Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Novel Fluorescence and MS-Active Labeling Reagent

Significance of the Topic

The detailed profiling of released N-glycans is a critical quality attribute in biopharmaceutical characterization, impacting drug efficacy, safety, and manufacturing consistency. Conventional glycan preparation methods for HILIC-FLR-MS analysis are often lengthy, laborious, and compromise sensitivity, limiting throughput and reliable detection of low-abundance species.

Objectives and Study Overview

This study aims to develop and validate a streamlined workflow that combines rapid deglycosylation, an innovative fluorescence and MS-active labeling reagent (RapiFluor-MS), and high-recovery HILIC SPE to deliver unbiased N-glycan profiling within 30 minutes, while dramatically enhancing both fluorescence and mass spectrometry sensitivity.

Methodology and Instrumentation

The three-step protocol comprises:

• Rapid Deglycosylation: Glycoproteins (e.g., mAb standard, fetuin, human IgG) are denatured with 1% RapiGest SF at ≥80 °C for 2 minutes, followed by 5 minutes at 50 °C with Rapid PNGase F to release N-glycosylamines.
• Rapid Labeling: Released glycosylamines are tagged in 5 minutes under aqueous conditions with RapiFluor-MS, featuring an NHS-carbamate rapid-tagging group, a quinoline fluorophore, and a tertiary amine charge tag.
• HILIC SPE Cleanup: Labeled glycans are purified using a GlycoWorks HILIC μElution Plate (aminopropyl sorbent), with conditioning (water), loading in high organic, formic acid wash, and elution in 200 mM ammonium acetate (pH 7) in 5% ACN.

The workflow is integrated on Waters instrumentation: ACQUITY UPLC H-Class Bio System, Glycan BEH Amide 130 Å columns (2.1×50 mm or 2.1×150 mm), ACQUITY QDa Mass Detector, Xevo G2-XS QTof MS, and SYNAPT G2-S HDMS.

Key Results and Discussion

• Fluorescence and MS Sensitivity: RapiFluor-MS labeled FA2 glycan yields 2× fluorescence and 780× MS signal versus Instant AB, and 14× fluorescence and 160× MS signal versus 2-AB.
• Rapid Deglycosylation Efficiency: SDS-PAGE assays confirmed complete deglycosylation of diverse glycoproteins in 10 minutes, matching conventional 30 minute protocols.
• SPE Recovery and Selectivity: GlycoWorks HILIC SPE delivers 70–80% absolute recovery across neutral to tetrasialylated glycans, with negligible bias in relative abundances after one or two SPE passes.

Benefits and Practical Applications of the Method

• Total sample-to-analysis time reduced to ~30 minutes, eliminating overnight incubations and lengthy labeling steps.
• Unbiased, quantitative glycan profiles across a wide structural range, supporting QA/QC and research settings.
• High sensitivity enables detection of low-abundance and highly sialylated species by both FLR and ESI-MS.
• 96-well μElution SPE format facilitates medium to high throughput workflows without dry-down.

Future Trends and Potential Applications

• Automation and integration into robotic platforms for routine glycomics and biopharmaceutical monitoring.
• Expansion to linkage-specific or multiplexed labeling strategies for in-depth structural characterization.
• Application to clinical glycomics, biomarker discovery, and personalized medicine.
• Adaptation to other post-translational modifications requiring rapid, sensitive tagging and analysis.

Conclusion

The GlycoWorks RapiFluor-MS N-Glycan Kit delivers a rapid, robust, and highly sensitive workflow for the preparation and analysis of released N-glycans by HILIC-FLR-MS. By integrating accelerated deglycosylation, innovative labeling chemistry, and efficient SPE cleanup, this approach transforms glycan profiling into a 30-minute procedure without sacrificing quantitative accuracy or detection sensitivity.

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