ROUTINE MONITORING OF N-GLYCANSUSING A NOVEL LABELING REAGENT WITH FLUORESCENCE AND MASS DETECTION

Importance of the Topic

Glycosylation is a critical quality attribute for biopharmaceuticals, affecting efficacy, safety, and consistency. Routine profiling of N-glycans ensures product quality during development and manufacturing. Traditional workflows often require lengthy sample preparation and separate fluorescence and mass analyses, limiting throughput and delaying confirmatory mass data.

Objectives and Study Overview

This study introduces a novel labeling reagent, RapiFluor-MS, designed to enhance both fluorescence and mass spectrometric responses of N-glycans. The goal is to demonstrate a streamlined UPLC-FLR-QDa workflow that enables rapid sample preparation, increased sensitivity, and reliable routine identification of glycans across a broad structural range.

Instrumentation Used

• Waters ACQUITY UPLC H-Class Bio system
• Fluorescence detector (FLR): excitation 260–265 nm, emission 425 nm, 5 Hz data rate
• QDa mass detector: cone 15 V, capillary 1.5 kV, source 600 °C, m/z 300–1250
• ACQUITY UPLC Glycan BEH Amide column (130 Å, 1.7 µm)
• Mobile phases: acetonitrile (A) and 50 mM ammonium formate pH 4.4 (B)
• RapiFluor-MS labeling reagent

Main Results and Discussion

RapiFluor-MS labeling achieved a 14-fold increase in fluorescence signal and a 160-fold boost in MS response compared to 2-AB. All glycans from neutral bi-antennary to tetra-sialylated structures were detected by both FLR and QDa, confirming coverage across the glycan spectrum.

High-throughput HILIC gradients on a shorter column reduced analysis time six-fold, although some isomeric peaks co-eluted. Application of selected ion recording (SIR) on the QDa resolved co-eluting species (e.g., M5 vs A2G1), enabling unambiguous identification of changes in glycan composition during simulated bioprocess development.

Benefits and Practical Applications

• Rapid sample preparation: from glycoprotein to labeled glycans in 30 minutes
• Simultaneous fluorescence and mass detection in a single run
• Enhanced sensitivity supports detection of low-abundance species (<0.5% RPA)
• High throughput suitable for QC and process monitoring labs
• Unambiguous identification of glycan structures using mass data

Future Trends and Applications

Integration of RapiFluor-MS labeling with advanced MS detectors and automated sample workflows will further accelerate glycan analysis. Emerging trends include real-time glycan monitoring in bioreactors, AI-driven data interpretation, and expansion to more complex glycoforms. Miniaturized columns and faster gradients may continue to boost throughput for large-scale screening.

Conclusion

The RapiFluor-MS reagent combined with UPLC-FLR-QDa detection provides a robust, sensitive, and high-throughput platform for routine N-glycan monitoring. This workflow simplifies sample preparation, delivers comprehensive structural data, and supports rapid decision-making in biopharmaceutical development and manufacturing.