GlycoWorks RapiFluor-MS N-Glycan Reagent Kits: Scalable Solutions for Low- to High-throughput N-Glycan Sample Preparation Using Manual, Semi-automated or Fully Automated Workflows

Significance of the Topic

The analysis of N-glycans is critical in biopharmaceutical development and quality control, as glycosylation impacts protein stability, efficacy, and safety.
Scalable sample preparation workflows enable laboratories of varying throughput demands to achieve reproducible and sensitive glycan profiling.

Study Objectives and Overview

This study demonstrates the flexibility and scalability of Waters GlycoWorks RapiFluor-MS N-Glycan reagent kits for manual, semi-automated, and fully automated workflows.
It compares performance across three approaches—manual preparation, Andrew Alliance pipetting robot, and Tecan automation—to validate precision and consistency in N-glycan profiling.

Methodology and Instrumentation

• Sample Preparation Workflows:
  • Manual: RapiFluor-MS N-Glycan Kit standard protocol.
  • Semi-automated: Integration with Andrew Alliance pipetting platform.
  • Fully automated: Validated Tecan script for high-throughput processing.
• Glycan Labeling: RapiFluor-MS reagent for rapid deglycosylation and enhanced fluorescence and mass detection sensitivity.
• Chromatography Conditions:
  • Instrument: ACQUITY UPLC I-Class or H-Class Bio with FLR detector.
  • Column: ACQUITY UPLC Glycan BEH Amide, 2.1×150 mm, 1.7 µm, 60 °C.
  • Mobile Phases: 50 mM ammonium formate (pH 4.4) in water; 100% acetonitrile, with a 55 min gradient.

Instrumentation Used

• Waters ACQUITY UPLC I-Class/H-Class Bio system with fluorescence detector.
• Tecan automated liquid handling platform with validated script.
• Andrew Alliance Pipetting Robot for semi-automation.
• Empower 3 chromatography data software for acquisition and analysis.

Results and Discussion

Automated workflows achieved high precision with coefficient of variation values below 2% for key glycan species, comparable to manual preparation.
Mean area percent distributions of G0F, G1Fa, G1Fb, and G2F glycans were consistent across methods, with minor bias within acceptable confidence intervals.
Statistical analysis confirmed equivalent performance in glycan profiling, demonstrating that automation does not compromise data quality.

Benefits and Practical Applications

• Throughput Flexibility: Easily scales from low to high sample volumes.
• Reproducibility: Automated protocols reduce manual variability and improve consistency.
• Sensitivity: RapiFluor-MS label enhances fluorescence and mass detection limits.
• Laboratory Efficiency: Validated scripts simplify method transfer and reduce hands-on time.

Future Trends and Opportunities

Advancements in liquid handling integration will further streamline glycan analysis, allowing broader adoption in regulated environments.
Combining automation with mass spectrometry and data analytics will enable rapid glycoform characterization and real-time monitoring.
Expansion of reagent compatibility across additional platforms will enhance accessibility for diverse laboratory settings.

Conclusion

Waters GlycoWorks RapiFluor-MS N-Glycan kits offer a unified solution for scalable N-glycan sample preparation across manual, semi-automated, and fully automated workflows.
Validated automation scripts and compatible reagent formats ensure high precision, reproducibility, and efficiency in glycan profiling.
This flexibility supports laboratories in meeting varying throughput demands without compromising analytical performance.

Reference

No external literature references were provided in the original application note.