A chemical and computational approach to comprehensive glycation characterization on antibodies

Non-enzymatic glycation is a challenging post-translational modification to characterize due to the structural heterogeneity it generates in proteins. Glycation has become increasingly recognized as an important product quality attribute to monitor, particularly for the biotechnology sector, which produces recombinant proteins under conditions that are amenable to protein glycation. The elucidation of sites of glycation can be problematic using conventional collision-induced dissociation (CID)-based mass spectrometry because of the predominance of neutral loss ions. A method to characterize glycation using an IgG1 monoclonal antibody (mAb) as a model is reported here. The sugars present on this mAb were derivatized using sodium borohydride chemistry to stabilize the linkage and identified using CID-based MS2 mass spectrometry and spectral search engines. Quantification of specific glycation sites was then done using a targeted MS1 based approach, which allowed the identification of a glycation hot spot in...