title: Approaches for recombinant human factor IX production in serum-free suspension cultures.
abstract: OBJECTIVE
  To establish a serum-free suspension process for production of recombinant human factor IX (rhFIX) based on the human cell line HEK 293T by evaluating two approaches: (1) serum-free suspension adaptation of previously genetic modified cells (293T-FIX); and (2) genetic modification of cells already adapted to such conditions (293T/SF-FIX).
  
  
  RESULTS
  After 10 months, 293T-FIX cells had become adapted to FreeStyle 293 serum-free medium (SFM) in Erlenmeyer flasks. After 48 and 72 h of culture, 2.1 µg rhFIX/ml and 3.3 µg rhFIX/ml were produced, respectively. However, no biological activity was detected. In the second approach, wild-type 293T cells were adapted to the same SFM (adaptation process took only 2 months) and then genetically modified for rhFIX production. After 48 h of culture, rhFIX reached 1.5 µg/ml with a biological activity of 0.2 IU/ml, while after 72 h, the production was 2.4 µg/ml with a biological activity of 0.3 IU/ml.
  
  
  CONCLUSIONS
  The findings demonstrate that the best approach to establish an rhFIX production process in suspension SFM involves the genetic modification of cells already adapted to the final conditions. This approach is time saving and may better ensure the quality of the produced protein.
authors.affiliation: School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Café Ave, w/n, Ribeirão Prêto, São Paulo State, CEP 14040-903, Brazil. amaral.r@usp.br.|Regional Blood Center of Ribeirão Preto, Tenente Catão Roxo Street, 2501, Ribeirão Prêto, São Paulo State, CEP 14051-140, Brazil. amaral.r@usp.br.
authors.forename: Robson Luis Ferraz
authors.initials: RL
authors.lastname: do Amaral
authors.affiliation: School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Café Ave, w/n, Ribeirão Prêto, São Paulo State, CEP 14040-903, Brazil. ni_bomfim@yahoo.com.br.
authors.forename: Aline
authors.initials: A
authors.lastname: de Sousa Bomfim
authors.affiliation: Regional Blood Center of Ribeirão Preto, Tenente Catão Roxo Street, 2501, Ribeirão Prêto, São Paulo State, CEP 14051-140, Brazil. mario.neto@hemocentro.fmrp.usp.br.
authors.forename: Mário Soares
authors.initials: MS
authors.lastname: de Abreu-Neto
authors.affiliation: Regional Blood Center of Ribeirão Preto, Tenente Catão Roxo Street, 2501, Ribeirão Prêto, São Paulo State, CEP 14051-140, Brazil. virginia.castro@hemocentro.fmrp.usp.br.
authors.forename: Virgínia
authors.initials: V
authors.lastname: Picanço-Castro
authors.affiliation: School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Café Ave, w/n, Ribeirão Prêto, São Paulo State, CEP 14040-903, Brazil. elisa@fcfrp.usp.br.
authors.forename: Elisa Maria
authors.initials: EM
authors.lastname: de Sousa Russo
authors.affiliation: Regional Blood Center of Ribeirão Preto, Tenente Catão Roxo Street, 2501, Ribeirão Prêto, São Paulo State, CEP 14051-140, Brazil. dimas@fmrp.usp.br.|Ribeirão Preto Medical School, University of São Paulo, Bandeirantes Ave, 3900, Ribeirão Prêto, São Paulo State, CEP 14049-900, Brazil. dimas@fmrp.usp.br.
authors.forename: Dimas Tadeu
authors.initials: DT
authors.lastname: Covas
authors.affiliation: School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Café Ave, w/n, Ribeirão Prêto, São Paulo State, CEP 14040-903, Brazil. kamilla@fcfrp.usp.br.
authors.forename: Kamilla
authors.initials: K
authors.lastname: Swiech
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D016895:Culture Media, Serum-Free; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D005164:Factor IX
keywords: Adaptation; Clotting factor; HEK293; Human cells; Recombinant factor IX; Serum-free suspension cultures