Laboratory Production of Human Prolactin from CHO Cells Adapted to Serum-Free Suspension Culture

Autor(es)

Arthuso, Fernanda Santos ^[1] ; Bartolini, Paolo ^[1] ; Jorge Soares, Carlos Roberto ^[1]

Afiliação do(s) autor(es)

^[1] Inst Pesquisas Energet & Nucl IPEN CNEN SP, Ctr Biotechnol, BR-05508900 Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Applied Biochemistry and Biotechnology; v. 167, n. 8, p. 2212-2224, AUG 2012.
• Citações Web of Science: 5

Resumo

Human prolactin (hPRL) is a polypeptide with 199 amino acids and a molecular mass of 23 kDa. Previously, a eukaryotic hPRL expression vector was used to transfect Chinese hamster ovary (CHO) cells: this work describes a fast and practical laboratory adaptation of these transfected cells, in similar to 40 days, to grow in suspension in serum-free medium. High cell densities of up to 4.0 x 10(6) cell/ml were obtained from spinner flask cultures and a stable and continuous production process was developed for at least 30 days. Two harvesting strategies were set up, 50 or 100 % of the total conditioned medium being collected daily and replaced by fresh culture medium. The volumetric productivity was 5-7 mu g hPRL/ml, as determined directly in the collected medium via reversed-phase HPLC (RP-HPLC). A two-step process based on a cationic exchanger followed by size exclusion chromatography was applied to obtain purified hPRL from conditioned medium. Two hPRL isoforms, non-glycosylated and glycosylated, could also be separated by high-performance size-exclusion chromatography (HPSEC) and, when analyzed by RP-HPLC, HPSEC, Western blotting, and bioassay, were found to be comparable to the World Health Organization International Reference Reagent of hPRL. These results are useful for the practical scale-up to the pilot and industrial scale of a bioprocess based on CHO cell culture. (AU)

• Processo FAPESP: 05/60826-3 - Desenvolvimento de bioprocesso para produção de larga escala de hormônios recombinantes e seus antagonistas em cultura de células CHO em biorreatores
• Beneficiário: Carlos Roberto Jorge Soares
• Modalidade de apoio: Auxílio à Pesquisa - Regular