Data-independent oxonium ion profiling of multi-glycosylated biotherapeutics

Abstract

The characterization of glycosylation is required for many protein therapeutics. The emergence of antibody and antibody-like molecules with multiple glycan attachment sites has rendered glycan analysis increasingly more complicated. Reliance on site-specific glycopeptide analysis is therefore necessary to fully analyze multi-glycosylated biotherapeutics. Established glycopeptide methodologies have generally utilized a priori knowledge of the glycosylation states of the investigated protein(s), database searching of results generated from data-dependent liquid chromatography–tandem mass spectrometry workflows, and extracted ion quantitation of the individual identified species. However, the inherent complexity of glycosylation makes predicting all glycoforms on all glycosylation sites extremely challenging, if not impossible. That is, only the “knowns” are assessed. Here, we describe an agnostic methodology to qualitatively and quantitatively assess both “known” and “unknown” site-specific glycosylation for biotherapeutics that contain multiple glycosylation sites. The workflow uses data-independent, all ion fragmentation to generate glycan oxonium ions, which are then extracted across the entirety of the chromatographic timeline to produce a glycan-specific “fingerprint” of the glycoprotein sample. We utilized both HexNAc and sialic acid oxonium ion profiles to quickly assess the presence of Fab glycosylation in a therapeutic monoclonal antibody, as well as for high-throughput comparisons of multi-glycosylated protein drugs derived from different clones to a reference product. An automated method was created to rapidly assess oxonium profiles between samples, and to provide a quantitative assessment of similarity.

The Role of Data-Independent Acquisition for Glycoproteomics

“…Madsen et al. ( 70 ) used a DIA method to generate diagnostic oxonium ion profiles for quantitative assessment of IgG glycosylation. Two types of highly abundant oxonium ions, HexNAc ( m/z 204.09, C 8 H 13 O 5 N 1 ) and SA ( m/z 274.09, NeuAc–H 2 O, C 11 H 15 O 7 N 1 ), were monitored using all ion fragmentation in which all precursors in the defined mass range were fragmented without mass filtering ( 71 ).…”

DIALib: an automated ion library generator for data independent acquisition mass spectrometry analysis of peptides and glycopeptides

“…During glycopeptide fragmentation in CID/HCD mode, glycans fragment into smaller ions called oxonium ions [20]. Oxonium ions not only signal the presence of a glycan, but can also provide some information on glycan composition and structure [21]. Thus, measurement of oxonium ion…”

DIALib: an automated ion library generator for data independent acquisition mass spectrometry analysis of peptides and glycopeptides

“…During glycopeptide fragmentation in CID/HCD mode, glycans fragment into smaller ions called oxonium ions [20]. Oxonium ions not only signal the presence of a glycan, but can also provide some information on glycan composition and structure [21]. Thus, measurement of oxonium ion Table S14).…”