Optimization of cell culture conditions for G-CSF (granulocyte-colony stimulating factor) production by genetically engineered namalwa KJM-1 cells

✍ Scribed by Shinji Hosoi; Kazunari Murosumi; Katsutoshi Sasaki; Mitsuo Satoh; Hiromasa Miyaji; Mamoru Hasegawa; Seiga Itoh; Tatsuya Tamaoki; Seiji Sato

• Publisher: Springer
• Year: 1991
• Tongue: English
• Weight: 637 KB
• Volume: 7
• Category: Article
• ISSN: 0920-9069
• DOI: 10.1007/bf00135635

✦ Synopsis

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 ~tg/ml/day using a 25 cm 2 tissue culture flask, even though the cell number was above 7 • 105 cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per celt under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter T M (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0-and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 4 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1 • 107 cells/ml), and the amount of G-CSF reached 41 ].tg/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.