GlycoWorks™ RapiFluor-MS™ N-Glycan Kits with the Andrew Pipetting Robot

Importance of the Topic

The detailed preparation and analysis of N-glycans is critical for biopharmaceutical quality control, structural elucidation and comparison of glycoprotein therapeutics.
High reproducibility, minimized manual error and increased throughput are essential to meet stringent regulatory and research requirements.

Objectives and Overview

This user manual establishes a fully integrated workflow for semi-automated N-glycan sample preparation by coupling Waters GlycoWorks™ RapiFluor-MS™ N-Glycan Kits with the Andrew Alliance pipetting robot.
It guides users through system installation, IQ/OQ validation and day-to-day operation for processing 8 to 24 samples per run.

Methodology

The protocol comprises:

1. Deck configuration: placement of Dominos™ (Peltier device, reagent and sample racks, vacuum manifold).
2. Reagent preparation: solubilization of RapiFluor-MS label and RapiGest SF surfactant, dilution of PNGase F enzyme, SPE buffer assembly.
3. IQ/OQ checks: pipette calibration, dead-volume assessment, Peltier temperature control and vacuum performance verification.
4. Automated sample processing:
   
   • Glycoprotein denaturation and enzymatic N-glycan release by Peltier-controlled heating.
   • Fluorescent labeling with RapiFluor-MS in DMF.
   • SPE clean-up on Waters μElution plates with conditioning, washing and elution steps.
   • Post-elution dilution to defined concentration for LC-MS analysis.
5. User interactions at key points to replace waste trays, load sample collection rack and confirm tip inventories.

Instrumentation

The core equipment includes:

• Andrew Alliance 1000G-XL or 1000R-XL pipetting robot with Andrew Lab software.
• Custom Dominos™: Peltier heating/cooling unit, GlycoWorks reagent and rack Dominos™, vacuum manifold assembly.
• Waters vacuum pump and SPE μElution™ manifold.
• Standard pipettes (20 µL–1250 µL) with extended-length tips.
• Additional labware: 0.5 mL septa caps, 15/50 mL conical tubes, 96-well PCR plates.

Main Results and Discussion

Implementation of this automated workflow achieves consistent sample handling across multiple runs, reducing hands-on time and variability.
IQ/OQ procedures ensure reliable pipetting accuracy, proper vacuum levels (2.5–4 Hg) and temperature control for enzyme reactions.
User prompts minimize risk during critical transitions (e.g., SPE cartridge swapping) while allowing continuous automation.

Benefits and Practical Applications

This integrated system delivers:

• Reproducible N-glycan profiles for QC of monoclonal antibodies and glycoproteins.
• Throughput of up to 96 samples per day by scaling with multiple runs.
• Reduced operator fatigue and error through standardized liquid-handling protocols.
• Compatibility with downstream LC-MS analysis for structural characterization and quantitation.

Future Trends and Potential Applications

Emerging developments may include higher-density sample plates, expanded enzyme chemistries for branching and sialylation variants, and integration with data-analysis pipelines.
AI-driven scheduling and remote monitoring will further streamline glycan workflows in regulated environments.
Enhanced compatibility with other labeling reagents and mass spectrometry platforms will broaden analytical scope.

Conclusion

The combination of Waters GlycoWorks™ RapiFluor-MS™ N-Glycan Kits and the Andrew Alliance pipetting robot offers a robust, high-throughput solution for N-glycan analysis.
The standardized protocol, detailed IQ/OQ checks and user prompts ensure accuracy, reproducibility and ease of use for routine biopharmaceutical applications.

References

• Andrew Lab User Guide, Andrew Alliance.
• GlycoWorks RapiFluor-MS N-Glycan Kit Care and Use Manuals (715004793EN, 715004903).
• RapiFluor-MS Quick Start Protocols (720005343EN, 720005470EN).
• Quality Control and Automation Friendly GlycoWorks RapiFluor-MS Application Note (720005506EN).