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

BMC Biotechnology

Research Article

Yves Durocher¹  Sven Ansorge¹  Amine Kamen¹  Virgínia Picanço-Castro²  Mário SA Neto²  Dimas T Covas³  Elisa MS Russo-Carbolante⁴  Kamilla Swiech⁵ 

[1] National Research Council Canada, Biotechnology Research Institute, Montreal, Quebec, Canada;Regional Blood Center of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, Brazil;Regional Blood Center of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, Brazil;Department of Clinical Medicine, Faculty of Medicine of Ribeirrão Preto, University of São Paulo (USP), Ribeirão Preto, Brazil;Regional Blood Center of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, Brazil;Department of Clinical, Toxicological and Food Science Analysis, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil;Regional Blood Center of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, Brazil;Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil;

关键词: HEK293 Cell;    Enhanced Green Fluorescent Protein;    Transient Transfection;    Recombinant Factor VIII;    Coagulation Factor VIII;   

DOI  :  10.1186/1472-6750-11-114

 received in 2011-08-16, accepted in 2011-11-24,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundHemophilia 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 μg/106 cells) and repeated transfections done at 34° and 37°C.ResultsWe 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°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection.ConclusionSerum-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.

【 授权许可】

CC BY   
© Swiech et al; licensee BioMed Central Ltd. 2011