A Rapid Method for Identifying Glycation Sites in Therapeutic Proteins by Isotopic Labeling with 13C6- glucose

RP-128 During the production of recombinant proteins, they are exposed to cell culture medium containing glucose and some times galactose. Through a non-enzymatic process known as glycation, the reducing sugars can react with amino groups on the protein. Recombinant antibodies purified from mammalian cell cultures, typically exhibit low levels (~5%) of glycation at protein level. 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 regular peptide maps. We developed a novel method for rapid and robust identification of site specific glycation at levels below 1%. We used a recombinant humanized monoclonal antibody A (rhuMAb A) as a model and subjected it to in-vitro glycation with a mixture of 1:1 (v/v) 12C6:13C6 D-glucose. We assumed that glycation happened at the same sites but at higher levels than in the native protein. This in-vitro glycated protein was digested with trypsin and analyzed by lc/ms/ms. A unique feature of mass spectra of glycated peptides generated in this way is the presence of molecular ion peaks at 1:1 ratio that differ by 6.018 Da resulting from differential isotopic labeling in the glycation process. An in-house developed script processes the high resolution Orbitrap lc/ms data and automatically flags the glycated peptides based on this unique signature. Accurate mass capabilities of Orbitrap allowed these values to readily match with a list of all predicted glycated peptide masses in a rapid fashion. A list of observed glycated peptides was then used to (a) verify their presence in the native digest; (b) estimate the percent glycation at each site in the native protein; (c) confirm the glycation sites by targeted multi-stage ms/ms analysis. We found the glycation sites to be distributed across the antibody, most of them at levels <1%.