β-elimination and peptide bond hydrolysis:: Two distinct mechanisms of human IgG1 hinge fragmentation upon storage

We expand the understanding of hinge-region degradation of human IgG1 monoclonal antibodies. First, our data more accurately confirm previously proposed hydrolysis of the CO-NH peptide bonds within the IgG1 heavy chain C(220)DKTHTC hinge sequence. Second, we provide evidence that the adjacent heavy chain S219-C220 bond is cleaved by a different mechanism. The cleavage of S219-C220 becomes more pronounced with elevated pH (pH > 7) as does the formation of light-chain-heavy-chain thioether linkage, another IgG1 degradation product whose mechanism has remained unknown. Careful application of bioanalytical methods and mass spectrometry provide strong evidence for a central role of a beta-elimination mechanism to account for the S219-C220 cleavage as well as the formation of the thioether linkage product.