化學添加劑提升動物細胞重組蛋白生產之研究

Improvement of Recombinant Proteins Production in NS0 Myeloma Cells under Serum-Free Conditions: Effects of Stimulatory Agents and Antioxidants.

張順芳(Shuen-Fang Chang)

指導教授 ： 余琬琴

國立臺北科技大學/工程學院/有機高分子研究所/碩士(2006年)

https://doi.org/10.6841/NTUT.2006.00152

摘要

動物細胞大量培養是近代生技產業生產高複雜度蛋白質藥物的重要技術之 一。由於單株抗體等蛋白質藥物的蓬勃發展，進入臨床前與臨床試驗的蛋白質藥物與日俱增，核准上市的蛋白質藥物也快速增加，因此如何快速增加產量並降低製造成本來滿足這些需求成為重要課題。在培養基中添加誘導劑是快速提高蛋白質產量的方法，常見的誘導劑有sodium butyrate、DMSO等，但這些誘導劑常造成細胞凋亡，限制了蛋白質的最終產量。本論文針對一生產基因重組干擾素的骨髓瘤細胞(NS0)，研發利用誘導劑與抗氧化劑的添加來提升蛋白質產量的方法，此細胞株已被馴化至一化學組成無血清培養基，本實驗室先前比較數種誘導劑，發現丙酸(sodium propionate)對NS0細胞具有獨特的效用，重組蛋白質產量高於文獻中常用的丁酸。以轉瓶批次培養，丁酸的最適濃度約為0.5mM，此濃度不會造成細胞死亡，且蛋白質產量提升了三分之ㄧ；丙酸適合濃度為1.0-2.0mM，蛋白質產量提高一倍。
本論文首先以2L的生物反應器驗證丙酸的效果，結果與轉瓶的實驗吻合。接著測試四種抗氧化劑L-ascorbic acid、N-acetyl L-cysteine、Aurintricarboxylic acid、Pyrrolidine dithiocarbamate 對NS0細胞的影響，以T型培養瓶為實驗系統，找出合適的添加濃度後，在與誘導劑搭配使用，期能將誘導劑造成的凋亡減到最低，而進一步提升蛋白質產量。結果顯示N-acetyl L-cysteine與誘導劑搭配使用，可進一步提升蛋白質產量。

關鍵字

骨髓瘤細胞 ； 誘導劑 ； 抗氧化劑 ； 化學合成培養基

並列摘要

The large-scale culture of mammalian cells has become an important part of the biotechnology industry, for the production of many therapeutic and diagnostic proteins. Because high protein dosages are often required for therapeutic efficacy, it is highly desirable to increase the yield and lower the manufacturing cost of therapeutic proteins. A simple way to rapidly enhance protein productivity is the addition of stimulatory agents to the growth media, such as sodium butyrate (NaBu) and DMSO. However, the beneficial effects of these stimulatory agents are often compromised by their growth inhibiting and apoptosis inducing effects, thus limiting the final protein yield. In order to improve the low protein productivity in a recombinant NS0 cell producing α-interferon, we first compared various stimulatory agents for their protein production enhancing effects and then examined several antioxidants for their ability to reduce the cytotoxic effects of stimulatory agents. In previous studies we found that sodium propionate is a better protein production
ii i
enhancing agent than the well known NaBu. Sodium propionate is more effective and less toxic. Under batch culture conditions in spinner flasks, the optimal NaBu concentration was about 0.5mM, and the final protein titer was increased 30%. On the other hand, the optimal concentration for sodium propionate was about 1-2mM, and the final protein titer was doubled.
In this thesis, the protein production stimulatory effect of sodium propionate was confirmed using 2L bioreactor and the result agree well with those of spinner flask batch cultures. In order to further increase the protein yield, we investigated the effect of four antioxidants: L-ascorbic acid, N-acetyl L-cysteine (NAC), Aurintricarboxylic acid (ATA), and Pyrrolidine dithiocarbamate (PDTC) on cell growth and protein production. Results indicate that NAC is the only antioxidant tested with a positive effect on protein production. When added simultaneously with sodium propionate, NAC further increased cell viability, culture longevity and protein yield. The other antioxidant tested all had negative impacts on protein production.

並列關鍵字

Mammalian cells ； stimulatory agents ； a-interferon

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