UHPLC Analysis of 2-Aminobenzamide- Labeled Glycans with the Vanquish Flex System

Significance of the Topic

Glycan analysis is a cornerstone in biotherapeutic development and disease biomarker discovery. N-linked glycans modulate protein efficacy, stability, and immunogenicity, influencing the performance of monoclonal antibodies and recombinant proteins. Robust profiling of glycan heterogeneity supports quality control in biopharmaceutical manufacturing and aids the identification of glycan-based disease markers.

Study Objectives and Overview

This technical evaluation demonstrates the separation, detection, and quantitation of 2-aminobenzamide (2-AB)-labeled N-glycan libraries using a Thermo Scientific™ Vanquish™ Flex UHPLC system with a Vanquish Fluorescence Detector F. The study reports retention time precision for complex glycan libraries and a single-glycan standard, assesses area precision and linearity for three glycan standards, and provides guidance on selecting between 2 µL and 8 µL flow cells.

Methodology and Instrumentation

Glycans from bovine fetuin and human IgG were labeled with 2-AB and separated under hydrophilic interaction chromatography conditions. Two columns were employed: a GlycanPac™ AXR-1 (1.9 µm, 2.1 × 250 mm) with a ternary gradient of acetonitrile, water, and 100 mM ammonium formate (pH 4.4), and an Accucore™ 150 Amide-HILIC column (2.6 µm, 2.1 × 150 mm) with a binary gradient of acetonitrile and 50 mM ammonium formate (pH 4.4). The Vanquish Flex UHPLC system comprised a quaternary pump, split sampler FT, active-preheater column compartment, and a fluorescence detector fitted with either a 2 µL or 8 µL flow cell. Buffers were prepared at pH 4.4, and excitation/emission wavelengths were set at 250/430 nm or 320/420 nm depending on the method.

Main Results and Discussion

The bovine fetuin library yielded over 60 resolved peaks in 50 minutes, while the IgG library produced more than 20 peaks within 30 minutes. Retention time precision (n = 6) was ≤ 0.1 % RSD for fetuin glycans and < 0.04 % RSD for IgG glycans; a single NA2 standard achieved 0.008 % RSD. Area precision for five injections of 1 pmol NA2 glycan was 0.92 % RSD. Linearity tests for NA2, A1, and A2 standards over concentration ranges up to 10 pmol delivered r2 values above 0.99 in both flow cells. Comparing flow cells, the 8 µL cell provided higher sensitivity at low analyte levels, while the 2 µL cell offered superior resolution for complex mixtures.

Benefits and Practical Applications

• Exceptional retention time stability supports reliable peak identification via database matching without co-injection of standards.
• High linearity and sensitivity enable quantitation at low femtomole levels.
• Flow cell choice tailors the method to analytical priorities: sensitivity or resolution.
• Compatibility with MS coupling allows further structural elucidation and MS/MS fragmentation.

Future Trends and Opportunities

Advances may include integrated UHPLC-MS workflows for automated glycan profiling, expanded glycan libraries for biomarker research, and enhanced detector technologies for multiplexed fluorescence detection. Computational tools and machine learning could further streamline glycan identification and quantitation.

Conclusion

The Vanquish Flex UHPLC system with Fluorescence Detector F delivers high-precision separation and quantitation of 2-AB-labeled glycans. Its excellent retention time reproducibility, robust linearity, and flexible flow cell options make it an effective platform for both routine glycan analysis in biopharmaceutical QC and advanced glycomics research.

Reference

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