Development of a Rapid APTS Sample Preparation Workflow for N-Glycan Release and Labeling

Significance of the topic

N-Glycan structures on biotherapeutic proteins play a pivotal role in modulating immunogenicity, pharmacokinetics, and pharmacodynamics. Fluorescent labeling with 8-aminopyrene-1,3,6-trisulfonate (APTS) followed by capillary electrophoresis (CE) or UHPLC-HILIC enables high-resolution profiling of released N-glycans. Conventional APTS workflows often require extensive sample drying and multi-hour cleanup, delaying analytical turnaround and throughput.

Objectives and study overview

This work aimed to develop and validate a rapid APTS labeling workflow by integrating the Agilent AdvanceBio Gly-X N-Glycan Prep platform with APTS Express. The goal was to reduce total sample preparation time to approximately 2.5 hours while maintaining compatibility with historical APTS data and ensuring broad applicability across CE and UHPLC-HILIC platforms.

Methodology and instrumentation

• Sample preparation workflow:
  
  • In-solution denaturation and PNGase F release in a 96-well PCR plate (3 min denaturation, 5 min deglycosylation).
  • On-matrix reductive amination with APTS Express (60 min labeling) without drying steps.
  • Vacuum-driven solid-phase cleanup (15–30 min) to remove >99% free APTS dye.
• Instrumentation:
  
  • Capillary electrophoresis:
    • Agilent Gly-Q Glycan Analysis System with LED-induced fluorescence (LEDIF), 310/>385 nm;
    • Sciex PA800 plus CE with laser-induced fluorescence (488/520 nm).
  • UHPLC-HILIC on an amide column (2.1 × 150 mm, 1.7 µm) at 60 °C with gradient elution (30–60% 50 mM ammonium formate, pH 4.4).

Main results and discussion

• Total sample prep time was reduced to ~2.5 hours compared to 4–5 hours in prior GlykoPrep workflows.
• Cleanup efficiency exceeded 99% removal of unreacted APTS, yielding sharp early peaks in CE separations.
• Reproducibility for major glycan peaks showed %CV < 3% across six replicates of rituximab (MabThera).
• Input range of 1–200 µg protein produced consistent profiles for both MabThera and etanercept (Enbrel).
• APTS-labeled glycans separated effectively on both CE platforms and by UHPLC-HILIC, though APTS peaks were broader than those labeled with less polar dyes.
• Sialylation patterns were preserved through cleanup (50 ± 1% sialylated species), and a 10-minute Sialidase A digest enabled unambiguous assignment of α-linked sialic acids.

Benefits and practical applications

• Accelerates N-glycan profiling in biopharmaceutical R&D and QC laboratories.
• Maintains data continuity with historical APTS libraries and retention time databases.
• Supports high-throughput automation in a 96-well format.
• Compatible with multiple detection platforms, facilitating method transfer across laboratories.

Future trends and potential applications

Anticipated developments include integration with fully automated liquid-handling systems for end-to-end workflows, adaptation for multiplexed glycan labeling strategies, and coupling with mass spectrometry for structural confirmation. Advances in machine learning may leverage high-throughput APTS datasets to predict glycan profiles and refine biologic quality attributes across production batches.

Conclusion

The Agilent AdvanceBio Gly-X N-Glycan Prep with APTS Express offers a rapid, reproducible, and automation-ready solution for fluorescent labeling of released N-glycans. It streamlines traditional workflows without sacrificing data comparability, and its open format supports versatile separation techniques across CE and UHPLC platforms.

References

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• Szekrényes A.; et al. Multi-Site N-glycan Mapping Study 1: Capillary Electrophoresis – Laser Induced Fluorescence. MAbs 2016, 8(1), 56–64.
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