Type

Journal Article

Authors

Pauline M Rudd
Barry L Karger
Kieran Wynne
Mark Hilliard
Niaobh McLoughlin
Jonathan Bones

Subjects

Biochemistry

Topics
polysaccharides sialic acids standards chromatography ion exchange mass spectrometry asparagine glycosylation molecular sequence data poly n acetyllactosamine hydrophobic and hydrophilic interactions peptide mapping erythropoietin chemistry amino acid sequence chromatography high pressure liquid metabolism

2D-LC analysis of BRP 3 erythropoietin N-glycosylation using anion exchange fractionation and hydrophilic interaction UPLC reveals long poly-N-acetyl lactosamine extensions. (2011)

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.
Collections Ireland -> University College Dublin -> PubMed

Full list of authors on original publication

Pauline M Rudd, Barry L Karger, Kieran Wynne, Mark Hilliard, Niaobh McLoughlin, Jonathan Bones

Experts in our system

1
Pauline M Rudd
University College Dublin
Total Publications: 45
 
2
Kieran Wynne
Royal College of Surgeons in Ireland
Total Publications: 45
 
3
Jonathan Bones
University College Dublin
Total Publications: 28