Increasing Productivity and Confidence for N-linked Glycan Analysis of Biosimilars Using the BioAccord System

Significance of the Topic

Glycosylation is a critical quality attribute that influences the safety, efficacy, and stability of monoclonal antibody therapeutics. Detailed profiling of N-linked glycans is essential for biosimilar development to ensure high structural similarity to reference products and to satisfy regulatory requirements.

Objectives and Study Overview

This study evaluates the BioAccord System—a turnkey LC-FLR-MS platform with SmartMS technology—for released N-glycan analysis of innovator and biosimilar infliximab. It demonstrates an end-to-end workflow from sample preparation to data reporting, aiming to enhance throughput, accuracy, and confidence in biosimilarity assessments.

Instrumentation Used

• ACQUITY UPLC I-Class PLUS System for high-performance HILIC separation
• ACQUITY FLR Detector (λex = 265 nm, λem = 425 nm) for fluorescence detection
• ACQUITY RDa Detector with built-in self-calibration for time-of-flight MS
• UNIFI Scientific Information Software 1.9.4 with Glycan Application Solution for automated data processing
• GlycoWorks RapiFluor-MS N-Glycan Kit for rapid glycan release and labeling

Methodology

Infliximab samples from both innovator and biosimilar sources were diluted and subjected to enzymatic release of N-glycans, followed by RapiFluor-MS labeling. Automated liquid handling ensured reproducible sample preparation. Glycans were separated on an ACQUITY Glycan BEH Amide column at 60 °C using a gradient of water with 50 mM ammonium formate (pH 4.4) and acetonitrile. Inline FLR detection and ESI+ MS (50–2000 m/z) acquisition at low and high collision energies enabled simultaneous mass confirmation and structural fragmentation without precursor selection.

Key Results and Discussion

Automated retention time calibration against a dextran ladder converted peaks to Glucose Units (GU), facilitating high-confidence library matching within a 10 ppm mass tolerance. A total of 26 glycans were identified in innovator infliximab and 27 in the biosimilar, confirming high sensitivity. Overlaid chromatograms revealed subtle differences in sialylated glycan abundances. MS fragmentation data enabled differentiation of isobaric species, such as FA2G2 versus its α-Gal epimer FA2G1Ga1, using a diagnostic m/z 528 fragment. Customizable UNIFI workflows streamlined data review, report generation, and direct comparison of glycan profiles.

Applications and Practical Benefits

• Comprehensive, compliance-ready solution for N-glycan profiling in biosimilar R&D and QA/QC
• Automated workflows reduce dependence on expert operators and minimize manual data handling
• Robust self-calibration and informatics streamline method deployment and scaling
• High throughput and accurate mass confirmation enhance laboratory productivity and confidence in results

Future Trends and Potential Applications

Integration of AI-driven data analysis may further accelerate glycan annotation and similarity assessments. Expanding the platform to additional biotherapeutic modalities, such as ADCs or fusion proteins, will enhance its utility. Advances in miniaturized LC-MS and microfluidic sample prep may enable even higher throughput and reduced reagent consumption.

Conclusion

The BioAccord System offers a unified, automated LC-FLR-MS workflow for detailed released glycan analysis. Its robust instrumentation, streamlined sample prep, and integrated informatics deliver rapid, reliable results for biosimilar comparability, reducing time and resource demands without sacrificing data quality.

References

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