Accurate and precise quantification of mAb-released N-glycans with an amide HILIC column

Importance of the Topic

Glycosylation of monoclonal antibodies represents a critical quality attribute in biotherapeutic development and manufacturing. Accurate and reproducible analysis of released N-glycans ensures product safety, efficacy, and regulatory compliance in quality control workflows.

Objectives and Study Overview

This study evaluates the robustness, reproducibility, accuracy, and precision of IgG N-glycan quantification using a Thermo Scientific Vanquish Horizon UHPLC system coupled with an Accucore 150 Amide HILIC column. The goal is to demonstrate consistent performance across column lots and extended use, as well as to benchmark quantification against a validated manufacturing method.

Methodology and Instrumentation

Sample preparation included protein denaturation, enzymatic release of N-glycans, fluorescent labeling with 2-AB or APTS, and removal of excess reagents. Chromatographic separation was performed on an Accucore 150 Amide HILIC column (2.1 × 150 mm, 2.6 µm) at 50 °C with a gradient of acetonitrile and 100 mM ammonium formate (pH 4.4). The flow rate was 0.45 mL/min. Detection was achieved by fluorescence at excitation/emission wavelengths of 320/420 nm (2-AB) and 455/500 nm (APTS). The Vanquish Horizon UHPLC system and Chromeleon CDS 7.2.5 software were employed for data acquisition and processing.

Main Results and Discussion

• Lot-to-lot and inter-column reproducibility: Retention time RSD < 2%, peak asymmetry RSD < 6.5%, efficiency RSD < 3%. Backpressure RSD < 3% between columns and < 7% across lots.
• Column lifetime: Over 500 consecutive injections, retention time RSD < 0.5%, G1Fa/G1Fb resolution remained between 2.07 and 2.26 (RSD 2.76%), and major glycan relative area RSD ≈ 1% (< 3% for low-abundance components).
• Quantification of human IgG N-glycans: Thirteen 2-AB labeled glycans were baseline separated within 22 minutes at moderate backpressure (< 200 bar), with peak widths < 7 s. Relative peak area RSDs were < 4% (n = 5). Absolute bias averaged ± 3% compared to manufacturer data. Deming regression yielded a slope of 1.017 and R² > 0.99, indicating excellent accuracy and correlation.

Benefits and Practical Applications

• The Accucore 150 Amide HILIC platform provides reliable, high-resolution glycan profiling critical for quality-by-design and QC in biopharmaceutical manufacturing.
• Moderate backpressures enable UHPLC efficiency without specialized high-pressure hardware.
• Accurate and precise quantification supports regulatory filings and batch release decisions.

Future Trends and Opportunities

Advancements in core-shell stationary phases, integration of mass spectrometry, and high-throughput automation are expected to further enhance glycan analysis speed and sensitivity. Emerging AI-driven data processing may enable real-time monitoring of glycosylation profiles during bioprocessing.

Conclusion

The Accucore 150 Amide HILIC column coupled with the Vanquish Horizon UHPLC system delivers robust, reproducible, and accurate quantification of released monoclonal antibody N-glycans. Its performance across multiple column lots and extended use supports reliable glycan profiling for biotherapeutic quality control and regulatory compliance.

Reference

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