Development of a Rapid 2-AB Sample Preparation Workflow for N-Glycan Release and Labeling

Importance of the topic

The structural characterization of N-linked glycans is a critical quality attribute in biotherapeutic development. Glycan patterns influence immunogenicity, pharmacokinetics, and pharmacodynamics of monoclonal antibodies and fusion proteins. Rapid and reliable workflows for glycan release and fluorescent labeling accelerate candidate screening, process development, and quality control in both pharmaceutical and biopharma laboratories.

Objectives and study overview

This study describes the development and evaluation of a rapid 2-aminobenzamide (2-AB) glycan labeling workflow based on two Agilent Technologies sample preparation kits. The goals were to reduce hands-on and total processing time while maintaining compatibility with established analytical methods. Two monoclonal antibodies (MabThera and NISTmAb) and one Fc fusion protein (Enbrel) were processed with:

• Agilent GlykoPrep Rapid N-Glycan Prep with 2-AB
• Agilent AdvanceBio Gly-X N-Glycan Prep with 2-AB Express

Comparative performance was assessed by HILIC-UHPLC-FLR and UHPLC/MS detection.

Methodology and instrumentation

Protein samples (50 µg) underwent denaturation followed by PNGase F–mediated deglycosylation. Two workflows were applied:

• GlykoPrep: on-matrix deglycosylation (15–60 min), in-solution 2-AB labeling (60 min), matrix cleanup (15–30 min), including a dry-down step
• Gly-X Express: in-solution deglycosylation (5 min), on-matrix 2-AB Express labeling and cleanup (60 min labeling, 10–15 min cleanup) without drying

Instrumentation used

• UHPLC system with FLR detector (Ex 360 nm/Em 428 nm)
• Amide HILIC column 2.1 × 150 mm, 1.7 µm particle size
• 50 mM ammonium formate, pH 4.4 mobile phase gradient
• Agilent GlykoPrep Rapid N-Glycan Prep with 2-AB kit
• Agilent AdvanceBio Gly-X N-Glycan Prep with 2-AB Express kit
• PNGase F enzyme, sodium cyanoborohydride reductant

Main results and discussion

Both workflows generated comparable glycan profiles for each therapeutic protein. Key findings include:

• MabThera and NISTmAb: dominance of core-fucosylated biantennary glycans (G0F, G1F, G2F) with minor high-mannose and sialylated species
• Enbrel: higher proportion of sialylated (A1F, A2F) and afucosylated glycans
• Total fluorescence response was equal or higher with the Gly-X Express workflow for MabThera and Enbrel; NISTmAb signals were comparable across methods
• Relative glycan abundances differed by less than 5% between methods for most major peaks, confirming quantitative agreement

Benefits and practical applications of the method

The Gly-X Express workflow reduces total sample preparation time to approximately two hours and eliminates drying steps, enabling higher throughput. It preserves established 2-AB labeling chemistry and produces data directly comparable to historic 2-AB results. This enhances laboratory efficiency in biopharma R&D, QC, and stability studies.

Future trends and applications

Emerging directions include integration of glycan labeling with automated liquid-handling platforms, development of alternative rapid reductive agents, and coupling with high-resolution mass spectrometry for detailed structural elucidation. Further miniaturization and multiplexing may support large-scale glycoform screening in biosimilar development and personalized medicine.

Conclusion

Agilent’s rapid 2-AB workflows deliver reproducible glycan profiles in significantly reduced time frames. The Gly-X Express method maintains compatibility with legacy 2-AB data while offering streamlined preparation and high throughput. Both approaches support robust biotherapeutic glycosylation analysis.

References

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