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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 the optimal CHO cells expressing rhHER2-mAb stably expressing recombinant humanized anti-HER2 monoclonal antibody (mAb), and to determine the activity of the purified antibody after purified by rotary shaking culture and small scale amplification. Affinity. Methods The cell strain was screened by using a one-time rotary shaker bioreactor -Tube-spin and then scaled up using a shake flask. The fusion protein was purified by Protein A affinity chromatography and analyzed by SDS-PAGE and HPLC The purity of the product, sandwich ELISA test product expression, MTT test product activity. Results By comparison, the # 38 cell clone with the highest yield was identified, and a sufficient amount of cell supernatant was obtained after the scale-up culture. The flask reached a volume of (152 ± 20) mg.L-1. After purification, the relative molecular mass of the tested protein was about 150 × 103, the purity was above 96%, which was consistent with the commercial Herceptin. The activity test and affinity test also showed that its activity and affinity were comparable to Herceptin. Conclusion The highly expressed cell strain was screened by cell line. The recombinant protein was successfully purified by rotary shaking culture. The purified protein was consistent with commercial drug, which laid the foundation for large-scale rotary shaking culture.