抗人表皮生长因子受体2人源化单克隆抗体在中国仓鼠卵巢细胞中的表达、纯化及活性测定

Expression,Purification and Activity Determination of Humanized Anti-HER2 Monoelonal Antibody in CHO

目的筛选得到稳定表达重组人源化抗HER2单克隆抗体(rhHER2-mAb)的中国仓鼠卵巢细胞(CHO)最优表达株,采用旋转振荡式培养及小规模放大,测定纯化后抗体蛋白的活性和亲和力。方法使用一次性旋转振荡生物反应器——Tube-spin对细胞株进行高通量筛选,然后使用摇瓶进行规模放大培养,利用Protein A亲和层析柱纯化融合蛋白,SDS-PAGE、HPLC检测产物的纯度,夹心ELISA检测产物的表达量,MTT检测产物的活性。结果通过比较,确定产量最高的#38细胞克隆株,经过规模放大培养得到足量细胞上清液,摇瓶表达量达到(152±20)mg.L-1。纯化后经检测目的蛋白的相对分子质量约为150×103,纯度在96%以上,与商品化的赫赛汀一致;活性检测和亲和力检测也显示其活性和亲和力与赫赛汀相当。结论通过细胞株筛选得到高表达细胞株,经旋转振荡式放大培养,成功纯化获得抗体蛋白,与商品化药物性质一致,为大规模旋转振荡培养发酵奠定基础。

OBJECTIVE To screen for the optimal CHO cell clone which stably expresses recombinant humanized anti-HER2 monoclonal antibody （rhHER2-mAb） anti to measure its bioactivity. METHODS Disposable orbital shaking bioreactor, Tuhespiu, was utilized for high-throughput screening, and then orbital shaking flask was used for seale-up. Protein A affinity chromatography was utilized to purify the fusion protein. SDS-PAGE and HPLC were utilized to detect its purity, ELISA was utilized to detect its production amount, and MTF was utilized to detect its bioactivity. RESULTS Through the cell clone selection, it was tound that # 38 cell clone had the best expression performance, with the productivity in shaking flask of （152 ±20） mg·L-1. After scale up, enough supernatant was got for the purification. The molecular weight of the purified protein was about 150 × 10^3 , and its purity was higher than 96%. Analys!s of bioactivity and binding affinity showed that its activity and bind affinky were close to the commercialized product, Herceptin. CONCLUSION A cell clone with high productivity was obtained by cell clone screening. The antibody protein was successfully obtained by orbital shaking scale-up cultivation and purification. Its bioactivity and binding affinity were consistent with the commercialized drug. This method has laid a foundation for large-scale fermentation of this recombinant protein.