肽
表位
鉴定(生物学)
生物化学
融合蛋白
亲和层析
免疫球蛋白轻链
肽序列
氨基酸
作者
Taylor Zhang,Jennifer Zhang,Daniel P. Hewitt,Ben Tran,Xiaoying Gao,Zhihua Julia Qiu,Max L. Tejada,Hélène Gazzano-Santoro,Yung-Hsiang Kao
摘要
The heterogeneity in therapeutic antibodies arising from buried unpaired cysteines has not been well studied. This paper describes the characterization of two unpaired cysteines in a recombinant humanized IgG1 monoclonal antibody (referred to as mAb A). The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis of mAb A samples showed three distinct peaks, indicating the presence of three species. The heterogeneities observed in the RP-HPLC have been determined to arise from unpaired cysteines (Cys-22 and Cys-96) that are buried in the VH domain. The Fab containing free thiols (referred to as “free-thiol Fab”) and the Fab containing the disulfide (referred to as “intact Fab”) of mAb A were generated through limited Lys-C digestion and purified with an ion exchange chromatography method. The binding of free-thiol Fab and intact Fab to its antigen was measured in a cell-based binding assay and an enzyme linked immunosorbent assay. The unpaired cysteines in the Fab of mAb A were found to ha...
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