High-level expression of recombinant human soluble thrombomodulin in serum-free medium by CHO-K1 cells

• Published in: Applied microbiology and biotechnology 1993, Vol.38 (4), p.520-525
• Main Authors: OGATA, M, WAKITA, K.-I, KIMURA, K, MARUMOTO, Y, OH-I, K, SHIMIZU, S
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Biotechnology; Cell Division; CHO Cells; Cloning, Molecular; Cricetinae; Culture Media, Serum-Free - pharmacology; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; Glucose - pharmacology; Humans; Methods. Procedures. Technologies; Microspheres; Modification of gene expression level; Oxygen - pharmacology; Receptors, Cell Surface - biosynthesis; Receptors, Cell Surface - genetics; Receptors, Thrombin; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Time Factors
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/bf00242948

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.