動物細胞の固定化及び培養環境の制御による有用物質の効率的生産

通过固定动物细胞和控制培养环境有效生产有用物质

• 批准号: 05555220
• 负责人: KOBAYASHI Takeshi
• 金额: $ 4.74万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05555220/
• 关键词: Mammalian cell culture; Immobilization; Dissolved oxygen concentration; Nutrients concentrations; Control of culture; Erythropoietin; L-929 cell; vero細胞; SV40; 温度感受性; 代謝制御

1.A photo-crosslinkable resin, BIX12, was selected from among various photo-crosslinkable resins for the immobilization of animal cells. BIX12 had no cytotoxic effect on the growth of hybridoma cells and the production of monoclonal antibody. A high viable cell density of more than 10^7 cells/ml-gel was attained, and the antibody productivity was improved 8.5-fold compared with conventional suspension culture using a spinner flask.2.By introduction of the positive charge to macroporous cellulosic support, anchorage-independent hybridoma cells were immobilized more than 10-fold compared with the unmodified support. Large amount of monoclonal antibody was continuously produced for long term. Recombinant L-929 cells transfected with the human erythropoietin (EPO) gene were also immobilized in the macroporous cellulosic support. A high cell density of more than 2x10^7 cells/ml-support and high EPO productivity were achieved and maintained for 50 d through the use of the inner-loop type air-lift bioreactor.The effects of dissolved oxygen concentration (DO) on anchorage-dependent cell growth, cell metabolism, and erythropoietin (EPO) or tissue plasminogen activator (tPA) production were investigated. The optimal DO for cell growth was 5mg/l, whereas the optimal EPO or tPA production was observed at higher DO such as 15mg/l.4.An on-line system using HPLC was developed for the measurement of glucose, glutamine and lactate in a culture broth. Using the system, the glucose and glutamine concentrations were controlled simultaneously by an adaptive-control algorithm within the ranges 0.2-2.0 and 0.1-0.6g/L,respectively. When the glucose or glutamine concentration was controlled at the low level of 0.2g/L,antibody production increased two-fold compared to the high glucose or glutamine level.

1.从多种光交联树脂中筛选出一种光交联树脂BIX 12，用于动物细胞的固定化。BIX 12对杂交瘤细胞的生长和单克隆抗体的产生无细胞毒作用。与传统的转瓶悬浮培养相比，抗体产量提高了8.5倍。2.通过在大孔纤维素载体上引入正电荷，固定化了非锚定依赖性杂交瘤细胞，固定化率提高了10倍以上。单克隆抗体的大量生产是长期持续的。用人红细胞生成素（EPO）基因转染的重组L-929细胞也固定在大孔纤维素载体中。采用内循环气升式生物反应器，在50 d内获得了2 × 10^7细胞/ml载体的高细胞密度和EPO的高产率，并研究了溶解氧浓度（DO）对细胞贴壁生长、细胞代谢和促红细胞生成素（EPO）或组织型纤溶酶原激活剂（tPA）产生的影响。细胞生长的最适DO为5 mg/l，而EPO或tPA的最佳产量在较高的DO（15 mg/l）下观察到。4.建立了高效液相色谱在线测定发酵液中葡萄糖、谷氨酰胺和乳酸的系统。利用该系统，通过自适应控制算法同时控制葡萄糖和谷氨酰胺的浓度分别在0.2-2.0和0.1-0.6g/L的范围内。当葡萄糖或谷氨酰胺浓度控制在0.2g/L的低水平时，抗体产量比高葡萄糖或谷氨酰胺水平增加两倍。

项目成果

Shuji Terashima: "Continuous production of human erythropoietin by immobilized recombinant L-929 cells" Journal of Fermentation and Bioengineering. 77(1). 52-56 (1994)

Shuji Terashima：“通过固定化重组 L-929 细胞连续生产人类促红细胞生成素”《发酵与生物工程杂志》。

Shuji Terashima: "Immobilization of hybridoma cells with macroporous cellulosic support and improved production of monoclonal antibody" Seibutsu-kogaku Kaishi. 71 (3). 165-170 (1993)

Shuji Terashima：“用大孔纤维素支持物固定杂交瘤细胞并改善单克隆抗体的生产”Seibutsu-kogaku Kaishi。

寺嶋 修司: "多孔性セルロース担体によるハイブリドーマ細胞の固定化とモノクローナル抗体の連続生産" 生物工学会誌. 71. 165-170 (1993)

Shuji Terashima：“使用多孔纤维素载体固定杂交瘤细胞并连续生产单克隆抗体”日本生物技术学会杂志71. 165-170（1993）。

Hideo Kurokawa: "Kinetic study of hybridoma metabolism and antibody production in continnous culture using serum-free medium" Journal of Fermentation and Bioengineering. 76. 128-133 (1993)

Hideo Kurokawa：“使用无血清培养基连续培养中杂交瘤代谢和抗体产生的动力学研究”发酵与生物工程杂志。

Hideo Kurokawa: "Kinetic study of hybridoma metabolism and antibody production in continuous culture using serum-free medium" Journal of Fermentation and Bioengineering. 76 (2). 128-133 (1993)

Hideo Kurokawa：“使用无血清培养基连续培养中杂交瘤代谢和抗体产生的动力学研究”发酵与生物工程杂志。