The development of comprehensive methods to characterize unpaired cysteines in monoclonal antibodies (mAbs) is very important for understanding structural heterogeneity, impurity, and stability. In this paper, unpaired cysteines observed in a therapeutic antibody (mAb1) were thoroughly studied by Liquid Chromatography-Mass Spectrometry (LC-MS) methods at the intact mAb, domain, and peptide levels. Three cysteine variants were observed at the intact mAb level with each variant containing two unpaired cysteines. Variants containing four or six unpaired cysteines were not observed. Domain analysis indicated that two Fab variants, each containing two unpaired cysteines, were present while the third variant contained two unpaired cysteines on the Fc region. Peptide mapping analysis localized the six unpaired cysteines to Cys22/Cys96, Cys146/Cys202, and Cys369/Cys427 in the heavy chain. No significant changes were observed for these unpaired cysteines in mAb1 under high pH and heat-stressed conditions. Structural analysis and molecular modeling revealed that these unpaired cysteines were buried inside the three-dimensional structure. The integrated LC-MS methods together with stress studies and structural analysis may potentially be applied to the analysis of unpaired cysteines in other mAbs.
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