Development of a Fed-Batch Culture Process for Enhanced Production of Recombinant Human Antithrombin by Chinese Hamster Ovary Cells(CELL AND TISSUE ENGINEERING)
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概要
- 論文の詳細を見る
Antithrombin is a serine protease inhibitor that inactivates several coagulation proteases, primarily thrombin and factor Xa. The Chinese hamster ovary (CHO) cell line transfected with a vector expressing recombinant human antithrombin (rAT) and a selectable marker, glutamine synthetase (GS), was cultivated in a 2-l fed-batch culture process using serum-free, glutamine-free medium. To maximize the rAT yield, effects of culture pH, balanced amino acid feeding, and an increased glutamate concentration on cell metabolism and rAT production were investigated. When cells were grown at pH values of 6.6, 6.8, 7.0, and 7.2, the maximum cell density and maximum lactate concentration decreased with decreasing pH. The highest production level of rAT was obtained at culture pH 6.8 due to the extended culture lifetime. Compared to the imbalanced amino acid feeding at culture pH 6.8, the balanced amino acid feeding increased the amount of rAT activity by 30% as a result of an increased viable cell number. A decrease in the specific glucose consumption rate (q_<Glc>) with increasing culture time was observed in all the above-mentioned experiments, while the glucose concentration was maintained above 0.7gl^<-1>. In addition, a decrease in the specific rAT production rate (q_<rAT>) was observed after the depletion of lactate in the late cultivation stage. Taken together, these results suggest that the reduced availability of cellular energy caused by the decrease in q_<Glc> and depletion of lactate led to the decrease in q_<rAT>. This decrease in q_<rAT> was partially prevented by increasing the residual glutamate concentration from 1mM to 7mM, thus resulting in an additional 30% increase in the amount of rAT activity. The optimized fed-batch culture process yielded 1.0gl^<-1> rAT at 287 h of cultivation.
- 社団法人日本生物工学会の論文
- 2005-11-25
著者
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Miki Hideo
Protein Research Laboratory, Mitsubishi Tanabe Pharma Corporation
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Kobayashi Kaoru
Protein Research Laboratory, Mitsubishi Tanabe Pharma Corporation
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Ohya Tomoshi
Protein Research Laboratory Pharmaceutical Research Division Mitsubishi Pharma Corporation
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Ohya Tomoshi
大阪大学 工学研究科生命先端工学専攻
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Ohya Tomoshi
Protein Reserach Laboratories Pharmaceuticals Research Division Mitsubishi Pharma Corporation
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Kobayashi K
Department Of Chemical And Biological Sciences Faculty Of Science Japan Women's University
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Kobayashi K
Laboratory Of Molecular Biotechnology Graduate School Of Biological & Agricultural Sciences Nago
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Kuwae Shinobu
Protein Research Laboratory, Pharmaceutical Research Unit, Mitsubishi Pharma Corporation
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Ohda Toyoo
Protein Research Laboratory, Pharmaceutical Research Unit, Mitsubishi Pharma Corporation
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Tamashima Hiroshi
Protein Research Laboratory, Pharmaceutical Research Unit, Mitsubishi Pharma Corporation
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Ohda Toyoo
Bioscience Research Drug Discovery Laboratories Pharmaceutical Research Division Yoshitomi Pharmaceu
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Tamashima Hiroshi
Protein Research Laboratory Pharmaceutical Research Unit Mitsubishi Pharma Corporation
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Kobayashi Kaoru
Protein Research Laboratory Pharmaceutical Research Division Mitsubishi Pharma Corporation
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Kobayashi K
Shizuoka Univ. Shizuoka Jpn
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Miki Hideo
Protein Res. Lab. Mitsubishi Tanabe Pharma Corp.
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Kuwae Shinobu
Protein Research Laboratory Pharmaceutical Research Division Mitsubishi Pharma Corporation
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Kuwae Shinobu
Research Center For Cell And Tissue Culture Faculty Of Agriculture Kyoto University
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Kobayashi Kaoru
Protein Research Laboratory Mitsubishi Tanabe Pharma Corporation
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Ohya Tomoshi
Protein Research Laboratory Mitsubishi Tanabe Pharma Corporation
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