A comprehensive characterization of biotherapeutics, mandated by regulatory authorities, requires analyses of a protein drug at multiple structure levels. Such multilevel characterization can be performed by mass spectrometry (MS), with established conventional MS-based assays of product quality attributes (PQAs) comprising intact protein and subunit middle-up MS with analytes resolved on a C4 column, and bottom-up peptide mapping with analytes resolved on a C18 column. Recent advances in MS...

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 (mAbs), 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...

Antibody-based pharmaceuticals are the leading biologic drug platform (> $75B/year).^([1]) Despite a wealth of information collected on them, there is still a lack of knowledge on their inter-domain structural distributions, which impedes innovation and development. To address this measurement gap, we have developed a new methodology to derive biomolecular structure ensembles from distance distribution measurements via a library of tagged proteins bound to an unlabeled and otherwise unmodified...

The characterization of the impurities of pharmaceutical monoclonal antibodies (mAbs) is crucial for their function and safety. Capillary zone electrophoresis (CZE) is one of the most efficient tools to separate charge variants of mAbs; however, peak characterization remains difficult, since the hereby used background electrolytes (BGEs) are not compatible with electrospray ionization-mass spectrometry (ESI-MS). Here, a method that allows the separation of intact mAb charge variants is presented...

Investigating the structural heterogeneity of monoclonal antibodies is crucial to achieving optimal therapeutic outcomes. We show that tandem-trapped ion mobility spectrometry enables collision-induced unfolding measurements of subpopulations of a humanised IgGk NIST monoclonal antibody (NISTmAb). Our results indicate that differential glycosylation of NISTmAb does not modulate its conformational heterogeneity.