Robson Luis Ferraz do Amaral1,2, Aline de Sousa Bomfim3, Mário Soares de Abreu-Neto4, Virgínia Picanço-Castro5, Elisa Maria de Sousa Russo6, Dimas Tadeu Covas7,8, Kamilla Swiech9. 1. 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. 2. Regional Blood Center of Ribeirão Preto, Tenente Catão Roxo Street, 2501, Ribeirão Prêto, São Paulo State, CEP 14051-140, Brazil. amaral.r@usp.br. 3. 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. 4. 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. 5. 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. 6. 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. 7. Regional Blood Center of Ribeirão Preto, Tenente Catão Roxo Street, 2501, Ribeirão Prêto, São Paulo State, CEP 14051-140, Brazil. dimas@fmrp.usp.br. 8. Ribeirão Preto Medical School, University of São Paulo, Bandeirantes Ave, 3900, Ribeirão Prêto, São Paulo State, CEP 14049-900, Brazil. dimas@fmrp.usp.br. 9. 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.
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. 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.
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. 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.
Authors: Elianet Lorenzo; Lidice Méndez; Elsa Rodríguez; Nemecio Gonzalez; Gleysin Cabrera; Carlos Pérez; Rafael Pimentel; Yusmel Sordo; Maria P Molto; Talia Sardina; Alina Rodríguez-Mallon; Mario P Estrada Journal: AMB Express Date: 2019-09-05 Impact factor: 3.298
Authors: Lucia F Zacchi; Dinora Roche-Recinos; Cassandra L Pegg; Toan K Phung; Mark Napoli; Campbell Aitken; Vanessa Sandford; Stephen M Mahler; Yih Yean Lee; Benjamin L Schulz; Christopher B Howard Journal: Commun Biol Date: 2021-03-23