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, 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.
Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares IPEN-CNEN/SP, Av. Prof. Lineu Prestes 2242, Cidade Universitária, 05508-900 São Paulo, Brazil. fernandaarthuso@yahoo.com.br
Fernanda Santos
FS
Arthuso
Paolo
P
Bartolini
Carlos Roberto Jorge
CR
Soares
D000818:Animals; D016466:CHO Cells / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods*; D006224:Cricetinae; D016895:Culture Media, Serum-Free / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D011388:Prolactin / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D016895:Culture Media, Serum-Free; D011388:Prolactin