N-glycan Profiling of monoclonal Antibody (mAb) on Nexera Bio UHPLC Coupled with Fluorescence Detector and Q-TOF Mass Spectrometer

Importance of the Topic

Glycosylation of monoclonal antibodies (mAbs) critically influences their stability, biological activity and immunogenicity. Regulatory requirements for biosimilar mAbs demand thorough N-glycan profiling to ensure consistent product quality and therapeutic efficacy.

Objectives and Study Overview

This study aimed to establish a robust, sensitive and reproducible platform for N-glycan profiling of a bevacizumab biosimilar. The system integrates a Nexera Bio UHPLC, Shimadzu RF-20A fluorescence detector and LCMS-9030 Q-TOF mass spectrometer.

Methodology and Instrumentation

Sample Preparation

• Protein solubilization in Tris buffer and desalting via 10 kDa MWCO filtration.
• Reduction with DTT and alkylation with iodoacetamide.
• Enzymatic release of N-glycans using PNGase F at 37 °C overnight.
• Extraction and cleanup of released glycans using LudgerClean™ EB10 cartridges.
• Labeling with 2-aminobenzamide (2-AB) and purification using LudgerClean™ S cartridges.

Used Instrumentation

UHPLC and Detection

• Shimadzu Nexera Bio UHPLC system
• HALO® Glycan column (2.7 µm, 150 × 2.1 mm) at 60 °C, flow rate 0.4 mL/min
• Fluorescence detector RF-20A (Ex 330 nm, Em 420 nm)

MS Analysis

• Shimadzu LCMS-9030 Q-TOF with heated ESI source
• MS scan range 500–2500 m/z; MS/MS 100–2500 m/z with 50 ± 17 V collision energy

Key Results and Discussion

Reproducibility

• Injection-to-injection RSD for peak area and retention time below 2% across nine major N-glycans.

Identification

• Accurate mass assignment (< 2 ppm error) combined with MS/MS fragment patterns provided high confidence in glycan identification (Man3, G0F-2GN, G0-GN, G0F-GN, G0, Man5, G0F, G1Fa, G1Fb).

Quantitation

• Relative abundance profiling revealed G0F as the dominant glycoform (87.23% of total N-glycans).

Benefits and Practical Applications

This platform delivers high sensitivity, accurate mass identification and excellent repeatability, making it suitable for routine glycan profiling in biosimilar development, quality control and regulatory compliance.

Future Trends and Opportunities

Advances may include higher throughput workflows, automated sample preparation, integration with data-processing pipelines and extension to other therapeutic glycoproteins for comprehensive biopharmaceutical characterization.

Conclusion

The combination of Nexera Bio UHPLC, RF-20A fluorescence detection and LCMS-9030 Q-TOF mass spectrometry provides a reliable, high-performance solution for N-glycan profiling of monoclonal antibody biosimilars, meeting stringent accuracy and reproducibility requirements.

Reference

• Keser T, Pavic T, Lauc G, Gornik O. Comparison of 2-Aminobenzamide, Procainamide and RapiFluor-MS as Derivatizing Agents for High-Throughput HILIC-UPLC-FLR-MS N-glycan Analysis. Front Chem. 2018;6:324.
• LudgerClean™ EB10 LC cleanup guide.
• LudgerClean™ S cartridge cleanup protocol.