IMPROVING CONFIDENCE AND PRODUCTIVITY FOR N-LINKED GLYCAN ANALYSIS IN BIOTHERAPEUTICS DEVELOPMENT USING AN INTEGRATED UPLC-FLR-HRMS SYSTEM

Significance of the Topic

The glycosylation pattern of biotherapeutic proteins critically affects their efficacy, safety and immunogenicity. Reliable monitoring of N-linked glycans is essential throughout drug development and manufacturing. Combining fluorescence detection with high-resolution mass spectrometry addresses the need for both sensitive quantitation and detailed structural characterization.

Objectives and Study Overview

This study introduces a streamlined workflow for released N-linked glycan analysis using an integrated UPLC-FLR-HRMS platform (BioAccord System). A head-to-head comparison of innovator Remicade™ and a biosimilar infliximab glycan profile demonstrates the system’s performance in a real-world biosimilarity assessment.

Methodology

The analytical procedure comprises:

• Automated sample preparation: released glycans from 15 µg of mAb are labeled using RapiFluor-MS and processed via an automated platform.
• Chromatographic separation: HILIC on a BEH Amide column with a 50 mM ammonium formate/water and acetonitrile gradient at 60 °C.
• Detection: fluorescence monitoring (λex = 265 nm, λem = 425 nm, 2 Hz) synchronized with high-resolution TOF MS data acquisition.
• Data processing: retention times converted to Glucose Units using a dextran ladder; library searches with ±10 ppm mass tolerance and ±0.2 GU tolerance for robust peak annotation.

Used Instrumentation

The BioAccord System components include:

• ACQUITY UPLC I-Class PLUS
• ACQUITY FLR detector (excitation 265 nm, emission 425 nm)
• ACQUITY RDa compact TOF MS with SmartMS™ auto-calibration and dual-energy fragmentation (70–90 V)
• Software: UNIFI 1.9.4 with Glycan FLR workflow and RFMS glycan GU library

Main Results and Discussion

The integrated workflow enabled:

• Automated glycan identification and reporting via clickable chromatograms and structure annotations.
• Clear detection of key glycans such as FA2G2 and its isomer FA2G1Ga1, supported by MS2 diagnostic ions (m/z 528 for α-1,3 galactose).
• Comparative profiling showing elevated FA2G1Sg1 in the biosimilar sample, highlighting subtle differences in sialylation patterns.
• Efficient review of overlaid FLR chromatograms and summary plots for rapid assessment of glycan identity and abundance.

Benefits and Practical Applications

The BioAccord System offers:

• Streamlined HILIC-FLR-HRMS workflow suitable for process development and QC laboratories.
• High confidence in glycan assignments through orthogonal FLR and MS confirmation.
• Quick turnaround for biosimilarity studies, enabling fast detection of glycan profile deviations.
• Customizable reporting for different regulatory and operational requirements.

Future Trends and Applications

Emerging developments may include:

• Deeper integration with automated QA/QC pipelines and laboratory information systems.
• Expansion of glycan libraries to cover rare and modified structures.
• Application of machine learning to refine peak annotation and reduce manual review.
• Miniaturized and multiplexed workflows for higher throughput and sample economy.

Conclusion

The introduction of a compact, integrated UPLC-FLR-TOF system significantly enhances productivity and analytical confidence in released N-linked glycan profiling. This workflow supports robust biosimilarity assessments and is readily deployable in biopharmaceutical development and manufacturing environments.

References

1. Alley W.R.J. and Yu Y.Q. Combining RapiFluor-MS and UNIFI Scientific Information System for a total N-linked glycan solution for innovator vs. biosimilar infliximab comparisons. Waters Application Note 720005753EN (2016).
2. Yu Y.Q. A holistic workflow for acquiring, processing, and reporting fluorescent-labeled glycans. Waters Application Note 720004619EN (2016).