Sensitive and Reproducible Glycan Analysis of Human Immunoglobulin G

Importance of the topic

Glycosylation of immunoglobulin G critically affects antibody stability, activity, and clinical performance. Detailed profiling of N-linked glycans supports quality control in biopharmaceutical production, ensures batch-to-batch consistency of monoclonal antibody drugs, and can serve as a biomarker of disease in clinical samples.

Objectives and overview of the study

This work evaluates the sensitivity, reproducibility, and quantitative performance of the Agilent 1260 Infinity Bio-inert Quaternary LC System equipped with an AdvanceBio 2.7 µm Glycan Mapping column and fluorescence detection for analysis of 2-AB-labeled N-glycans released from human IgG. Key goals include assessment of retention time and peak area precision, linearity over a low-pmol range, and limits of detection and quantification.

Methodology and Instrumentation

Sample preparation and chromatographic method

• N-Glycans released from human IgG and labeled with 2-aminobenzamide (2-AB).
• Separation by HILIC on a 2.1 × 150 mm, 2.7 µm Agilent AdvanceBio Glycan Mapping column.
• Mobile phases: A—acetonitrile; B—100 mM ammonium formate, pH 4.5; gradient from 15 % B to 50 % B over 42 min.
• Flow rate: 0.5 mL/min initial, reduced to 0.25 mL/min during gradient hold; column temperature 60 °C; sample cooler at 6 °C.

Instrumentation

• Agilent 1260 Infinity Bio-inert Quaternary Pump
• Agilent 1260 Infinity Bio-inert High-Performance Autosampler
• Agilent 1290 Infinity Thermostat and Thermostatted Column Compartment with bio-inert solvent heat exchangers
• Agilent 1260 Infinity Fluorescence Detector (Ex 260 nm, Em 430 nm)
• Data acquired and processed with Agilent OpenLAB CDS ChemStation Edition.

Main results and discussion

Precision

• Retention time RSD < 0.27 % (15 dominant glycan peaks, n = 6).
• Peak area RSD < 3.5 % under identical injection conditions.

Linearity and sensitivity

• Calibration over 0.016–1 pmol for five glycan standards (M5, A2G2, A2G2S1, FA2G2S1, A2G2S2).
• Correlation coefficients ≥ 0.997, most ≥ 0.999.
• Limits of detection: 9–12 fmol (S/N = 3); limits of quantification: 30–40 fmol (S/N = 10).

Enhanced fluorescence detection at Ex 260 nm/Em 430 nm provided improved signal-to-noise compared with conventional settings.

Benefits and practical applications of the method

This LC-FLD platform delivers robust, high-resolution glycan profiling with low sample requirements. It is suited for routine QA/QC of therapeutic antibodies, comparability and biosimilarity studies, and biomarker discovery in clinical research.

Future trends and opportunities

Further enhancements may include multiplexed fluorescence detection, integration with automated sample preparation, coupling to high-resolution mass spectrometry for structural elucidation, and advanced data processing workflows for real-time glycoform monitoring in biomanufacturing.

Conclusion

The Agilent 1260 Infinity Bio-inert Quaternary LC System combined with the AdvanceBio Glycan Mapping column and optimized fluorescence detection achieves sensitive, precise, and linear analysis of 2-AB-labeled human IgG N-glycans, supporting critical applications in biotherapeutic development and clinical glycomics.

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