化学
亲和层析
色谱法
蛋白质纯化
钙
钙螯合素
抗体
生物化学
酶
有机化学
生物
免疫学
兰尼定受体
作者
Hee Sun Park,Hyungsu Jeon,Hyung Gon Jin,Jin Ho Bang,Young-Woo Song,Mi-Hyun Choi,Daekyung Sung,Won Suk Choi,Jin Yong Lee,Jae Sung Woo,Sangyong Jon,Sunghyun Kim
标识
DOI:10.1021/acs.analchem.2c00026
摘要
Affinity chromatography utilizing specific interactions between therapeutic proteins and bead-immobilized capturing agents is a standard method for protein purification, but its scalability is limited by long purification times, activity loss by the capturing molecules and/or purified protein, and high costs. Here, we report a platform for purifying therapeutic antibodies via affinity precipitation using the endogenous calcium ion-binding protein, calsequestrin (CSQ), which undergoes a calcium ion-dependent phase transition. In this method, ZZ-CSQ fusion proteins with CSQ and an affinity protein (Z domain of protein A) capture antibodies and undergo multimerization and subsequent aggregation in response to calcium ions, enabling the antibody to be collected by affinity precipitation. After robustly validating and optimizing the performance of the platform, the ZZ-CSQ platform can rapidly purify therapeutic antibodies from industrial harvest feedstock with high purity (>97%) and recovery yield (95% ± 3%). In addition, the ZZ-CSQ platform outperforms protein A-based affinity chromatography (PAC) in removing impurities, yielding ∼20-fold less DNA and ∼4.8-fold less host cell protein (HCP) contamination. Taken together, this platform is rapid, recyclable, scalable, and cost-effective, and it shows antibody-purification performance superior or comparable to that of the standard affinity chromatography method.
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