Comprehensive protein glycosylation comparison of an innovator monoclonal antibody to a candidate biosimilar by HILIC UHPLC analysis

Importance of the Topic

Glycosylation is a pivotal quality attribute for monoclonal antibody (mAb) therapeutics, directly affecting safety, efficacy and immunogenicity. Rapid and reliable glycan profiling is essential during biosimilar development to ensure comparability with the reference product.

Study Objectives and Overview

The study compared the N-glycan profiles of an innovator IgG1 mAb (trastuzumab) and a CHO-derived biosimilar candidate by applying a fast hydrophilic interaction ultra-high performance liquid chromatography (HILIC UHPLC) method. Key aims were:

• Comprehensive glycan profiling of 2-aminobenzoic acid (2-AA) labelled glycans by HILIC UHPLC-FLD.
• Detection of profile differences between innovator and biosimilar.
• Confirmation of glycan structures using exoglycosidase digestion and high-resolution accurate-mass (HRAM) Orbitrap mass spectrometry.

Methodology and Applied Instrumentation

Released N-glycans were enzymatically cleaved from denatured and reduced mAb samples using PNGase F, labelled with 2-AA, purified by HILIC SPE and separated on an Accucore 150-Amide-HILIC column with fluorescence detection. Structural confirmation employed sequential exoglycosidase digestions (sialidase, α-fucosidase, β-galactosidase, β-hexosaminidase) followed by Orbitrap MS analysis.

Applied Instrumentation:

• Thermo Scientific Vanquish Horizon UHPLC with Accucore 150-Amide-HILIC column.
• Fluorescence detector (λex = 350 nm, λem = 425 nm).
• Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap MS, HESI negative mode.
• Exoglycosidase panel: ABS sialidase, BKF α-fucosidase, SPG β-galactosidase, GUH β-hexosaminidase.

Main Results and Discussion

Mirror chromatograms revealed 13 glycan features in the innovator and 16 in the biosimilar. Key differences included:

• Innovator product: 92.5% core-fucosylated bi-antennary structures, 5.6% sialylation.
• Biosimilar candidate: 80.9% core fucosylation, elevated high-mannose species (14.5%), no detectable sialylation.

Exoglycosidase digestion shifts and MS accurate-mass data enabled unambiguous structural assignments of all major glycoforms. The workflow demonstrated high resolution and specificity in distinguishing subtle glycan variations.

Benefits and Practical Applications

The described HILIC UHPLC-FLD/MS approach offers:

• Rapid, high-throughput glycan profiling for comparability studies.
• Accurate structural confirmation via targeted exoglycosidase arrays and HRAM MS.
• Enhanced confidence in biosimilar development and quality control environments.

Future Trends and Applications

Advances in glycoanalytics are likely to focus on:

• Integration of automated sample preparation and data processing workflows.
• Higher-throughput MS platforms with real-time glycan identification.
• Machine learning–driven glycan pattern recognition for predictive analytics in biotherapeutic characterization.

Conclusion

This study established a comprehensive, integrated UHPLC-FLD and HRAM MS workflow for detailed glycosylation comparison between an innovator mAb and a biosimilar candidate. The method’s sensitivity and structural clarity support rigorous biosimilar assessment and ongoing quality monitoring.

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