Rapid Identification of Low Level Glycation Sites in Recombinant Antibodies by Isotopic Labeling with ¹³C₆-Reducing Sugars

Recombinant antibodies exhibit low levels of glycation from exposure to reducing sugars during production. As the glycation sites are typically distributed across the entire antibody, the levels at any one site are low and it becomes difficult to detect them in the conventional peptide maps. A model antibody was subjected to forced glycation by incubating with a high concentration of a 1:1 mixture of ¹²C₆/¹³C₆ reducing sugars with the assumption that the same sites in the native antibody will be glycated but to a lower extent. This approach simplified the detection of glycated tryptic peptide elution in the LC/MS analysis by giving a unique signature of two molecular ions with equal intensity and differing by 6.018 Da. An in-house developed script automatically processed large data files to generate a list of such peptide mass pairs. The high mass accuracy of the Orbitrap allowed us to assign the sequences unambiguously by comparison with all possible glycated peptide masses. This sequence list was subsequently used to verify their presence/absence in the digest of the native antibody. This work flow enabled rapid and confident identification of site-specific glycation even when levels are below 0.5%. We found the glycation sites to be distributed across the entire antibody studied.