Innovator and Biosimilar Monoclonal Antibody-Peptide Map Comparison Using the Agilent 7100 Capillary Electrophoresis System and Agilent MatchCompare Software

Importance of the Topic

Understanding the detailed peptide fingerprint of monoclonal antibodies is essential for ensuring the safety, efficacy, and regulatory compliance of both innovator and biosimilar products. Capillary electrophoresis (CE) peptide mapping offers high-resolution separation of complex digests and sensitive detection of post-translational modifications (PTMs) that can alter charge, structure, and biological activity.

Study Objectives and Overview

This work demonstrates a robust workflow to compare the peptide maps of innovator and biosimilar rituximab using the Agilent 7100 Capillary Electrophoresis system. The goal is to assess comparability through high-efficiency separation, reproducible performance, and automated data analysis with Agilent MatchCompare software.

Methodology and Instrumentation

Sample Preparation

• Denaturation in 6 M urea with 25 mM ammonium bicarbonate, pH 8.0
• Reduction using 10 mM DTT at 37 °C, 1 hour
• Alkylation with 40 mM iodoacetamide in the dark, 1 hour
• Quench with DTT, trypsin digestion (1 : 20 enzyme:protein) overnight at 37 °C
• Acidification with TFA and desalting via ZipTip C18

Instrumentation and CE Conditions

• Agilent 7100 CE system with bare fused-silica capillary (65 cm total, 56 cm effective, 50 µm id)
• Background electrolyte: 50 mM phosphate buffer pH 2.5 + 0.05 % hydroxyethyl cellulose
• Separation voltage: +25 kV at 25 °C
• Hydrodynamic injection: 25 s at 50 mbar
• UV detection: DAD at 200 nm/16 nm and 214 nm/4 nm bandwidth
• Data acquisition: Agilent OpenLAB CDS ChemStation
• Automated comparison: Agilent MatchCompare software

Key Results and Discussion

Approximately 60 peptide peaks were resolved between 10 and 35 minutes, demonstrating high separation efficiency. Overlay of innovator and biosimilar maps revealed variations in peak intensities and presence of unique peaks in the biosimilar, indicative of PTMs such as lysine truncation, oxidation, and deamidation. Migration time reproducibility showed RSD ≤ 2.1 % and peak area RSD ≤ 12.1 % for selected peaks. MatchCompare achieved a similarity score of 0.9782, highlighting minor but significant differences.

Benefits and Practical Applications

This CE-based peptide mapping approach offers

• High resolution of charged and low-molecular-weight peptides
• Low sample and solvent consumption
• Rapid and reproducible separations
• Automated, objective data comparison for batch-to-batch and biosimilar characterization

Future Trends and Applications

Emerging directions include coupling CE to mass spectrometry for structural identification of modified peptides, integrating high-throughput automation in quality control workflows, and expanding software capabilities for multidimensional data analysis. Advances in capillary coatings and microfluidics may further enhance separation performance and reduce runtime.

Conclusion

The Agilent 7100 CE system, combined with MatchCompare software, provides a robust platform for detailed peptide mapping of innovator and biosimilar monoclonal antibodies. The method achieves excellent reproducibility, high separation efficiency, and objective comparison metrics, supporting its adoption in biopharmaceutical comparability studies and quality control.

References

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3. OpenLAB CDS Match Compare for Chromatographic Peak Matching and Comparison Agilent Technologies Data Sheet 5991-2553EN 2013
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9. OpenLAB CDS Match Compare tech flyer Agilent Technologies Data Sheet 5991-6346EN 2015