title: 2D-LC analysis of BRP 3 erythropoietin N-glycosylation using anion exchange fractionation and hydrophilic interaction UPLC reveals long poly-N-acetyl lactosamine extensions.
abstract: Post-translational modifications, in particular glycosylation, represent critical structural attributes that govern both the pharmacodynamic and pharmacokinetic properties of therapeutic glycoproteins. To guarantee safety and efficacy of recombinant therapeutics, characterization of glycosylation present is a regulatory requirement. In the current paper, we applied a multidimensional strategy comprising a shallow anion exchange gradient in the first dimension, followed by analysis using the recently introduced 1.7 μm HILIC phase in the second dimension for the comprehensive separation of complex N-glycans present on the European Biological Reference Preparation (BRP) 3 erythropoietin standard. Tetra-antennary glycans with multiple sialic acids and poly-N-acetyl lactosamine extensions were the most abundant oligosaccharides present on the molecule. Site-specific glycan analysis was performed to examine microheterogeneity. Tetra-antennary glycans with up to four sialic acids and up to five poly-N-acetyl lactosamine extensions were observed at asparagine 24 and 83, while biantennary glycans were the major structures at asparagine 38. The combined AEC x UPLC HILIC allows for the rapid and comprehensive analysis of complex N-glycosylation present on therapeutic glycoproteins, such as BRP3 erythropoietin.
authors.affiliation: NIBRT Dublin-Oxford Glycobiology Laboratory, NIBRT-The National Institute for Bioprocessing Research and Training, UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Bones
authors.forename: Niaobh
authors.initials: N
authors.lastname: McLoughlin
authors.forename: Mark
authors.initials: M
authors.lastname: Hilliard
authors.forename: Kieran
authors.initials: K
authors.lastname: Wynne
authors.forename: Barry L
authors.initials: BL
authors.lastname: Karger
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Rudd
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D002852:Chromatography, Ion Exchange / Q000379:methods / Q000592:standards; D004921:Erythropoietin / Q000737:chemistry* / Q000378:metabolism / Q000592:standards; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011134:Polysaccharides / Q000032:analysis*; D012794:Sialic Acids / Q000032:analysis
chemical_list: D011134:Polysaccharides; D012794:Sialic Acids; D004921:Erythropoietin; D001216:Asparagine; C037199:poly-N-acetyllactosamine