Comprehensive assessment of N-glycans derived from a murine monoclonal antibody: A case for multimethodological approach

Yehia Mechref, Jan Muzikar, Milos V. Novotny

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61 Scopus citations

Abstract

Highly efficient separation techniques, laser-induced fluorescence (LIF) detection, and different mass-spectrometric (MS) measurements were combined in a multi-methodological scheme to perform a comprehensive structural characterization of N-linked oligosaccharides in a murine monoclonal antibody (immunoglobulin G (IgGκ)). Monosaccharide compositional analysis was carried out through a capillary electrophoresis (CE)-LIF method, in which the chemically and enzymatically released sugars were fluorescently labeled. This analysis provides a preliminary assessment of certain structures, being followed by CE-LIF and matrix-assisted laser desorption/ionization (MALDI)-MS profiling of the intact glycan structures. Linkages and monosaccharide residues were confirmed by MALDI-MS in conjunction with exoglycosidase digestion. MALDI-MS and CE data were effectively combined to reveal the overall structural diversity of both acidic and neutral glycans. Finally, the sites of glycosylation and site occupancies were deduced through the measurements performed with microcolumn liquid chromatography coupled via electrospray to a quadrupole/time-of-flight instrument.

Original languageEnglish
Pages (from-to)2034-2046
Number of pages13
JournalELECTROPHORESIS
Volume26
Issue number10
DOIs
StatePublished - May 2005

Keywords

  • Glycoproteins
  • Glycosylation sites
  • N-Glycans
  • Recombinant monoclonal antibody

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