Parallel development of chromatographic and mass-spectrometric methods for quantitative analysis of glycation on an IgG1 monoclonal antibody

•BAC and MS methods have been developed simultaneously to quantify glycation on an IgG1 mAb.•Data from the two orthogonal methods correlate to each other within experimental error.•Glycation can be underestimated by overcompanseting for non-specific interactions.•In chromatography, contamination of the retained peak with non-glycated species has been quantified with mass spectrometry.•We have designed a workflow that can be utilized to customize BAC according to the individual properties of different mAbs. Monitoring post-translational modifications (PTMs) in biotherapeutics is of paramount importance. In pharmaceutical industry, chromatography with optical detection is the standard choice of quantitation of product related impurities; and mass spectrometry is used only for characterization. Parallel development of a boronate affinity chromatographic (BAC) and a mass spectrometric methods for quantitative measurement of glycation on a monoclonal antibody (mAb) shed light on the importance of certain characteristics of the individual methods. Non-specific interactions in BAC has to be suppressed with the so-called shielding reagent. We have found that excessive amount of shielding reagents in the chromatographic solvents may cause significant underestimation of glycation. Although contamination of the retained peak with the non-glycated isoforms in BAC is unavoidable, our work shows that it can be characterized and quantitated by mass spectrometry. It has been demonstrated that glycation can be measured by mass spectrometry at the intact protein level with an LOQ value of 3.0% and error bar of ±0.5%. The BAC and MS methods have been found to provide equivalent results. These methods have not been compared from these points of view before.