HPAE-PAD N-linked oligosaccharide profiling of IgG

Significance of the Topic

Glycoprotein characterization is critical in biotherapeutics development. IgG antibodies carry conserved N-linked oligosaccharides that influence effector functions, serum clearance, and immunogenicity. Detailed profiling of these glycans supports quality control, ensures batch-to-batch consistency, and meets regulatory requirements for therapeutic antibodies.

Aims and Study Overview

The goal of this work was to develop and validate an HPAE-PAD method for comprehensive profiling of the major neutral and charged N-linked oligosaccharides released from IgG, including monoclonal antibodies. The method seeks to separate 14 common neutral structures and key charged species, resolve high-mannose variants, and provide reproducible retention index values across different systems.

Methodology

The workflow combines enzymatic release and exoglycosidase digestions with high-resolution chromatography:

• PNGase F digestion to release N-linked glycans from IgG (20 μL PNGase F with 200 μL protein at 37 °C for 20 h).
• Sequential exoglycosidase treatments (neuraminidase, β-galactosidase, α-L-fucosidase) for structural confirmation and isomer assignment.
• Chromatography at 0.5 mL/min, 30 °C, with partial-loop injection (5 μL) on a CarboPac PA200 column.
• Eluent A: deionized water; B: 100 mM NaOH; C: 200 mM sodium acetate in 100 mM NaOH. A linear gradient from 50 mM to 100 mM NaOH (0–30 min) followed by 200 mM acetate gradient (30–50 min) separates neutral and charged species.
• Pulsed amperometric detection using a gold working electrode under a four-potential waveform.
• Retention index calibration with maltose (RI 200), maltotriose (RI 300), and maltotetraose (RI 400) to correct for system-to-system variability.

Used Instrumentation

• Thermo Scientific Dionex ICS-5000+ HPIC system (or ICS-3000 alternative).
• Dionex SP single pump or DP dual pump module; DC detector/Chromatography module.
• Dionex AS-AP autosampler with 10 μL PEEK sample loop.
• Dionex ED electrochemical detector with gold working electrode and Ag/AgCl reference electrode.
• Thermo Scientific CarboPac PA200 analytical column (3 × 250 mm) with PA200 guard (3 × 50 mm).

Main Results and Discussion

The optimized method successfully separated the 14 major neutral and six charged IgG oligosaccharides, plus high-mannose forms. Baseline shifts associated with the hydroxide-to-acetate gradient were managed by robust eluent preparation. Exoglycosidase digests confirmed peak identities, and retention index values varied by less than 2% across multiple systems, analysts, eluent batches, and column lots. Representative peaks include G0F, G0, G1F isomers, G0bF, G2F, G1bF, and sialylated A1F/A2F species. Microgram-level sample loads yielded clear chromatograms, demonstrating high sensitivity.

Practical Benefits and Applications

• Orthogonal glycan profiling complementing mass spectrometry and fluorescence methods.
• Low sample consumption suitable for limited therapeutic antibody material.
• Retention index calibration enhances interlaboratory consistency.
• Applicable to lot release testing and comparability studies in QC labs.

Future Trends and Potential Applications

Advances may include coupling HPAE-PAD with mass spectrometry for structural elucidation, further automation for high-throughput screening, optimized gradients to resolve sialic acid linkage isomers, and application to glycoengineered biotherapeutics. Online monitoring of glycosylation during cell culture and integration into process analytical technology (PAT) platforms are promising developments.

Conclusion

The presented HPAE-PAD method provides a robust, reproducible platform for detailed N-linked oligosaccharide profiling of IgG. Retention index calibration ensures reliable peak assignment across different systems. Low sample requirements, combined with comprehensive structural confirmation via exoglycosidases, make this approach well suited for biopharmaceutical development and quality control.

References

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