ueda EK, Gout PW, Morganti L (2003) Current and prospective applications of metal ion–protein binding. J Chromatogr A 988:1–23CrossRefGoogle Scholar
  17. 17.
    Porath J, Olin B (1983) Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions. Biochemistry 22:1621–1630CrossRefGoogle Scholar
  18. 18.
    Furlan M (1996) Von Willebrand factor: molecular size and functional activity. Ann Hematol 72:341–348CrossRefGoogle Scholar
  19. 19.
    Harper P, Favaloro EJ, Curtin J, Barnes C, Dunkley S (2016) Human plasma-derived FVIII/VWD concentrate (Biostate): a review of experimental and clinical pharmacokinetic, efficacy and safety data. Drugs Context 5:212292CrossRefGoogle Scholar
  20. 20.
    Valko M, Morris H, Cronin MT (2005) Metals, toxicity and oxidative stress. Curr Med Chem 12:1161–1208CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Claudia Iwashita Verinaud
    • 1
    • 2
  • Gabriel Pinna Feliciano
    • 1
    • 2
  • Roberta Rodrigues de Carvalho
    • 1
  • Alexandre Paulo Yague Lopes
    • 2
    • 3
  • Isaías Raw
    • 1
  • Elizabeth Angélica Leme Martins
    • 1
    • 2
  • Elisabeth Cheng
    • 1
    • 2
  1. 1.Laboratório de Desenvolvimento de ProcessosInstituto ButantanSão PauloBrazil
  2. 2.Programa de Pós-graduação Interunidades em BiotecnologiaUniversidade de São PauloSão PauloBrazil
  3. 3.Laboratório Especial de Desenvolvimento de VacinasInstituto ButantanSão PauloBrazil

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