A Modified GlycoWorks Procedure With Enhanced Detection Sensitivity

Significance of the Topic

Monitoring N-linked glycosylation is essential in biotherapeutic development because glycan structures influence serum half-life, effector function and immunogenicity. Sensitive detection of low-abundance glycoforms supports quality control, process optimization and regulatory compliance.

Objectives and Study Overview

This study introduces a modified Waters GlycoWorks RapiFluor-MS protocol designed to increase fluorescence (FLR) and mass spectrometry (MS) sensitivity for released N-glycans. The enhanced method is benchmarked against Agilent AdvanceBio Gly-X Prep with InstantPC labelling. Key aims include assessment of signal-to-noise improvements, sample cleanup recovery and evaluation of over-labelling artifacts.

Methodology

Samples of NISTmAb and bovine fetuin were used to cover a spectrum of neutral, high-mannose and sialylated N-glycans. The modified workflow increases the starting glycoprotein amount from 15 µg to 40 µg per reaction. N-glycans were enzymatically released with PNGase F, labelled with RapiFluor-MS or InstantPC, and purified by HILIC-SPE. HILIC-FLR and UPLC–MS analyses were performed without further dilution to directly compare detection sensitivity and glycan profiles.

Instrumentation Used

• UPLC Systems ACQUITY UPLC H-Class (FLR) and ACQUITY Premier UPLC (MS)
• Glycan Separation Column ACQUITY Premier Glycan BEH Amide 130 Å, 1.7 µm, 2.1×100 mm
• Detectors FLR detector with excitation 265 nm and emission 425 nm for RapiFluor-MS; excitation 285 nm and emission 345 nm for InstantPC
• Mass Spectrometer Xevo G2-XS QTof in ESI positive mode, mass range 50–2000 m/z, sensitivity mode, MSE acquisition

Main Results and Discussion

The modified GlycoWorks protocol achieved a threefold increase in FLR signal and a twofold increase in MS signal relative to the standard method. Glycan distribution remained consistent across sample loads, confirming reaction fidelity. Compared to the Agilent kit at 40 µg load, GlycoWorks offered 1.5× greater FLR sensitivity and 2.5× greater MS sensitivity. SPE recovery improved with higher sample amount, yielding 1.5× better cleanup efficiency than Agilent at 40 µg. Over-labelling analysis revealed that GlycoWorks produced 1.5–2× fewer doubly labelled glycans, reducing risks of misidentification.

Benefits and Practical Applications

• Enhanced detection of low-abundance glycoforms such as Man5
• Improved SPE recovery and consistent glycan profiling across concentration ranges
• Reduced over-labelling minimizes false peaks and quantification errors
• Rapid workflow (1–2 hours) compatible with automated and high-throughput laboratories

Future Trends and Opportunities

Advances in glycan labelling chemistry and automation will drive higher throughput and deeper sensitivity. Integration with real-time data analysis and AI-based peak identification may further streamline workflows. Emerging labels and SPE materials promise to expand dynamic range and reduce non specific reactions. Broader application to cell culture monitoring and glycoproteomics will enhance process control.

Conclusion

The modified GlycoWorks RapiFluor-MS protocol delivers substantially improved FLR and MS sensitivity without compromising glycan distribution or ease of use. Superior cleanup recovery and lower over-labelling compared to a leading alternative kit make this method well suited for rigorous N-glycan analysis in biotherapeutic research and quality control.

Reference

1. Zhang P et al. Drug Discovery Today 2016, 21(5):740–765
2. Reusch D, Tejada ML. Glycobiology 2015, 25(12):1325–1334
3. Goetze AM et al. Glycobiology 2011, 21(7):949–959
4. Lauber MA et al. Anal Chem 2015, 87(10):5401–5409
5. Koza SM et al. Waters Application Note 2020
6. Zhang X et al. SLAS Technology 2020, 24(4):380–387
7. Hanna CM et al. Waters Application Note 2023
8. Kalkhof S, Sinz A. Anal Bioanal Chem 2008, 392:305–312