Robust Automated High-Throughput N-Glycan Analysis Using the GlycoWorks RapiFluor-MS N-Glycan Kit for Automation

Importance of the Topic

The N-glycan profile of glycoproteins is a critical quality attribute in biopharmaceutical development, clinical diagnostics, and bioprocess monitoring. Rapid and reproducible analysis of N-glycans supports regulatory compliance, batch-to-batch consistency, and detailed structure–function studies. High-throughput automation addresses the need for standardized workflows, minimizes human error, and delivers robust data at scale.

Study Objectives and Overview

This work evaluates a fully automated N-glycan sample preparation workflow based on the Waters GlycoWorks RapiFluor-MS N-Glycan Kit for Automation coupled with a Tecan liquid-handling script. The objectives were to:

• Compare automated sample preparation performance with the established manual protocol.
• Characterize robustness against deliberate variations in reaction parameters, mixing, and SPE conditions.
• Demonstrate high-throughput capacity (up to 96 samples per run) without analytical compromise.

Instrumentation Used

The automated workflow integrates:

• Tecan EVO Freedom 100 platform with TELESH+ orbital mixing module and barcode scanning.
• GlycoWorks RapiFluor-MS N-Glycan Kit – Automation including reagents, HILIC µElution plate, and labware.
• ACQUITY UPLC I-Class or UPLC H-Class Bio system with FLR detector.
• ACQUITY UPLC Glycan BEH Amide column (130 Å, 1.7 μm, 2.1×150 mm).

Methodology

Key stages of the automated workflow:

• Sample normalization: glycoprotein solutions normalized to 1.5 mg/mL across 96 wells.
• Denaturation and deglycosylation: 90 °C for 3 min (with RapiGest SF), then 50 °C for 5 min (with PNGase F).
• RapiFluor-MS labeling: addition of label reagent in DMF, mixed via TELESH+ and incubated.
• HILIC SPE cleanup: conditioning, washing, and elution (five smaller aliquots) on µElution plate under controlled vacuum.
• Dilution and transfer: post-SPE samples diluted to 32:68 DMF/ACN for UPLC loading.
• UPLC-FLR analysis: gradient from 25% aqueous to 100% aqueous over 36.5 min; detection at Ex 265/Em 425 nm.

Main Results and Discussion

• Throughput and reproducibility: 48–96 samples processed in under 3 hours with <3% CV for major glycan peaks and equivalent area % results compared to manual prep.
• Robustness to reaction variations: denaturation and deglycosylation temperatures varied ±5 °C, mixing speeds adjusted widely, RapiGest SF concentration tested at 1.0% vs. 1.5%—no significant bias introduced.
• SPE performance: no discernible bias between pre- and post-SPE glycan distributions; SPE parameters (vacuum pressure, loading steps) tolerable to variation.
• On-deck and on-autosampler stability: reagents remain stable exposed on deck for an hour; prepared samples stable at 10 °C for at least 88 hours.
• Platform flexibility: kit quantities and protocol compatible with other automation platforms (Hamilton, Agilent Bravo) through script parameter adaptation.

Benefits and Practical Applications

The automated GlycoWorks RapiFluor-MS workflow delivers:

• Standardized, error-resistant sample preparation for routine QC and research.
• High throughput enabling large glycan mapping campaigns and process monitoring.
• Reduced hands-on time and improved method transferability between labs.
• Consistency across multiple operators, instruments, and reagent lots.

Future Trends and Potential Uses

• Integration with MS detection for deeper glycan structural elucidation.
• Shorter UPLC gradients or multiplexed injections to further increase throughput.
• AI-driven script optimization to adapt mixing and pipetting parameters on-the-fly.
• Expansion to other glycoprotein classes and complex biological matrices.
• Coupling with automated data processing pipelines for real-time QC decision making.

Conclusion

The GlycoWorks RapiFluor-MS N-Glycan Kit for Automation, combined with a validated Tecan script, provides a robust, high-throughput solution for N-glycan analysis. It matches manual protocol performance while significantly reducing labor and ensuring reproducibility under routine laboratory variations. This platform-agnostic approach supports scalable glycan profiling for biopharma, clinical research, and quality control environments.

References

• 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.
• Lauber MA, Fournier JL, Koza SM, Fountain KJ. GlycoWorks HILIC SPE Robust Glycan Sample Preparation. Waters Application Note 720005116EN, 2014.
• Koza SM, McCall SA, Lauber MA, Chambers EC. Quality Control and Automation Friendly GlycoWorks RapiFluor-MS N-Glycan Sample Preparation. Waters Application Note 720005506EN, 2016.
• Lauber MA et al. Rapid Preparation of Released N-glycans for HILIC Analysis Using a Labeling Reagent That Facilitates Sensitive Fluorescence and ESI-MS Detection. Anal. Chem. 2015, 97, 5401–5409.