检测并降低固定化蛋白A纯化的单克隆抗体制剂中蛋白A的污染

Detection and reduction of protein A contamination in immobilized protein A purified monoclonal antibody preparations.

作者信息

Bloom J W, Wong M F, Mitra G

机构信息

Protein Purification Department, Cutter Biological, Berkeley, CA 94710.

出版信息

J Immunol Methods. 1989 Feb 8;117(1):83-9. doi: 10.1016/0022-1759(89)90121-x.

DOI:10.1016/0022-1759(89)90121-x

PMID:2913162

Abstract

Protein A chromatography is an excellent technique for the purification of monoclonal antibodies. However, the presence of Protein A in therapeutic monoclonal antibody preparations due to leaching has been linked with toxicity in animals and humans. Two sandwich ELISAs were developed to monitor Protein A column leaching: (1) rabbit anti-Protein A for capture and anti-Protein A F(ab')2-HRP for detection; and (2) rabbit anti-Protein A for capture and anti-Protein A-biotin for detection. The biotin ELISA is sensitive to the subnanogram range. In addition, these assays were used to develop DEAE and gel filtration chromatography techniques that substantially reduce Protein A levels in monoclonal antibodies purified by Protein A chromatography.

摘要

蛋白A层析是纯化单克隆抗体的一项出色技术。然而，由于浸出导致治疗性单克隆抗体制剂中存在蛋白A，这已与动物和人类的毒性相关联。开发了两种夹心酶联免疫吸附测定法（ELISA）来监测蛋白A柱的浸出情况：（1）用兔抗蛋白A进行捕获，用抗蛋白A F(ab')2-辣根过氧化物酶（HRP）进行检测；（2）用兔抗蛋白A进行捕获，用抗蛋白A-生物素进行检测。生物素ELISA对亚纳克范围敏感。此外，这些检测方法被用于开发二乙氨基乙基（DEAE）和凝胶过滤层析技术，这些技术能大幅降低通过蛋白A层析纯化的单克隆抗体中的蛋白A水平。