Profiling and Characterization of Sialylated N-glycans by 2D-HPLC (HIAX/PGC) with Online Orbitrap MS/MS and Offline MSn

• Published in: Journal of pharmaceutical sciences 2014-02, Vol.103 (2), p.400-408
• Main Authors: Hanneman, Andrew J.S., Strand, James, Huang, Chi-Ting
• Format: Article
• Language: English
• Subjects: 2-AA; 2-AB; 2-aminobenzamide; 2-aminobenzoic acid; Acetylation; biotechnology; chinese hamster ovary; CHO; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; glycosylation; HIAX; high-pH anion-exchange chromatography; HILIC; HPAEC; HPLC; hydrophilic interaction anionexchange chromatography; hydrophilic interaction liquid chromatography; Indicators and Reagents; LC–MS; liquid chromatography–mass spectrometry; MALDITOF; Mass Spectrometry; matrix assisted laser desorption ionization-time of flight-mass spectrometry; MS/MS; MSn; N-Acetylneuraminic Acid - chemistry; N-glycan; nanospray ionization; NSI; PGC; Polysaccharides - chemistry; porous graphitized carbon; proteins; reverse-phase; sequential mass spectrometry; sialylation; Spectrometry, Mass, Electrospray Ionization; Sulfates - chemistry; Tandem Mass Spectrometry; TFA; trifluoroacetic acid
• ISSN: 0022-3549; 1520-6017
• DOI: 10.1002/jps.23792

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–MSn). 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–MSn investigations to characterize low-abundance species and specific moieties including O-acetylation and sulfation.