title: N- and O-glycosylation analysis of etanercept using liquid chromatography and quadrupole time-of-flight mass spectrometry equipped with electron-transfer dissociation functionality.
abstract: Etanercept is a highly glycosylated therapeutic Fc-fusion protein that contains multiple N- and O-glycosylation sites. An in-depth characterization of the glycosylation of etanercept was carried out using liquid chromatography/mass spectrometry (LC/MS) methods in a systematic approach in which we analyzed the N- and O-linked glycans and located the occupied O-glycosylation sites. Etanercept was first treated with peptide N-glycosidase F to release the N-glycans. The N-glycan pool was labeled with a 2-aminobenzamide (2-AB) fluorescence tag and separated using ultraperformance liquid chromatography-hydrophilic interaction liquid chromatography (UPLC-HILIC). Preliminary structures were assigned using Glycobase. These assignments, which included monosaccharide sequence and linkage information, were confirmed by exoglycosidase array digestions of aliquots of the N-glycan pool. The removal of the N-glycans from etanercept facilitated the selective characterization of O-glycopeptides and enabled the O-glycans to be identified. These were predominantly of the core 1 subtype (HexHexNAc O-structure) attached to Ser/Thr residues. α2→3,6,8,9 sialidase was used to remove the sialic acid residues on the O-glycans allowing the use of an automated LC/MS(E) protocol to identify the O-glycopeptides. Electron-transfer dissociation (ETD) was then used to pinpoint the 12 occupied O-glycosylation sites. The determination of N- and O-glycans and O-glycosylation sites in etanercept provides a basis for future studies addressing the biological importance of specific protein glycosylations in the production of safe and efficacious biotherapeutics.
authors.affiliation: Late Stage Development, Pharmaceutical Life Sciences, Waters Corporation , Milford, Massachusetts 01757, United States.
authors.forename: Stephane
authors.initials: S
authors.lastname: Houel
authors.forename: Mark
authors.initials: M
authors.lastname: Hilliard
authors.forename: Ying Qing
authors.initials: YQ
authors.lastname: Yu
authors.forename: Niaobh
authors.initials: N
authors.lastname: McLoughlin
authors.forename: Silvia Millan
authors.initials: SM
authors.lastname: Martin
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Rudd
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Williams
authors.forename: Weibin
authors.initials: W
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods; D004579:Electron Transport / Q000235:genetics*; D000068800:Etanercept; D006031:Glycosylation; D007074:Immunoglobulin G / Q000032:analysis* / Q000235:genetics*; D008969:Molecular Sequence Data; D018124:Receptors, Tumor Necrosis Factor / Q000032:analysis* / Q000235:genetics*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D007074:Immunoglobulin G; D018124:Receptors, Tumor Necrosis Factor; D000068800:Etanercept