“Small is beautiful” – the significance of reliable determination of low- abundant therapeutic antibody glycovariants

Glycans associated with biopharmaceutical drugs play crucial roles in drug safety and efficacy, and therefore, their reliable detection and quantification is essential. Our study introduces a multi-level quantification approach for glycosylation analysis in monoclonal antibodies, focusing on minor abundant glycovariants. Mass spectrometric data is evaluated mainly employing open-source software tools. Released glycan and glycopeptide data form the basis for integrating information across different molecular and structural levels up to intact glycoproteins. A comprehensive site-specific comparison showed that indeed, variations across structural levels were observed especially for minor abundant species. Utilizing MoFi, a tool for annotating mass peaks of intact proteins, we quantify isobaric glycosylation variants at the intact protein level. Our workflow's utility is demonstrated on NISTmAb, rituximab and adalimumab, profiling their minor abundant variants for the first time across diverse structural levels. This study enhances understanding and accessibility in glycosylation analysis, emphasizing the significance of minor abundant glycovariants in therapeutic antibodies.