Similis Bio - Streamlined mAb Subunit LC- MS Workflow for Multiple Attribute Monitoring of Biosimilar mAb Candidates During Bioprocessing and Development

Significance of the Topic

The ability to monitor multiple quality attributes of monoclonal antibodies at the subunit level is essential for efficient biosimilar development and process control. Rapid, automated workflows reduce analytical bottlenecks, enabling earlier clone selection and iterative process optimization. This approach supports critical assessments of glycosylation, charge variants, and other post-translational modifications that directly affect therapeutic performance.

Objectives and Study Overview

This study demonstrates an integrated at-line platform combining automated Protein A purification, targeted enzymatic digestion, and subunit LC-MS analysis to screen biosimilar mAb candidates against a reference product. The aim was to assess sensitivity and precision for key attributes including N-linked glycan profiles, unprocessed C-terminal lysine variants, glycation, and oxidation while ensuring comparability to established orthogonal assays.

Methodology

Cell culture samples from high-throughput bioreactors underwent automated Protein A capture followed by FabRICATOR digestion with partial reduction. Liberated subunits (Fc, light chain, Fdprime) were separated via reversed-phase chromatography and analyzed by time-of-flight mass spectrometry. Data acquisition and processing were fully streamlined using an integrated informatics platform, providing real-time deconvolution and quantitation.

Used Instrumentation

• Andrew+ Pipetting Robot with Protein A and Heater/Shaker modules for automated sample preparation
• BioAccord LC-MS System comprising UPLC, tunable UV detector, and high-mass time-of-flight analyzer
• Waters Connect Intact Mass Application for acquisition, processing, and visualization

Main Results and Discussion

Deconvoluted spectra enabled clear resolution of Fc subunit glycoforms and C-terminal lysine variants across innovator, biosimilar, and negative-control samples. Quantitation of high-mannose (Man5) and afucosylated species matched within 2 percent of values from a released glycan assay. Additional attributes such as light chain and Fdprime glycation and oxidation were determined in a single analysis. Cross-site reproducibility tests confirmed consistent results with manual or automated preparation workflows.

Benefits and Practical Applications

• High throughput with 15-minute analysis per sample
• Comprehensive multi-attribute monitoring in one assay
• Automated sample handling reduces hands-on time and variability
• Compliance-ready data management for QC environments
• Capability to localize modifications to Fc or Fab regions

Future Trends and Potential Applications

Further integration with advanced informatics and machine learning will enhance predictive process control. Adaptation to other therapeutic modalities such as fusion proteins or antibody fragments is feasible. Expansion into real-time release testing and continuous biomanufacturing workflows will drive next-generation quality assurance strategies.

Conclusion

The presented automated subunit LC-MS workflow delivers rapid, precise, and reproducible monitoring of critical mAb quality attributes. It offers a scalable solution for biosimilar screening, process development, and quality control, reducing reliance on multiple orthogonal assays and enabling faster decision making.

References

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