Journal Article

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

Covas, Dimas T, Picanço-Castro, Virgínia, Ansorge, Sven, Neto, Mário S. A, Durocher, Yves, Swiech, Kamilla, Russo-Carbolante, Elisa M. S, Kamen, Amine

2011

Overview

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 μg/106 cells) and repeated transfections done at 34° and 37°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°C using 0.4 μg DNA/106 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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Ammonia - analysis / Applied Microbiology / Biochemical Engineering / Biomedical Engineering/Biotechnology / Biotechnology / Biotechnology - methods / Blood Cell Count - methods / Blood coagulation factor VIII / Care and treatment / Cell Culture Techniques - methods / Chemistry / Chemistry and Materials Science / Cloning / Colleges & universities / Culture Media, Serum-Free / Deoxyribonucleic acid / Diagnosis / Dihydrofolate reductase / DNA / E coli / Enzyme-Linked Immunosorbent Assay / Erythrosine / Factor VIII - biosynthesis / Gene therapy / Genetic aspects / Genetic Engineering / Glucose - analysis / Health aspects / HEK293 Cells / Hemophilia / Humans / Lactic Acid - analysis / Manufacturing / Medical research / Methods / Physiological aspects / Plant Breeding/Biotechnology / Proteins / Recombinant Proteins - biosynthesis / Research Article / Transfection - methods