Tissue-type plasminogen activator를 생산하는 Chinese Hamster Ovary(CHO) 세포의 고농도 배양을 위한 전략 = Strategies for high density cultivation of CHO cells producing tissue-type plasminogen activator
저자
발행사항
춘천 : 江原大學校 , 2000
학위논문사항
학위논문(석사)-- 江原大學校 大學院 : 生物應用工學科 2000
발행연도
2000
작성언어
한국어
KDC
491.846 판사항(4)
발행국(도시)
강원특별자치도
형태사항
ii, 125 L. : 삽도 ; 26 cm.
소장기관
In animal cell culture, glutamine is essential amino acid used as the source of energy and precursors of other amino acids, nucleotides, purine or pyrimidine. The accumulating of ammonium ion in an animal cell culture media is inevitable because ammonium ion is excreted as a byproduct of glutamine. Ammonium ion has been shown to be inhibitory on cell growth and product formation according to many report. Therefore, ammonium ion removal strategy is required for high density cultivation of animal cells.
In this study, we make it a goal to develop a high tPA productivity system by establishing a high cell density culture system with ammonium ion removal and to set up an optimum operational strategy for the new system. Firstly, culture media for CHO cells was optimized for high density cultivation and cell density was increased by 2 times. The effects of ammonium ion on CHO cells were investigated to confirm the inhibitory characteristics of ammonium ion. Ammonium ion of 2mM inhibited cell growth by 60% and tPA productivity by 70%. Therefore, ammonium ion removal method was developed for application to high density cell culture system. Phillipsite-Gismondine synthetic zeolite(IW-85) with high selectivity and high NH_(4)^(+) adsorption capacity was selected and immobilized as several types, such as chip, bead, membrane, and agarose. The membrane type was selected as an optimum immobilized adsorbent. The application of membrane type immobilized adsorbent to the culture of anchorage dependent CHO cells resulted in the enhancement both cell growth and tPA productivity.
The gene amplification by DHFR-methotrexate cointegration system was also tried to enhance tPA productivity. After gene amplification, the maximum cell density decreased by 40% but specific productivity of tPA increased by 44 times. Lactate yield also increased with steps of amplification. The gene amplification was found to be quite stable because almost 80% of productivity could be maintained after reverse adaptation for 4 weeks. Development of anchorage-independent cells from both original cells and amplified cells were also tried to get high productivity cells. It was found that both suspension adapted cells showed better cell growth and tPA productivity than both anchorage- dependent cells.
In culture of suspension cells, a novel culture system coupled with membrane type immobilized adsorbent and batch operation mode was applied to high density culture. A novel culture system enhanced cell growth by 15% and tPA productivity by 17%. A novel culture system coupled with agarose type immobilized adsorbent and batch operation mode was also tried, and it enhanced cell growth by 13% and tPA productivity by 15%. A novel culture system coupled with membrane type immobilized adsorbent and fed-batch operation mode was also tried and showed increase of cell growth by 22% and tPA productivity by 25%. A coupling system of agarose type immobilized adsorbent and fed-batch operation mode also enhanced cell growth by 15% and tPA productivity by 18%.
A high cell density culture system for anchorage-dependent cells was also developed and an optimum operational strategy was suggested for the high tPA productivity of CHO cells. The glass microcarrier bead was selected as an optimum microcarrier in terms of cell growth for immobilized culture system of anchorage-dependent CHO cells. An integrated culture system coupled with fed-batch operation mode and packed bed of calcium -alginate zeolite beads enhanced cell growth by 20% and tPA productivity by 2.2 times. Ammonium ion concentration was reduced by 40%. A novel culture system coupled with fed-batch operation mode and membrane type immobilized adsorbent was also tried for anchorage-dependent CHO cells and the system enhanced cell growth by 30% and tPA productivity by 2.5 times.
The various novel culture systems coupled with immobilized adsorbent and optimum operation mode of fed-batch developed in this study can be applied to the production of not only tPA but also several high value products, such as EPO, ILN, INF, GM-CSF, in industrial animal cell culture process.
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