title: Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII.
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 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.
  
  
  CONCLUSIONS
  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.
authors: Regional Blood Center of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil. kamilla@hemocentro.fmrp.usp.br
authors: Kamilla
authors: K
authors: Swiech
authors: Amine
authors: A
authors: Kamen
authors: Sven
authors: S
authors: Ansorge
authors: Yves
authors: Y
authors: Durocher
authors: Virgínia
authors: V
authors: Picanço-Castro
authors: Elisa M S
authors: EM
authors: Russo-Carbolante
authors: Mário S A
authors: MS
authors: Neto
authors: Dimas T
authors: DT
authors: Covas
mesh_terms: D000641:Ammonia / Q000032:analysis; D001709:Biotechnology / Q000379:methods*; D001772:Blood Cell Count / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods*; D016895:Culture Media, Serum-Free; D004797:Enzyme-Linked Immunosorbent Assay; D004923:Erythrosine; D005169:Factor VIII / Q000096:biosynthesis*; D005947:Glucose / Q000032:analysis; D057809:HEK293 Cells; D006801:Humans; D019344:Lactic Acid / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection / Q000379:methods*
chemical_list: D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D019344:Lactic Acid; D000641:Ammonia; D005169:Factor VIII; D005947:Glucose; D004923:Erythrosine