Approaches for recombinant human factor IX production in serum-free suspension cultures
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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.
KeywordsAdaptation 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.
- Altamiro C, Berrios J, Vergara M, Becerra S (2013) Advances in improving mammalian cells metabolism for recombinant protein production. Eur J Biotechnol 16:1–14Google Scholar
- Bomfim A (2013) Cloning and expression of recombinant factor IX in 293T and SK-Hep-1 cells and characterization of producing cells. Master Dissertation, School of Pharmaceutical Sciences of Ribeirão Preto, University of Sao Paulo, Brazil). http://www.teses.usp.br/teses/disponiveis/60/60135/tde-13122013-111826/en.php
- Costa A, Withers J, Rodrigues M, McLoughlin N, Henriques M, Oliveira R, Rudd P, Azeredo J (2013) The impact of cell adaptation to serum-free conditions on the glycosylation profile of a monoclonal antibody produced by chinese hamster ovary cells. Nat Biotechnol 30:563–572Google Scholar
- Yekta A, Dalman A, Eftekhari-Yazdi P, Mohammad H, Shahverdi A, Fakheri R, Vazirinasab H, Daneshzadeh Z, Vojgani M, Zomorodipour A, Fatemi N, Vahabi Z, Mirshahvaladi S, Ataei F (2013) Production of transgenic goats expressing human coagulation factor IX in the mammary glands after nuclear transfer using transfected fetal fibroblast cells. Transgenic Res 22:1131–1142Google Scholar