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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

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Author(s):
Swiech, Kamilla [1, 2] ; Kamen, Amine [3] ; Ansorge, Sven [3] ; Durocher, Yves [3] ; Picanco-Castro, Virginia [2] ; Russo-Carbolante, Elisa M. S. [2, 4] ; Neto, Mario S. A. [2] ; Covas, Dimas T. [2, 5]
Total Authors: 8
Affiliation:
[1] Univ Sao Paulo, Dept Pharmaceut Sci, Fac Pharmaceut Sci Ribeirao Preto, BR-14049 Ribeirao Preto - Brazil
[2] Univ Sao Paulo, Reg Blood Ctr Ribeirao Preto, BR-14049 Ribeirao Preto - Brazil
[3] Natl Res Council Canada, Biotechnol Res Inst, Montreal, PQ H4P 2R2 - Canada
[4] Univ Sao Paulo, Fac Pharmaceut Sci Ribeirao, Dept Clin Toxicol & Food Sci Anal, BR-14049 Ribeirao Preto - Brazil
[5] Univ Sao Paulo, Fac Med Ribeirrao Preto, Dept Clin Med, BR-14049 Ribeirao Preto - Brazil
Total Affiliations: 5
Document type: Journal article
Source: BMC Biotechnology; v. 11, NOV 24 2011.
Web of Science Citations: 21
Abstract

Background: Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 mu g/10(6) cells) and repeated transfections done at 34 degrees and 37 degrees C. Results: We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34 degrees C using 0.4 mu g DNA/10(6) cells through which almost 50 IU of active rFVIII was produced six days post-transfection. Conclusion: Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability. (AU)