title: Profiling and characterization of sialylated N-glycans by 2D-HPLC (HIAX/PGC) with online orbitrap MS/MS and offline MSn.
abstract: Glycosylation is a critical parameter used to evaluate protein quality and consistency. N-linked glycan profiling is fundamental to the support of biotherapeutic protein manufacturing from early stage process development through drug product commercialization. Sialylated glycans impact the serum half-life of receptor-Fc fusion proteins (RFPs), making their quality and consistency a concern during the production of fusion proteins. Here, we describe an analytical approach providing both quantitative profiling and in-depth mass spectrometry (MS)-based structural characterization of sialylated RFP N-glycans. Aiming to efficiently link routine comparability studies with detailed structural characterization, an integrated workflow was implemented employing fluorescence detection, online positive and negative ion tandem mass spectrometry (MS/MS), and offline static nanospray ionization-sequential mass spectrometry (NSI-MS(n)). For routine use, high-performance liquid chromatography profiling employs established fluorescence detection of 2-aminobenzoic acid derivatives (2AA) and hydrophilic interaction anion-exchange chromatography (HIAX) charge class separation. Further characterization of HIAX peak fractions is achieved by online (-) ion orbitrap MS/MS, offering the advantages of high mass accuracy and data-dependent MS/MS. As required, additional characterization uses porous graphitized carbon in the second chromatographic dimension to provide orthogonal (+) ion MS/MS spectra and buffer-free liquid chromatography peak eluants that are optimum for offline (+)/(-) NSI-MS(n) investigations to characterize low-abundance species and specific moieties including O-acetylation and sulfation.
authors.affiliation: Glycan Connections, LLC, Lee, New Hampshire.
authors.forename: Andrew J S
authors.initials: AJ
authors.lastname: Hanneman
authors.forename: James
authors.initials: J
authors.lastname: Strand
authors.forename: Chi-Ting
authors.initials: CT
authors.lastname: Huang
mesh_terms: D000107:Acetylation; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013431:Sulfates / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D007202:Indicators and Reagents; D011134:Polysaccharides; D013431:Sulfates; D019158:N-Acetylneuraminic Acid
keywords: HIAX; HPLC; N-glycan; biotechnology; glycosylation; mass spectrometry; proteins; sialylation