Released N-linked Glycan Analysis Using the Glycan Application Solution with UNIFI

Importance of the topic

N linked glycans critically influence the pharmacokinetics, efficacy and safety of biotherapeutic proteins. Precise profiling and structural elucidation of these glycans are essential in biopharmaceutical development and quality control.

Objectives and overview of the study

This study demonstrates two complementary workflows within the Waters Glycan Application Solution and UNIFI Scientific Information System for comprehensive N linked glycan analysis. Workflow one integrates fluorescent detection with mass confirmation for profiling and quantitation. Workflow two employs data dependent acquisition MS/MS for in depth structural elucidation via external software.

Methodology and instrumentation

Sample preparation uses the GlycoWorks RapiFluor MS N Glycan Kit for enzymatic release, fluorescent labeling and cleanup. Chromatographic separation is performed on an ACQUITY UPLC H Class Bio System with Column Manager and Glycan BEH Amide column under HILIC conditions. Fluorescent detection is achieved with an ACQUITY UPLC FLR detector (excitation 265 nm, emission 425 nm). Mass analysis uses a Xevo G2 XS QTof MS with optimized capillary voltage cone gas and desolvation settings. Data acquisition, processing and reporting are managed by the UNIFI Scientific Information System. For structural workflow two, processed MS/MS data are exported to SimGlycan.

Main results and discussion

Workflow one matches calibrated retention times in glucose units and accurate MS masses against a scientific glycan library for rapid identification of over 300 N linked structures. Relative abundances are quantitated from integrated fluorescence intensities. Workflow two acquires CID MS/MS in DDA mode, converts fragment ions to singly charged spectra and exports them for fragment ion annotation and structural assignment in SimGlycan. Both approaches deliver robust, reproducible profiles and confirm low abundant or immunogenic glycan species.

Benefits and practical applications

• Integrated optical and MS detection in a single UPLC FLR QTof platform
• High sensitivity labeling with RapiFluor MS for low abundance glycans
• Comprehensive scientific libraries with customizable entries for rapid identification
• Automated quantitation of glycan profiles for QC and comparability studies
• Flexible structural elucidation via external software export

Future trends and opportunities

Ongoing development of a RapiFluor MS based GU library will expand structure coverage. Integration of machine learning for automated glycan annotation and improved MS data interpretation is emerging. Higher throughput workflows, deeper structural resolution with alternative fragmentation methods and cloud based informatics promise to accelerate biopharmaceutical glycoanalysis in research and quality control.

Conclusion

The Glycan Application Solution with UNIFI delivers two fit for purpose workflows that combine advanced sample preparation, high resolution chromatography, sensitive detection and powerful informatics. It enables reliable profiling, quantitation and structural elucidation of N linked glycans, supporting biotherapeutic development and regulatory compliance.

References

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2. Rapid Preparation of Released N Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI MS Detection Anal Chem 2015 87 10 5401-5409
3. Applying a Novel Glycan Tagging Reagent RapiFluor MS and an Integrated UPLC FLR QTof MS System for Low Abundant N Glycan Analysis Waters Application Note p n 720005383EN
4. UNIFI Scientific Information System Waters literature p n 720004686EN
5. Mattew P Campbell Louise Royle Catherine M Radcliffe Raymond A Dwek and Pauline M Rudd GlycoBase and autoGU tools for HPLC based glycan analysis Bioinformatics Applications Note 24 9 2008 1214-1216
6. Biopharmaceutical Platform Solution with UNIFI A Holistic Workflow for Acquiring Processing and Reporting Fluorescent Labeled Glycans Waters Application Note p n 720004619EN