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

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 ~40 days...

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Veröffentlicht in:Applied biochemistry and biotechnology 2012-08, Vol.167 (8), p.2212-2224
Hauptverfasser: Arthuso, Fernanda Santos, Bartolini, Paolo, Soares, Carlos Roberto Jorge
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Sprache:eng
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Zusammenfassung: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 ~40 days, to grow in suspension in serum-free medium. High cell densities of up to 4.0 × 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 μg 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.
ISSN:0273-2289
1559-0291
DOI:10.1007/s12010-012-9745-1