A Comprehensive N-Glycan Profiling Analysis of Bevacizumab Biosimilar by UHPLC with Fluorescence Detection and Q-TOF Mass Spectrometry

Significance of the Topic

The accurate profiling of N-glycans on monoclonal antibodies is critical for ensuring product safety, efficacy and consistency in biopharmaceutical manufacturing. Glycosylation profoundly influences antibody stability, immune activity and pharmacokinetics. As biosimilars gain approval and market share, robust analytical workflows are essential to demonstrate glycan similarity to reference products and meet regulatory requirements.

Objectives and Study Overview

This work aimed to develop and validate a reliable, high-resolution method for comprehensive N-glycan profiling of a bevacizumab biosimilar. Key goals included release and tagging of glycans, chromatographic separation, structural identification by mass spectrometry, and relative quantitation of glycan species to assess similarity to the innovator product.

Methodology and Instrumentation

• Glycan Release and Labeling: N-glycans were enzymatically liberated using PNGase F, purified via solid-phase clean-up, and labeled with 2-aminobenzamide (2-AB) for fluorescence detection.
• Chromatography: Separation was performed on a HALO® Glycan column (2.7 µm, 150 × 2.1 mm) at 60 °C using a gradient of 50 mM ammonium formate and acetonitrile at 0.4 mL/min.
• Detection: Fluorescence detection employed a Shimadzu RF-20A (excitation 330 nm, emission 420 nm) coupled in-line with a Shimadzu Nexera Bio UHPLC.
• Mass Spectrometry: Structural confirmation and accurate mass measurement utilized the Shimadzu LCMS-9030 Q-TOF with heated electrospray ionization (+), mass range 500–2500 m/z, and MS/MS fragmentation.

Results and Discussion

• Nine 2-AB-labeled N-glycans were resolved and identified: Man3, G0-GN, G0F-2GN, G0F-GN, G0, Man5, G0F, G1Fa and G1Fb.
• Accurate masses matched theoretical values within ±1 ppm, and MS/MS spectra confirmed structural assignments.
• Relative quantitation showed G0F comprised 87.23% of total glycan pool; Man3 was the least abundant at 0.7%.
• System precision was demonstrated by retention time and peak area RSDs below 2% across triplicate injections, confirming method repeatability and stability.

Benefits and Practical Applications

This UHPLC-fluorescence/Q-TOF workflow provides high sensitivity and specificity for N-glycan profiling while delivering rapid turnaround and robust quantitation. It supports biosimilar development by enabling detailed glycosylation comparisons, batch-to-batch consistency monitoring, and regulatory submissions.

Future Trends and Applications

• Automation of sample preparation and derivatization to increase throughput.
• Integration with ion mobility or higher-resolution MS to differentiate isomeric glycan forms.
• Application to other therapeutic antibodies and Fc-fusion proteins for broad biosimilar characterization.
• Development of label-free or accelerated labeling strategies to shorten analysis time.
• Exploration of glycan heterogeneity impacts on clinical performance and quality control.

Conclusion

A robust and reproducible UHPLC-fluorescence coupled with Q-TOF MS method was established for detailed N-glycan profiling of a bevacizumab biosimilar. The protocol achieved low variability, high mass accuracy and confident glycan identification, making it highly suitable for biosimilar quality assessment and comparability studies.

References

• LudgerClean™ EB10 user guide. Ludger Ltd.
• Keser T, Pavić 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™ S user guide. Ludger Ltd.