Identification and Localization of Unpaired Cysteine Residues in Monoclonal Antibodies by Fluorescence Labeling and Mass Spectrometry

Each human IgG1 antibody contains a total of thirty-two cysteine residues. In theory, all of them are involved in disulfide bonds, and no free sulfhydryl should be detected. However, literature has suggested that the presence of low levels of free sulfhydryl groups is likely a common feature of recombinant and wild type IgG1 antibodies. Currently, there is little information correlating the presence of free sulfhydryl to specific cysteine residues. The presence of free sulfhydryl groups in five recombinant monoclonal antibodies and their locations were further investigated in the current study. Free sulfhydryl groups were first modified using 5-idoacetamidofluorescein (5-IAF), which was followed by reduction of disulfide bonds and alkylation with iodoacetic acid (IAA). This procedure allowed differentiation of free cysteine residues from cysteine residues that were involved in disulfide bonding. In addition, it allowed a sensitive fluorescence detection of peptides with free sulfhydryl groups. The locations of the free sulfhydryl groups were determined using mass spectrometry after fraction collection. The results indicated that different antibodies had different levels of free sulfhydryl due to multiple unpaired cysteine residues commonly in the constant domains. Furthermore, free sulfhydryl due to unpaired cysteine residues in the variable domains varied for different antibodies. Interestingly, free sulfhydryl was rarely associated with cysteine residues that were involved in interchain disulfide bonds.