Localization and Quantitation of Free Sulfhydryl in Recombinant Monoclonal Antibodies by Differential Labeling with 12C and 13C lodoacetic Acid and LC-MS Analysis

A low percentage of free sulfhydryl is a common feature of recombinant monoclonal antibodies although, in theory, all cysteine residues should be involved in disulfide bonds. A differential alkylation method was developed to determine the percentage of free sulfhydryl at each cysteine residue of four recombinant monoclonal antibodies. Free sulfhydryl was first alkylated with C-12 iodoacetic acid. Free sulfhydryl, resulting from the reduction of disulfide bonds, was then alkylated with C-13 iodoacetic acid. Cysteine containing peptides that were modified by C-13 iodoacetic acid showed a molecular weight that was 2 Da higher than the same peptide that was modified by C-12 iodoacetic acid. Peptides, containing the same cysteine residues that were modified with both alkylation reagents, coeluted on reversed-phase chromatography. Analysis by mass spectrometry resulted in two partially overlapped m/z series for each cysteine containing peptide, corresponding to modification by iodoacetic acid with C-12 or C-13. The percentage of free sulfhydryl was then calculated using the two m/z series at each cysteine site. A low percentage of free sulfhydryl was detected at every cysteine residue in the four antibodies studied. Although different antibodies contained different levels of free sulfhydryl, similar distribution of free sulfhydryl in the domain structures was observed in the four antibodies.