Abstract
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.
Highlights
Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII
Transient Transfection The constructs used in this study were based on the replication-deficient bicistronic lentiviral vector cPPT-C (FVIIIdelB)IGWS, which codes for human B-domain deleted factor VIII (FVIII)(FVIIIdelB), the deleted region of B domain was from from 2428 to 5067 nucleotides and this vector has the IRES element followed by the enhanced green fluorescent protein (EGFP)
After 4 days of transfection, 200, 109, and 49 ng/mL of Recombinant factor VIII (rFVIII) were produced with DNA concentrations of 0.4, 0.6, and 0.8 μg DNA/106 cells, respectively
Summary
Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. 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. Hemophilia A is a bleeding disorder caused by a deficient coagulation factor VIII (FVIII) that affects one in 5,000 to 10,000 males. Treatment for hemophilia A patients consists of replacement therapy using human plasma-derived FVIII (pdFVIII) or recombinant FVIII (rFVIII). Recombinant factor VIII (rFVIII) emerges as an alternative to pdFVIII for the treatment of hemophilia A. These include amplification of the FVIII transgene using dihydrofolate reductase/methotrexate selection, addition of FVIII stabilizing agents such as bovine/human albumin, or coexpression of the von Willebrand factor (vWf), and use of continuous-perfusion fermentation to maximize cell growth, cell density, and product recovery [4]
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