蛋白A亲和层析法纯化单克隆抗体工艺的优化

Improved protein-A chromatography for monoclonal antibody purification

摘要:

治疗性单克隆抗体药物已成为生物医药领域市场最主要的产品类别。蛋白A亲和层析作为第一步捕获抗体蛋白最为有效的手段仍然在现有单克隆抗体纯化平台中占据主导地位。在本研究中，首先开发了一种基于低pH处理抗体细胞回收液的新型细胞液回收技术，该技术能有效去除宿主相关污染物 (非组蛋白宿主杂质蛋白、组蛋白、DNA、蛋白聚合物等)，同时保证较高的抗体回收率。通过该技术有效预处理后，蛋白A纯化效率可提高10倍左右，并且有效避免了抗体洗脱液中和后浊度的上升，大大减轻了后续蛋白纯化的压力。同时我们也对酸性处理中各种宿主杂质去除机制进行了研究。然后，预处理的洗脱液再经一步Capto adhere色谱纯化，非组蛋白宿主杂质蛋白降低至5 ppm、DNA小于1 ppb、组蛋白降低至检测限以下、蛋白聚合物小于0.01%。总过程抗体蛋白收率87%。该两步法抗体纯化技术可有效集成至当前主流抗体纯化平台，具有良好的大规模应用价值。

Abstract:

Therapeutic monoclonal antibodies become the major product class within the biopharmaceutical market. Protein A as the first capture step is still dominant in current platforms for purification of monoclonal antibodies. In this study, we developed a new antibody harvest process that incorporates acidic treatment of cell harvest, demonstrating high process yield, improved clearance of host cell associated contaminants, like non-histone host cell protein, histone, DNA and heteroaggregates. Host protein contamination was reduced about 10-fold compared to protein A loaded with harvest clarified by centrifugation and microfiltration. Turbidity increase of eluted IgG upon pH neutralization was nearly eliminated. Residual levels of impurities in the protein A eluate were achieved that potentially meet requirements of drug substance and thus alleviate the burden for further impurities removal in subsequent chromatography steps. The mechanism of host cell associated contaminants removal during acidic treatment was also explored. After a polishing step by Capto adhere, host cell protein was reduced to less than 5 ppm, DNA less than 1 ppb, histone to undetectable level, heteroaggregates less than 0.01% with total IgG recovery around 87%. This efficient process can be easily integrated into current IgG purification platforms, and may overcome downstream processing challenges.