開發可高效率純化抗體之多重複鏈球菌蛋白G

Development of High Capacity Multiple Protein G for The Antibody Purification

黃奕中(Yi-Jung Huang)

指導教授 ： 鄭添祿

高雄醫學大學/生命科學院/生物科技學系碩士班/碩士(2016年)

摘要

治療性單株抗體藥需求逐年成長，因此如何有效地降低生產成本，將會是抗體藥廠面臨的問題，針對此問題我們提出了解決策略，挑選出蛋白質G-C2區域進行重複延長，進而得到多重複蛋白C2，此方式能透過提升抗體結合數，藉以增加單株抗體的產量，以降低生產成本。接著透過2C2，4C2及 8C2基因建構、蛋白質表現、純化及功能性測試，我們發現8C2結合抗體數多於4C2, 2C2及蛋白質G。除此之外，在管柱純化系統上也驗證了相同的結果，綜觀上述8C2純化系統具有1.獲取抗體數多於市售Cys-protein G 2.大幅降低純化成本3.能快速的合併至現有標準、規模化的抗體純化平台，另外透過8C2氨基酸組成及結構分析，也發現此蛋白具有1.耐高溫2.免疫訊號放大的特性，此特性可應用至加熱法製造及優化免疫沉澱法，最後進行各大蛋白質藥廠的市占率及成長率評估，以提高媒合至抗體生產線的速度，以上的研究表明8C2極具產業應用性。

關鍵字

單株抗體藥 ； 蛋白質G-C2區域

並列摘要

The monoclonal antibody drugs are wildly used in clinical, and the market has been 10% increased year by year. Therefore, it is necessary to have a good approach for the purification of antibody. The protein G column is often used in the production of antibody drug in pharmaceutical factory. However, the multiple purification process lead to an unstable environment which reduced the yield of antibody and increased the cost in the protein G based system. In our study, we developed a high efficiency antibody purification system which utilized the multiple C2 domain from streptococcus G protein. The C2 domain had the advantage to bind the Fc portion of antibody. We constructed the multiple C2 (2C2, 4C2, 8C2) gene into the PET-22b vector. The multiple C2 protein were successfully expressed by the BL21 and examined by SDS PAGE. The large scale multiple C2 protein were purified by Ni-column based system and S200 size exclusion column. The binding ability to antibody of purified multiple C2 protein were investigated by ELISA. The 8C2 had higher optical density than 2C2 and 4C2 protein, it suggests that the binding capacity were increased with the repeating of C2 protein. Therefore, the multiple C2 protein could be used in many aspects, such as (1) To prepare the high capacity column for the purification of antibody with high yield and low cost. (2). To prepare the immune-beads for immunoprecipitation experiment. (3) To prepare signal amplification bead to improve the sensitivity of ELISA system.

並列關鍵字

monoclonal antibodies ； protein G - C2 domain

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