New Capabilities for Monitoring Released N-Glycans through the Combined Use of RapiFluor-MS Labeling, ACQUITY UPLC H-Class Bio System, and Serial Fluorescence/ACQUITY QDa Mass Detection

Significance of the Topic

The glycosylation patterns of biotherapeutic proteins are critical quality attributes that influence safety, efficacy, and stability. Reliable and routine monitoring of released N-glycans supports process development, quality control, and regulatory compliance. Traditional workflows often require lengthy sample preparation and high-end mass spectrometers operated by specialists, limiting throughput and accessibility.

Study Objectives and Overview

This work evaluates an integrated workflow combining Rapi Fluor-MS labeling, ACQUITY UPLC H-Class Bio separation, and serial fluorescence plus ACQUITY QDa mass detection. The goal is to reduce sample preparation time, enhance glycan detection sensitivity, and enable routine mass confirmation for each peak, demonstrated on IgG N-glycans.

Methodology and Instrumentation

Sample preparation exploited the GlycoWorks Rapi Fluor-MS N-Glycan Kit to label released glycans in under 5 minutes. Separation was achieved on an ACQUITY UPLC Glycan BEH Amide column (2.1×150 mm, 1.7 µm) at 60 °C. The ACQUITY UPLC H-Class Bio System delivered a binary gradient of acetonitrile and 50 mM ammonium formate (pH 4.4). Fluorescence detection used 265 nm excitation and 425 nm emission. Mass detection employed the ACQUITY QDa (ESI+, 500–1250 Da, cone voltage 15 V, capillary 1.5 kV, probe temperature 500 °C) with a 5 points/s acquisition rate. High-resolution comparison was performed on a SYNAPT G2-S.

Main Findings and Discussion

Rapi Fluor-MS labeling increased MS sensitivity by one to two orders of magnitude compared to 2-AB and promoted higher charge states, bringing key glycan ions into the QDa mass window. In IgG glycan profiling, every fluorescent peak was matched by a clean, high-quality QDa spectrum, facilitating unambiguous identification. Charge state shifts (e.g., [M+2H]2+ and [M+3H]3+) and relative peak areas were consistent across fluorescent and mass traces.

Benefits and Practical Applications

• End-to-end sample prep to analysis in ~30 minutes
• Sensitive fluorescence quantitation coupled with mass confirmation for each glycan peak
• User-friendly, cost-effective mass detection without specialized MS expertise
• Enhanced confidence in glycoprofile monitoring for biopharmaceutical development and QC

Future Trends and Opportunities

• Extension of rapid labeling strategies to other glycan classes and complex biomolecules
• Automation of sample preparation and data processing for high-throughput glycomics
• Integration with biosimilar comparability and batch-release testing workflows
• Development of enhanced detectors and software for real-time glycan monitoring

Conclusion

The combination of Rapi Fluor-MS labeling with ACQUITY UPLC H-Class Bio and QDa detection offers a streamlined, highly sensitive platform for routine N-glycan profiling. This workflow significantly reduces analysis time, lowers barriers to mass detection, and improves the reliability of glycan assignment in biopharmaceutical contexts.

References

1. Lauber MA, Brousmiche DW, Hua Z, Koza SM, Guthrie E, Magnelli P, Taron CH, Fountain KJ. Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Novel Fluorescence and MS-Active Labeling Reagent. Waters Application Note 720005275EN, 2015.