Instrument configuration for native N-linked oligosaccharide characterization by HPAE-PAD/MS

Importance of the topic

The analysis of native N-linked oligosaccharides is essential for rigorous characterization of biotherapeutics. Traditional derivatization approaches can introduce bias, while native glycan profiling by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) coupled to high-resolution mass spectrometry offers unbiased, high-throughput insights into glycosylation patterns required by regulatory guidelines.

Objectives and study overview

This work details the integration of a Thermo Scientific Dionex ICS-5000+ HPIC system with PAD detection to a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer. The goal is to establish a rapid, robust workflow for in-depth profiling of native, released N-linked glycans from glycoproteins including monoclonal antibodies and biosimilars.

Methodology and Instrumentation

• In-solution glycoprotein digestion using heat denaturation and PNGase F
• Purification on graphitized carbon in 96-well format for removal of proteins, detergents, salts
• Anion-exchange separation on CarboPac PA200 with PAD detection at 0.5 mL/min
• Post-column flow split: ~45% to PAD, ~55% to MS via ERD 500 electrolytic desalter
• Electrospray ionization in negative mode on Q Exactive Orbitrap, data dependent MS/MS acquisition
• Continuous monitoring of pH and column temperature through virtual channels in Chromeleon software

Main results and discussion

The combined HPAE-PAD/MS approach resolved 53 distinct N-glycans from human α-1-acid glycoprotein with mass errors below 2 ppm. Monoclonal antibody glycoprofiling (Humira, Inflectra) identified 28–33 glycans including sialylated, fucosylated, α-galactosylated, and N-glycolyl-modified structures. MS/MS fragmentation yielded diagnostic glycosidic and cross-ring ions for confident isomer annotation. The ERD 500 desalter maintained stable sodium removal over multiple runs without degrading neutral glycan resolution.

Benefits and practical applications

• Bias-free native glycan analysis without labeling artifacts
• Processing of 96 samples in ~2 hours supports high throughput
• Detailed structural elucidation including positional isomers
• Compliance with ICH Q6b for biopharmaceutical QC
• Seamless integration into existing HPAE-PAD systems with minimal hardware additions

Future trends and potential applications

Integration of ion mobility separation could enhance isomer resolution. Automated data processing with machine learning will accelerate glycan annotation. Expanding applications to complex glycoprotein mixtures and cellular secretomes may drive biomarker discovery and biosimilar development.

Conclusion

This HPAE-PAD/MS platform offers reproducible, high-resolution, and high-throughput profiling of native N-linked glycans. It delivers detailed structural information and meets regulatory requirements for biotherapeutic characterization, serving as a robust tool for glycoprofiling in research and QC environments.

Used instrumentation

• Thermo Scientific Dionex ICS-5000+ HPIC system with DP pump and PAD detector
• CarboPac PA200 guard and analytical columns
• Thermo Scientific ERD 500 electrolytic desalter
• Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer
• Thermo Scientific Dionex WPS TBPL thermostatted autosampler
• Chromeleon CM7.2 SR4 CDS and SimGlycan v5.0 software

Reference

• ICH Q6b guidelines on biological product characterization
• Thermo Scientific Technical Note 71 on eluent preparation for HPAE-PAD
• Thermo Scientific Technical Note 21 on carbohydrate detection settings
• Thermo Scientific Electrochemical Detection user compendium
• Thermo Scientific Technical Note 110 on disposable Au on PTFE electrodes
• Szabo Z et al. Anal Bioanal Chem 2017 409 3089–3101
• Eavenson M et al. Glycobiology 2015 25 66–73