Relative quantitation of glycans in cetuximab using ultra-high-performance liquid chromatography-high-resolution mass spectrometry by Pronase E digestion.

Glycans play important roles in the activity and function of monoclonal antibodies (mAbs). In this study, an isotope labeling method for the relative quantitative analysis of glycans in cetuximab, a chimeric human/mouse IgG1 monoclonal antibody that specifically targets epidermal growth factor receptor, via hydrophilic interaction LC-ultra-high-performance LC-HRMS was established based on Pronase E digestion. To this aim, novel isotope MS probes, i.e., 3-benzoyl-2-oxothiazolidine-4-carboxylic acid (d0-BOTC) and 3-(2,3,4,5,6-pentadeuterio-benzoyl)-2-oxothiazolidine-4-carboxylate acid (d5-BOTC), which include a carboxyl group to target the amino functional group in glycosylamine, were developed. The nonspecific Pronase E enzyme could simultaneously digest the peptide bound to the N- and O-glycans into glycosylamine having only one amino acid. Since the mass difference between the light- and heavy-labeled glycans was 5.0 Da, the relative abundance of their MS peaks was used to achieve the qualitative and relative quantitative analysis of glycans. Sialylglycopeptide was used as a complex glycan model to validate the accuracy of the method. The results demonstrated the good linearity (R

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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.