, Volume 68, Issue 5, pp 1687–1696 | Cite as

Effect of transmembrane pressure on Factor VIII yield in ATF perfusion culture for the production of recombinant human Factor VIII co-expressed with von Willebrand factor

  • Seung-Chul Kim
  • Sora An
  • Hyun-Ki Kim
  • Beom-Soo Park
  • Kyu-Heum Na
  • Byung-Gee Kim
Original Article


In this study, we evaluated three cell retention devices, an alternating tangential flow (ATF) system, a spin-filter, and a Centritech Lab III centrifuge, for the production of recombinant human Factor VIII co-expressed with von Willebrand factor. From the results, it was found that the FVIII activity in bioreactor was significantly higher in the ATF perfusion culture than two other perfusion cultures. Moreover, the FVIII activity yield was unexpectedly low in the ATF perfusion culture. We have, therefore, studied the reasons for this low FVIII activity yield. It was revealed that the inactivation and the surface adsorption of FVIII onto the harvest bag were not the main reasons for the low yield in the ATF perfusion culture. The FVIII activity yield was not increased by the use of a hollow fiber filter with 0.5 μm pore size instead of 0.2 μm pore size. Additionally, the retention of FVIII molecules by the hollow fiber filter was a dominant factor in the low FVIII activity yield in the ATF perfusion culture. We demonstrated that FVIII yield was significantly improved by controlling transmembrane pressure (TMP) across the hollow fiber filter membrane. Taken together, these results suggest that TMP control could be an efficient method for the enhancement of FVIII yield in an ATF perfusion culture.


Transmembrane pressure ATF perfusion culture FVIII activity yield FVIII/vWF complex Hollow fiber filter membrane Molecular sieving 


