Biotechnology Letters

, Volume 38, Issue 3, pp 385–394 | Cite as

Approaches for recombinant human factor IX production in serum-free suspension cultures

  • Robson Luis Ferraz do Amaral
  • Aline de Sousa Bomfim
  • Mário Soares de Abreu-Neto
  • Virgínia Picanço-Castro
  • Elisa Maria de Sousa Russo
  • Dimas Tadeu Covas
  • Kamilla SwiechEmail author
Original Research Paper



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).


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.


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.


Adaptation HEK293 Clotting factor Human cells Recombinant factor IX Serum-free suspension cultures 



The authors acknowledge financial support from Sao Paulo Research Foundation (FAPESP). Process numbers 2012/04629-8 and 2011/10778-3.


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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Robson Luis Ferraz do Amaral
    • 1
    • 2
  • Aline de Sousa Bomfim
    • 1
  • Mário Soares de Abreu-Neto
    • 2
  • Virgínia Picanço-Castro
    • 2
  • Elisa Maria de Sousa Russo
    • 1
  • Dimas Tadeu Covas
    • 2
    • 3
  • Kamilla Swiech
    • 1
    Email author
  1. 1.School of Pharmaceutical Sciences of Ribeirão PretoUniversity of São PauloRibeirão PrêtoBrazil
  2. 2.Regional Blood Center of Ribeirão PretoRibeirão PrêtoBrazil
  3. 3.Ribeirão Preto Medical SchoolUniversity of São PauloRibeirão PrêtoBrazil

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