LC-MS Analysis of Highly Sialylated Glycans Using MS Compatible Mixed Mode Chromatographic Separation

Význam tématu

The sialylation level of N-glycans significantly influences the pharmacokinetics and biological activity of glycoprotein therapeutics. Accurate analysis of highly sialylated structures is challenging using conventional hydrophilic interaction chromatography (HILIC) coupled with fluorescence detection and mass spectrometry (FLR-MS).

Cíle a přehled studie / článku

This work evaluates a mixed-mode reversed-phase/anionic exchange (RP/AX) LC-FLR-MS method on the Waters BioAccord System for improved separation and characterization of fluorescently labeled, highly sialylated N-glycans released from recombinant erythropoietin (rEPO).

Použitá metodika a instrumentace

• Sample: rEPO from European Pharmacopeia and commercial supplier, labeled by GlycoWorks RFMS Kit.
• Chromatography: Mixed-mode separation on ACQUITY Premier Glycan BEH C18 AX column (1.7 μm, 2.1×150 mm); comparison to ACQUITY Premier Glycan Amide HILIC column.
• Instrumentation: Waters BioAccord System with ACQUITY Premier LC, FLR detector (Ex 265 nm, Em 425 nm) and integrated bench-top MS.
• Mobile phases: 100 mM ammonium formate/formic acid in water/ACN for RP/AX; 50 mM ammonium formate and acetonitrile for HILIC.
• Data analysis: waters_connect with UNIFI software.

Hlavní výsledky a diskuse

Mixed-mode RP/AX separation achieved approximately 20 % narrower peak widths and superior resolution of isomeric sialylated glycans compared to HILIC. Charge-driven retention differences allowed grouping by the number of sialic acids, enabling distinct elution of glycan species such as tetra-antennary FA4S3 and FA4G4S4. Fragment ion spectra provided complementary compositional confirmation, including sialic acid count and monosaccharide sequence.

Přínosy a praktické využití metody

The optimized method improves resolving power for complex and highly sialylated glycan mixtures and streamlines compositional assignments in biopharmaceutical characterization workflows. It supports robust glycan profiling for quality control and structure–function studies.

Budoucí trendy a možnosti využití

Future developments may integrate higher-resolution MS detection, automated data processing, and application to a broader range of glycoproteins. The approach is well-suited for advanced biopharmaceutical QC and glycoengineering research.

Závěr

The mixed-mode RP/AX LC-FLR-MS method on BioAccord with an ACQUITY Premier Glycan C18 AX column provides enhanced separation and characterization of highly sialylated N-glycans, offering improved peak capacity, charge-based selectivity, and reliable compositional analysis.

Reference

Birdsall R., Yu Y. Q. LC-MS Analysis of Highly Sialylated Glycans Using MS Compatible Mixed Mode Chromatographic Separation. Waters Corporation, Milford MA, 2021.