  1. Berntorp E (1997) Second generation, B-domain deleted recombinant factor VIII. Thromb Haemost 78:256–260Google Scholar
  2. Boedeker BG (2001) Production processes of licensed recombinant factor VIII preparations. Semin Thromb Hemost 27:385–394CrossRefGoogle Scholar
  3. Casademunt E, Martinelle K, Jernberg M, Winge S, Tiemeyer M, Biesert L, Knaub S, Walter O, Schroder C (2012) The first recombinant human coagulation factor VIII of human origin: human cell line and manufacturing characteristics. Eur J Haematol 89:165–176CrossRefGoogle Scholar
  4. Clincke MF, Molleryd C, Zhang Y, Lindskog E, Walsh K, Chotteau V (2013) Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor. Part I. Effect of the cell density on the process. Biotechnol Prog 29:754–767CrossRefGoogle Scholar
  5. Crowley J, Wubben M, Martin JMC (2012) Process for cell culturing by continuous perfusion and alternating tangential flow. US Patent 8206981 B1Google Scholar
  6. Himmelfarb P, Thayer PS, Martin HE (1969) Spin filter culture: the propagation of mammalian cells in suspension. Science 164:555–557CrossRefGoogle Scholar
  7. Hoyer LW, Shainoff JR (1980) Factor VIII-related protein circulates in normal human plasma as high molecular weight multimers. Blood 55:1056–1059Google Scholar
  8. Ishaque A, Thrift J, Murphy JE, Konstantinov K (2007) Over-expression of Hsp70 in BHK-21 cells engineered to produce recombinant factor VIII promotes resistance to apoptosis and enhances secretion. Biotechnol Bioeng 97:144–155CrossRefGoogle Scholar
  9. Jiang R, Monroe T, McRogers R, Larson PJ (2002) Manufacturing challenges in the commercial production of recombinant coagulation factor VIII. Haemophilia 8(Suppl 2):1–5CrossRefGoogle Scholar
  10. Johnson M, Lanthier S, Massie B, Lefebvre G, Kamen AA (1996) Use of the Centritech Lab centrifuge for perfusion culture of hybridoma cells in protein-free medium. Biotechnol Prog 12:855–864CrossRefGoogle Scholar
  11. Kaufman RJ, Pipe SW (1999) Regulation of factor VIII expression and activity by von Willebrand factor. Thromb Haemost 82:201–208Google Scholar
  12. Kaufman RJ, Wasley LC, Dorner AJ (1988) Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells. J Biol Chem 263:6352–6362Google Scholar
  13. Kaufman RJ, Wasley LC, Davies MV, Wise RJ, Israel DI, Dorner AJ (1989) Effect of von Willebrand factor coexpression on the synthesis and secretion of factor VIII in Chinese hamster ovary cells. Mol Cell Biol 9:1233–1242CrossRefGoogle Scholar
  14. Kelley B, Jankowski M, Booth J (2010) An improved manufacturing process for Xyntha/ReFacto AF. Haemophilia 16:717–725CrossRefGoogle Scholar
  15. Kim BJ, Oh DJ, Chang HN (2008) Limited use of Centritech Lab II centrifuge in perfusion culture of rCHO cells for the production of recombinant antibody. Biotechnol Prog 24:166–174CrossRefGoogle Scholar
  16. McLeod A, Walker I, Zheng S, Hayward C (2000) Loss of factor VIII activity during storage in PVC containers due to adsorption. Haemophilia 6:89–92CrossRefGoogle Scholar
  17. Mei B, Chen Y, Chen J, Pan CQ, Murphy JE (2006) Expression of human coagulation factor VIII in a human hybrid cell line, HKB11. Mol Biotechnol 34:165–178CrossRefGoogle Scholar
  18. Mercille S, Johnson M, Lemieux R, Massie B (1994) Filtration-based perfusion of hybridoma cultures in protein-free medium: reduction of membrane fouling by medium supplementation with DNase I. Biotechnol Bioeng 43:833–846CrossRefGoogle Scholar
  19. Mufarrege EF, Antuña S, Etcheverrigaray M, Kratje R, Prieto C (2014) Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells. A new strategy for recombinant human FVIII (rhFVIII) production. Protein Expr Purif 95:50–56CrossRefGoogle Scholar
  20. Selvaraj SR, Scheller AN, Miao HZ, Kaufman RJ, Pipe SW (2012) Bioengineering of coagulation factor VIII for efficient expression through elimination of a dispensable disulfide loop. J Thromb Haemost 10:107–115CrossRefGoogle Scholar
  21. Seyfried BK, Friedbacher G, Rottensteiner H, Schwarz HP, Ehrlich H, Allmaier G, Turecek PL (2010) Comparison of plasma-derived and recombinant von Willebrand factor by atomic force microscopy. Thromb Haemost 104:523–530CrossRefGoogle Scholar
  22. Shankaran H, Alexandridis P, Neelamegham S (2003) Aspects of hydrodynamic shear regulating shear-induced platelet activation and self-association of von Willebrand factor in suspension. Blood 101:2637–2645CrossRefGoogle Scholar
  23. Shevitz J (2003) Fluid filtration system. US 6544424 B1Google Scholar
  24. Tokashiki M, Arai T, Hamamoto K, Ishimaru K (1990) High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge. Cytotechnology 3:239–244CrossRefGoogle Scholar
  25. Vlot AJ, Koppelman SJ, van den Berg MH, Bouma BN, Sixma JJ (1995) The affinity and stoichiometry of binding of human factor VIII to von Willebrand factor. Blood 85:3150–3157Google Scholar
  26. Ward NJ, Buckley SM, Waddington SN, VandenDriessche T, Chuah MK, Nathwani AC, McIntosh J, Tuddenham EG, Kinnon C, Thrasher AJ (2011) Codon optimization of human factor VIII cDNAs leads to high-level expression. Blood 117:798–807CrossRefGoogle Scholar
  27. Weiss HJ, Sussman II, Hoyer LW (1977) Stabilization of factor VIII in plasma by the von Willebrand factor. Studies on posttransfusion and dissociated factor VIII and in patients with von Willebrand’s disease. J Clin Invest 60:390–404CrossRefGoogle Scholar
  28. Wise RJ, Dorner A, Krane M, Pittman D, Kaufman R (1991) The role of von Willebrand factor multimers and propeptide cleavage in binding and stabilization of factor VIII. J Biol Chem 266:21948–21955Google Scholar
  29. Yabannavar VM, Singh V, Connelly NV (1992) Mammalian cell retention in a spinfilter perfusion bioreactor. Biotechnol Bioeng 40:925–933CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Seung-Chul Kim
    • 1
    • 2
  • Sora An
    • 1
  • Hyun-Ki Kim
    • 1
  • Beom-Soo Park
    • 1
  • Kyu-Heum Na
    • 1
  • Byung-Gee Kim
    • 2
    • 3
  1. 1.Research InstituteDong-A Socio-Holdings Co., Ltd.Yong-inRepublic of Korea
  2. 2.Interdisciplinary Program for BioengineeringSeoul National UniversitySeoulRepublic of Korea
  3. 3.School of Chemical and Biological Engineering, College of Engineering, Institute of BioengineeringSeoul National UniversitySeoulRepublic of Korea

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