Optimizing HILIC-based Analyses of RapiFluor-MS Labeled Sialylated N-Glycans

Importance of the Topic

Terminal sialic acids on N-glycans critically influence the stability, serum half-life, and immunogenic profile of glycoprotein therapeutics. Accurate profiling of sialylated glycan species is essential in biopharmaceutical development, quality control, and cell-culture monitoring.

Study Objectives and Overview

This application note describes a systematic investigation to enhance hydrophilic interaction chromatography (HILIC)–fluorescence detection (FLR)–mass spectrometry (MS) analysis of RapiFluor-MS labeled sialylated N-glycans. Key aims were to optimize MS source parameters, refine HILIC separation using high ionic strength mobile phases on a wide-pore amide bonded phase, and integrate neuraminidase treatments for linkage-specific sialic acid profiling.

Methodology and Instrumentation

Samples: RapiFluor-MS Sialylated Glycan Performance Test Standard (bovine fetuin N-glycans)

HILIC Separation:

• ACQUITY UPLC Glycoprotein BEH Amide Column, 300 Å, 1.7 µm, 2.1×150 mm
• Mobile phases: high-strength ammonium formate (50–200 mM, pH 4.4) in water, acetonitrile as organic modifier
• Gradient optimized to balance retention and resolution for up to tetrasialylated species

Detection:

• FLR: Ex 265 nm/Em 425 nm
• MS: Xevo G2-XS QTof, positive-ion ESI, m/z 700–2000 (narrowed to 715–2000 for high salt buffers)
• Software: MassLynx 4.1, UNIFI 1.8, LockSpray calibration

Neuraminidase Digestions:

• α2-3 Neuraminidase S for selective removal of α2-3 sialic acids
• α2-3,6,8,9 Neuraminidase A for complete sialic acid release

Key Results and Discussion

MS Source Parameter Optimization:

• Sampling cone voltage: 75 V yielded highest protonated tri-charged ion ([A3G3S3+3H]³⁺) while minimizing in-source fragmentation
• Desolvation temperature: 500 °C balanced signal intensity and fragmentation control
• Capillary voltage (2200 V), source temperature (120 °C), and desolvation gas flow (600 L/Hr) were confirmed as optimal

HILIC Resolution Enhancement:

• Increasing ammonium formate from 50 to 200 mM extended retention times and sharpened peaks for highly sialylated glycans
• Baseline resolution achieved for isomeric tri- and tetrasialylated species (e.g., A3G3S3 variants)
• High-salt mobile phases introduced low-mass background; narrowing MS acquisition window restored sensitivity

Sialic Acid Linkage Profiling:

• Native profile: predominantly tri- (72 %) and tetrasialylated (17 %) triantennary glycans plus disialylated biantennaries (11 %)
• After α2-3 digestion: disappearance of tetrasialylated peaks, indicating all α2-3 linkages in S4 species; quantitative ratios of α2-3 vs. α2-6 in di- and tri-sialylated glycans were inferred
• After broad-specificity digestion: only asialo bi- and triantennary cores remained, confirming complete sialic acid removal

Benefits and Practical Applications

• Enhanced MS sensitivity for sialylated glycan species through fine-tuned ionization settings
• Improved chromatographic resolution supports accurate quantification of isomeric glycans
• Rapid, fluorescence-MS compatible workflow suitable for routine glycan profiling in QC labs
• Neuraminidase-based linkage assay provides fast, cursory insight into sialic acid linkage distribution without extensive MS/MS

Future Trends and Applications

Advances could include integration with glycan retention libraries for automated identification, deployment of ion mobility for enhanced isomer separation, and coupling with high-throughput automation to accelerate glycan analysis in bioprocess monitoring and personalized medicine.

Conclusion

This work demonstrates a robust HILIC-FLR-MS platform for high-fidelity analysis of sialylated N-glycans. Optimized MS source parameters and high-strength mobile phases on a wide-pore amide column yield superior sensitivity and resolution, while neuraminidase treatments enable simple linkage interrogation. Together, these methods support comprehensive glycan characterization in therapeutic development and quality control.

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