LC/MS/MS Peptide Mapping Comparison of Innovator and Biosimilars of Rituximab

Significance of the Topic

Monoclonal antibodies are a leading class of biotherapeutics whose patents are expiring, opening the way for biosimilars. Ensuring that biosimilars match innovator products in safety, efficacy, and purity demands detailed analytical comparability. LC/MS/MS peptide mapping is a critical tool for confirming primary sequence and monitoring post-translational modifications that can impact biological function.

Objectives and Study Overview

This study compares an innovator rituximab with two biosimilar versions using a unified peptide mapping workflow. Goals included achieving high sequence coverage, identifying differences in peptide abundances, and quantifying key modifications such as oxidation, deamidation, lysine truncation, and pyroglutamate formation.

Methodology

Each mAb sample underwent reduction, alkylation, and tryptic digestion using an automated Bravo liquid-handling system. Peptides were cleaned by C18 desalting and separated on an AdvanceBio Peptide Mapping column with a 30-minute gradient at 60 °C. Data-dependent acquisition employed iterative MS/MS to capture low-abundance species.

Used Instrumentation

• Agilent AssayMAP Bravo for automated sample preparation
• Agilent 1290 Infinity II LC System with high-speed pump and multisampler
• Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer in positive ESI mode
• Agilent MassHunter BioConfirm software for feature extraction, peptide matching, and PTM quantitation

Main Results and Discussion

Sequence coverage exceeded 97% for both heavy and light chains across all samples. Chromatograms revealed differential peptide levels and truncation forms. Quantitative highlights include:

• Met256 oxidation: 1.48% in innovator, 1.27% in biosimilar 1, and 2.50% in biosimilar 2.
• Asn388 deamidation: elevated in biosimilars (~20%) vs. innovator (10.9%).
• Lys451 truncation and N-terminal pyroGlu formation varied consistently among samples.

These results illustrate the method’s sensitivity to detect subtle structural variations.

Benefits and Practical Applications

The presented workflow offers:

• Comprehensive sequence validation and PTM profiling in a single run.
• Automated sample handling and data analysis for higher throughput.
• Quantitative histograms that facilitate batch-to-batch and product comparability.

Future Trends and Opportunities

Next-generation approaches may integrate ion mobility for isomer separation, machine-learning algorithms for spectral interpretation, and cloud-based reporting for regulatory submissions. Combining peptide mapping with multi-attribute methods could further streamline QC in biomanufacturing.

Conclusion

This work demonstrates that an Agilent LC/MS/MS peptide mapping workflow delivers high sequence coverage and precise PTM quantitation, making it well suited for analytical comparability of innovator and biosimilar monoclonal antibodies.

References

1. Ecker DM, Jones SD, Levine HL. The Therapeutic Monoclonal Antibody Market. MAbs. 7(1):9-14 (2015).
2. Agilent Technologies. Agilent MassHunter BioConfirm Software, publication 5991-8552EN.