The Road to Glycan Analysis Without Compromise

Importance of the Topic

Glycosylation is a critical quality attribute of biotherapeutics, influencing effector functions, serum half-life and immunogenicity. Monitoring released N-glycans provides essential insights for quality control, biosimilarity assessments and process optimization. Conventional labeling via reductive amination is laborious, requires harsh conditions and yields limited sensitivity, motivating the need for faster, more sensitive workflows.

Study Objectives and Overview

This work introduces a streamlined GlycoWorks RapiFluor-MS N-Glycan Kit that integrates rapid enzymatic deglycosylation, a novel fluorescent and MS‐active labeling reagent and robust SPE cleanup, reducing total sample preparation to 30 minutes. The study demonstrates improvements in reaction speed, detection sensitivity and method robustness, and outlines compatibility with UPLC and HPLC platforms and various informatics tools.

Methodology and Instrumentation

• Rapid PNGase F deglycosylation assisted by RapiGest SF surfactant (80 °C for 2 min; 50 °C for 5 min)
• Instant labeling using RapiFluor-MS reagent (NHS carbamate with quinoline fluorophore and tertiary amine), adding a 312 Da tag in seconds
• HILIC SPE cleanup of neutral to tetrasialylated glycans via GlycoWorks cartridges
• Chromatographic separation on ACQUITY UPLC Glycan BEH Amide columns
• Detection with ACQUITY UPLC FLR detector and Xevo G2-XS QTof or ACQUITY QDa mass analyzers
• Data analysis using UNIFI Glycan Application or Empower/MassLynx with dedicated GU libraries and MS/MS workflows

Main Results and Discussion

• Complete deglycosylation and labeling workflow in 30 min vs 5 h–2 days for 2-AB methods, achieving >95% labeling yield
• FLR sensitivity up to 2× greater than 2-AB and comparable to Instant AB; MS sensitivity enhancement up to 100–1000× over 2-AB
• Quantitative SPE recovery (~74% experimental yield; minimal bias in glycan profiles)
• Method transferability demonstrated between UPLC and HPLC with similar resolution and sensitivity
• 10 min rapid LC method validated for glycan profiling and monitoring (e.g., Man5/A2G1 ratio with <6% RSD)

Benefits and Practical Applications

• High-throughput, sensitive glycan profiling for biopharmaceutical QA/QC and process development
• Improved detection of low-abundance and highly sialylated structures
• Seamless integration with existing chromatographic platforms and software infrastructures
• Unified workflow supports both quantitative fluorescence and qualitative MS/MS analyses

Future Trends and Opportunities

Anticipated advances include automation through robotics, expansion of MS-active tagging chemistries, integration with in-line process analytical technology (PAT) and AI-driven data analysis to accelerate glycoform screening and comparability studies.

Conclusion

The GlycoWorks RapiFluor-MS N-Glycan Kit delivers a rapid, sensitive and robust solution for released glycan analysis, enabling high-throughput glycan profiling with fluorescence and mass detection in a 30-minute workflow, addressing the stringent demands of biotherapeutic development and quality control.

Reference

• Analytical Chemistry 2013, 85 (2), 715–736