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.

A case for protein-level and site-level specificity in glycoproteomic studies of disease

“…Peptide standards of the same sequence were heavy isotope-labeled and spiked into the mixture to allow for quantification of the total protein concentration and sitespecific non-occupancy. Reproduced with permission from Zhu et al [39], copyright 2014 American Society for Mass Spectrometry new approaches to effective and efficient analysis of intact glycopeptides [50][51][52][53][54][55][56].…”

Mass spectrometry for glycosylation analysis of biopharmaceuticals

“…While CID preferably results in the fragmentation of the glycan portion, ETD provides information on the amino-acid sequence, while leaving the glycan moiety intact [56]. Similarly, higher-energy C-trap dissociation (HCD) performed on an Orbitrap instrument has proved useful in characterizing both the glycan and peptide moiety, while also providing ultra-high resolution and mass-accuracy MS and MS/MS spectra [57].…”

“…These advanced fragmentation techniques are particularly useful in biopharmaceutical research. Moreover, they facilitate site-specific O-glycan analysis, which is more challenging than N-glycans due to the lack of a single consensus sequence and thus higher heterogeneity [41,56]. These approaches may also be extended for a distinction between isomeric N-or O-glycan structures by using the same instrumentation [59].…”

“…Alternatively, a data-independent, so-called MS E , peptide-mapping approach with ramping collision energies on a high-resolution and accuracy mass spectrometer has been applied to N-and O-,glycopeptides to obtain both site-specific glycan and peptide sequence information within the same run [31,45,56]. The generation of multiple fragment spectra during (glyco)peptide mapping results in vast amounts of data, particularly in dataindependent analysis, leading to a major bottleneck.…”

“…In addition, in clone selection, medium development, pharmacokinetic and pharmacodynamic studies, process development and characterization, and lot release, a high sample throughput can be useful. This can be facilitated, e.g., by the use of immobilized protein A for mAb purification in 96-well plate format [39] or by exoglycosidase arrays for obtaining linkage information [56].…”