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

Autor(es)

Ferraz do Amaral, Robson Luis ^{[1, 2]} ; Bomfim, Aline de Sousa ^[1] ; de Abreu-Neto, Mario Soares ^[2] ; Picanco-Castro, Virginia ^[2] ; de Sousa Russo, Elisa Maria ^[1] ; Covas, Dimas Tadeu ^{[2, 3]} ; Swiech, Kamilla ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Sch Pharmaceut Sci Ribeiro Preto, Cafe Ave W-N, BR-14040903 Ribeirao Preto, SP - Brazil

^[2] Reg Blood Ctr Ribeirao Preto, Tenente Catao Roxo St 2501, BR-14051140 Ribeirao Preto, SP - Brazil

^[3] Univ Sao Paulo, Ribeirao Preto Med Sch, Bandeirantes Ave 3900, BR-14049900 Ribeirao Preto, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Biotechnology Letters; v. 38, n. 3, p. 385-394, MAR 2016.
• Citações Web of Science: 4

Resumo

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. (AU)

• Processo FAPESP: 12/04629-8 - Estabelecimento de uma plataforma de produção de proteínas recombinantes terapêuticas em células humanas
• Beneficiário: Kamilla Swiech Antonietto
• Linha de fomento: Auxílio à Pesquisa - Apoio a Jovens Pesquisadores
• Processo FAPESP: 11/10778-3 - Adaptação para cultivo em suspensão e meio de cultura livre de soro fetal bovino da linhagem celular 293-FIX-GFP para produção de FIX recombinante
• Beneficiário: Robson Luis Ferraz do Amaral
• Linha de fomento: Bolsas no Brasil - Iniciação Científica