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

作者：Ferraz do Amaral Robson Luis; Bomfim Aline de Sousa; de Abreu Neto Mario Soares; Picanco Castro Virginia; de Sousa Russo Elisa Maria; Covas Dimas Tadeu; Swiech Kamilla^*

来源：Biotechnology Letters, 2016, 38(3): 385-394.

DOI：10.1007/s10529-015-1991-1

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

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 A mu g rhFIX/ml and 3.3 A mu g 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 A mu g/ml with a biological activity of 0.2 IU/ml, while after 72 h, the production was 2.4 A mu g/ml with a biological activity of 0.3 IU/ml.
Conclusion 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.

• 出版日期2016-3