title: High-level expression of recombinant human soluble thrombomodulin in serum-free medium by CHO-K1 cells.
abstract: Cultivation of gene-engineered Chinese hamster ovary (CHO-K1) cells that produce recombinant human soluble thrombomodulin (rsTM) was investigated to optimize conditions for high-level expression of the protein in a serum-free medium. For economic protein production, oxygenation of cultures with pure O2 permitted sufficient cell growth for high rsTM production with only 1 g/l of microcarriers and a low foetal bovine serum concentration. A longer growth phase (over 5 days) with serum was important to establish sufficient growth of this cell line on the microcarriers for subsequent serum-free culture, and to support a long-term production phase (about 2 months). In the production phase, a high glucose concentration (6.15 g/l) in the serum-free medium was very effective for prolonging the harvest cycle interval. Under these conditions, up to 100 mg/l rsTM was expressed in the conditioned medium. The rates of glucose consumption (G) and lactate production (L) were measured periodically and their ratio (L/G ratio) correlated with rsTM productivity. When the average L/G ratio was lower, reflecting a lower lactate production rate due to appropriate oxygenation of the culture, the specific rsTM production rate increased. Thus it may be possible to estimate protein productivity from L/G ratios calculated from the glucose and lactate measurements.
authors.affiliation: Production Technology Research Laboratories, Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Ogata
authors.forename: K
authors.initials: K
authors.lastname: Wakita
authors.forename: K
authors.initials: K
authors.lastname: Kimura
authors.forename: Y
authors.initials: Y
authors.lastname: Marumoto
authors.forename: K
authors.initials: K
authors.lastname: Oh-i
authors.forename: S
authors.initials: S
authors.lastname: Shimizu
mesh_terms: D000818:Animals; D016466:CHO Cells; D002455:Cell Division; D003001:Cloning, Molecular; D006224:Cricetinae; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D008863:Microspheres; D010100:Oxygen / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics; D018179:Receptors, Thrombin; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013997:Time Factors
chemical_list: D016895:Culture Media, Serum-Free; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D005947:Glucose; D010100:Oxygen