Quality Control and Automation Friendly GlycoWorks RapiFluor-MS N-Glycan Sample Preparation

Significance of the Topic

The precise and reproducible profiling of N-glycans is essential in biopharmaceutical development and quality control, as glycosylation critically influences efficacy, safety, and consistency of glycoprotein therapeutics. A streamlined sample preparation workflow that minimizes pipetting errors and suits automated platforms enhances throughput, data reliability, and regulatory compliance.

Objectives and Study Overview

This work aimed to adapt the Waters GlycoWorks RapiFluor-MS N-glycan labeling procedure to use pipetting volumes ≥10 µL, thereby reducing systematic and random errors inherent at lower volumes. The redesigned protocol was evaluated against the original flexible-volume method by preparing and analyzing released N-glycans from a monoclonal antibody standard, comparing chromatographic and mass spectrometric performance.

Methodology

The revised sample preparation eliminates the water dilution step and increases RapiGest SF concentration from 1% to 1.5% for denaturation. A 10 µL aliquot of 1.5 mg/mL glycoprotein is combined with 10 µL RapiGest and 10 µL diluted PNGase F for deglycosylation (30 µL total). Labeling employs 10 µL of the RapiFluor-MS reagent, adjusted in concentration to maintain the original reagent-to-protein ratio. Following labeling, samples are diluted in acetonitrile and loaded onto HILIC SPE cartridges for cleanup prior to analysis.

Instrumentation

• Waters ACQUITY UPLC H-Class Bio System
• ACQUITY UPLC FLR Detector with analytical flow cell (265/425 nm)
• ACQUITY UPLC Glycan BEH Amide Column, 2.1 × 150 mm, 1.7 µm, 130 Å
• Xevo G2-XS QTof Mass Spectrometer (ESI+, resolution ~40 000)
• MassLynx Software v4.1
• GlycoWorks RapiFluor-MS N-Glycan Kit and associated buffers

Key Results and Discussion

Chromatograms and fluorescence peak area distributions for the QC-friendly protocol closely matched those of the original flexible-volume workflow. Both methods resolved over 25 glycan species with relative abundances down to 0.06 %. Quantitative comparisons demonstrated minimal deviation (<5 %) across major and minor peaks, confirming that larger pipetting volumes did not compromise labeling efficiency or chromatographic resolution.

Benefits and Practical Applications

The optimized protocol enhances pipetting precision and supports single-channel and multichannel automation in QC laboratories. By standardizing on ≥10 µL transfers, it reduces operator variability and improves inter- and intra-laboratory reproducibility. The simplified workflow shortens hands-on time and is readily integrated into regulated environments for routine glycan profiling.

Future Trends and Applications

Integration of this QC-friendly RapiFluor-MS protocol into fully automated platforms, including robotic liquid handlers and high-throughput screening systems, will accelerate glycan analysis in process development and batch release testing. Further advances may include multiplexed labeling reagents, microfluidic SPE cartridges, and AI-driven data analysis for deeper glycan characterization and real-time monitoring.

Conclusion

The revised GlycoWorks RapiFluor-MS N-glycan sample preparation protocol successfully uses ≥10 µL pipetting volumes without sacrificing analytical performance. It offers enhanced precision, reproducibility, and compatibility with automated workflows, making it well suited for quality control and high-throughput glycan profiling in biopharmaceutical environments.

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).
2. Lauber MA, Yu YQ, Brousmiche DW, Hua Z, Koza SM, Magnelli P, Guthrie E, Taron CH, Fountain KJ. Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection. Analytical Chemistry 2015, 87, 5401–5409.
3. Waters. GlycoWorks RapiFluor-MS N-Glycan Kit Care and Use Manual. Document 715004793.
4. Lauber MA, Morris MF, Brousmiche DW, Koza SM. Robustness of RapiFluor-MS N-Glycan Sample Preparations and Glycan BEH Amide HILIC Chromatographic Separations. Waters Application Note 720005370EN (2015).
5. ISO 8655-2:2002. Piston-operated volumetric apparatus. Part 2: Piston pipettes.