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

Author(s)

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]

Affiliation

^[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

• Document type: Journal article
• Source: Biotechnology Letters; v. 38, n. 3, p. 385-394, MAR 2016.
• Web of Science Citations: 4

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 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)

• FAPESP's process: 11/10778-3 - Serum-free suspension culture adaptation of the 293-FIX-GFP cell line for recombinant factor IX production
• Grantee: Robson Luis Ferraz do Amaral
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 12/04629-8 - Establishment of a production platform for recombinant therapeutic proteins in human cells
• Grantee: Kamilla Swiech Antonietto
• Support Opportunities: Research Grants - Young Investigators Grants