Available online 26 April 2024, 100982

Quantification of minor abundant glycovariants occurring in monoclonal antibodies.

Integration of quantitative data across protein structural levels.

Quantification of MS data at the raw data level by open-source software tools.

Site-specific discrepancies exist at diverse structural levels for minor species.

Comprehensive meta-data study on glycosylation analyses in literature.

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 N-glycan and glycopeptide data form the basis for integrating information across different structural levels up to intact glycoproteins. Comprehensive comparison showed that indeed, variations across structural levels were observed especially for minor abundant species. Utilizing MoFi (short for modification finder), a tool for annotating mass spectra 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, spotlighting minor abundant glycovariants in therapeutic antibodies.

Glycosylation

Mass spectrometry

Monoclonal antibodies

Abundance profiling

Minor glycovariants

Multi-level analysis

Data integration

© 2024 The Author(s). Published by Elsevier B.V. on behalf of Xi’an Jiaotong University.