title: Comparison of two approaches for quantitative O-linked glycan analysis used in characterization of recombinant proteins.
abstract: The principal aim of this study was to demonstrate the optimization and fine-tuning of quantitative and nonselective analysis of O-linked glycans released from therapeutic glycoproteins. Two approaches for quantitative release of O-linked glycans were examined: ammonia-based β-elimination and hydrazinolysis deglycosylation strategies. A significant discrepancy in deglycosylation activity was observed between the ammonia-based and hydrazinolysis procedures. Specifically, the release of O-glycans from glycoproteins was approximately 20 to 30 times more efficient with hydrazine compared with ammonia-based β-elimination reagent. In addition, the ammonia-based reagent demonstrated bias in the release of particular glycan species. A robust quantitative hydrazinolysis procedure was developed for characterization of O-glycans. The method performance parameters were evaluated. It was shown that this procedure is superior for quantitative nonselective release of O-glycans. Identity confirmation and structure elucidation of O-glycans from hydrophilic interaction chromatography (HILIC) fractions was also demonstrated using linear ion trap Fourier transform mass spectrometry (LTQ FT MS) with mass accuracy below 1ppm.
authors.affiliation: Analytical Development, Biogen Idec, Cambridge, MA 02142, USA. Electronic address: iva.turyan@biogenidec.com.
authors.forename: Iva
authors.initials: I
authors.lastname: Turyan
authors.affiliation: Analytical Biochemistry, Biogen Idec, Cambridge, MA 02142, USA.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Hronowski
authors.affiliation: Analytical Development, Biogen Idec, Cambridge, MA 02142, USA.
authors.forename: Zoran
authors.initials: Z
authors.lastname: Sosic
authors.affiliation: Analytical Development, Biogen Idec, Cambridge, MA 02142, USA.
authors.forename: Yelena
authors.initials: Y
authors.lastname: Lyubarskaya
mesh_terms: D000641:Ammonia / Q000737:chemistry; D002845:Chromatography / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D010100:Oxygen / Q000737:chemistry*; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry*
chemical_list: D006023:Glycoproteins; D006834:Hydrazines; D011134:Polysaccharides; D011994:Recombinant Proteins; C029424:hydrazine; D000641:Ammonia; D010100:Oxygen
keywords: HILIC; LTQ FT MS; O-linked glycan analysis