本論文的主要目標為開發適合中國倉鼠卵巢細胞(CHO)的無血清培養基與提高CHO之重組蛋白質產量。
首先我們馴化生產重組蛋白質之CHO細胞，使其適應無血清培養條件，並以統計的方法篩選並開發血清取代成分，所開發出的血清取代物之成分為1 % (v/v) SITE, 0.3g/L 酵母抽出物與 0.09% (v/v)亞油酸-牛血清球蛋白混合物。此配方與JRH公司之無血清產品EX-CELLTM301，對CHO細胞生長及重組蛋白質生產效果相當。

為進一步降低培養基成本﹐重新分析所有添加的無血清取代成分，發現胰島素是影響CHO細胞生長最重要的成分，而ATA在無血清或無蛋白質培養的條件下，有促進CHO細胞生長之作用，並可以取代胰島素。ATA還會影響細胞生長的方式，50mg/L濃度以下的ATA會誘使部分CHO以貼附方式生長；當ATA添加濃度大於50 mg/L，則所有細胞以懸浮方式生長。因為ATA可以取代胰島素，所以我們利用不同kinase抑制物，討論其對胰島素或ATA誘導的CHO細胞增殖之影響，結果發現二者抑制趨勢完全相同，由此推測ATA與胰島素可能分享共同的作用目標或機制，且此作用機制是與kinase的磷酸化有關。

在提高CHO細胞之重組蛋白質產量方面，本研究探討在發酵液中添加不同化學物質及改變發酵操作條件的方式，來促進重組蛋白質之生產。

探討一系列不同碳鏈長度之有機酸對細胞生長及重組蛋白質生產之影響，發現戊酸較一般文獻常用的丁酸對細胞生長抑制性較低，且有高的重組蛋白質生產促進效果(產量約提高2.2倍)，其最適的添加濃度是1 mM。此外作者亦發現DMSO對CHO細胞生產重組蛋白質有促進效果(產量約提高2倍)﹐同時會抑制細胞生長，其最適的添加濃度是1 %；然而DMSO對融合瘤細胞生長也有抑制作用，但對融合瘤細胞之單株抗體生產則無促進效果。戊酸與DMSO同時添加會較單獨添加，有更好的重組蛋白質生產促進效果(產量約提高2.8倍)。

The aims of this thesis are to develop new serum-free media for recombinant Chinese hamster ovary (CHO) cells and to increase the recombinant protein production of CHO cells.
First of all, CHO NTHU 108 cells were adapted to grow in serum-free media. A serum free medium for recombinant CHO NTHU 108 cell growth and fusion protein (CD20 linked to a human IgG-Fc γ4 fragment) synthesis were systematically developed using factorial designs combined with the steepest ascent method. Experimental results indicate that the optimal composition of serum replacement for specific fusion protein production was 1% SITE (selenium, insulin, transferrin, ethanolamine), 0.3g/L yeast extract, and 0.09% linoleic acid-BSA. Cell growth and fusion protein production of the adapted CHO NTHU 108 cultured in Iscove’s modified Dulbecco’s medium supplemented with these serum substitutes were comparable to those in the Ex-CellTM 301 commercial serum-free medium. These serum substitutes can also promote CHO cell growth and fusion protein production in nine kinds of commercial media. The low protein content of the developed medium facilitates downstream processing and product purification.

We analyzed all these serum replacement ingredients and found that insulin is the most important ingredient to support the proliferation of CHO cells in serum-free conditions. Aurintricarboxylic acid (ATA) was the chemical that can replace insulin and support the proliferation of CHO cells in serum-free conditions. When ATA was added at a concentration of 30 mg/l in DME/F12 medium to CHO cells in static six-well plates, some of the cells exhibited adherent growth while others grew in suspension. Beyond the critical concentration of ATA, CHO cells grew in single-cell suspension. In the same serum-free medium, insulin at a level of 0.5mg/l was found to be the most important protein ingredient promoting cell growth. We used the respective kinase inhibitors to investigate their influence on the cell proliferation induced by ATA and insulin. It is interesting that the inhibition by seven kinase inhibitors of ATA-induced proliferation is similar to that of insulin-induced proliferation. It is possible that ATA mimics insulin and influences the mitogen activated signal transduction to induce the proliferation of CHO cells. Although the actual mechanism of the proliferation of CHO cells by ATA is unclear, ATA supported the long-term proliferation of CHO cells under serum-free conditions and thus could be used as a good substitute for insulin in the formulation of protein-free media.

In order to elevate the recombinant protein production of CHO cells, we evaluate several chemicals. Nine carboxylic acids were tested to evaluate their effects on recombinant fusion protein production and cell growth of Chinese Hamster Ovary (CHO) cells. Pentanoic acid was demonstrated to have the highest enhancement effect on the protein biosynthesis of CHO cells among the acids tested. Pentanoic acid also had less growth suppression effects compared with butyrate. The optimal induction time and concentration of pentanoic acid for a 120-h batch culture were 72 h and 1 mM, respectively. Apoptosis (programmed cell death) was observed in the serum-free batch culture of CHO cells using a cell death detection ELISA kit. The addition of butyrate accelerated the rate of apoptosis of CHO cells whereas the addition of pentanoate did not. These results confirmed that pentanoic acid was a better stimulant for protein biosynthesis in animal cell culture than butyrate.

Dimethyl sulfoxide (DMSO) was found to stimulate stable transformed CHO cells to synthesize different recombinant proteins and repress the proliferation rate. The expressions of fusion protein andβ-galactosidase were increased 2.0- and 1.4–fold following the addition of DMSO. The expression of fusion protein could be elevated up to 2.8–fold that of uninduced control by the simultaneous addition of DMSO and pentanoic acid. However, DMSO could not elevate the production of the monoclonal antibody of three tested hybridoma cell lines, although it could inhibit the growth rates of hybridoma. The optimal induction concentration of DMSO for protein stimulation of CHO cells was 1%. Whereas, the inducing timing of DMSO for CHO cells was not so critical for 8-day batch culture.

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