Peptide Mapping of Innovator and Biosimilar Monoclonal Antibody Using an Agilent 1290 Infinity UHPLC Coupled to an Agilent 6550 iFunnel Q-TOF LC/MS System

Importance of Topic

Monoclonal antibodies (mAbs) like Rituximab are vital therapeutics in oncology and autoimmune disorders. Comparing innovator and biosimilar mAbs at the amino acid level ensures safety, efficacy, and regulatory compliance. Peptide mapping via LC/MS is essential to confirm sequence identity and characterize post-translational modifications (PTMs) that impact product performance.

Objectives and Study Overview

This study aims to evaluate molecular similarity between innovator and biosimilar Rituximab by performing peptide mapping. Trypsin digestion followed by UHPLC-QTOF MS analysis was conducted to compare sequence coverage, oxidation, and deamidation patterns in both antibody versions.

Methods and Instrumentation

The antibodies were reduced, alkylated, and digested with trypsin under denaturing conditions. Peptides were separated on an Agilent AdvanceBio Peptide Mapping column using a segmented gradient of formic acid in water and acetonitrile. Data were acquired in positive ion mode with MS and MS/MS scans collected for peptide identification and PTM analysis.

Used Instrumentation

• Agilent 1290 Infinity Binary Pump
• Agilent 1290 Infinity Autosampler
• Agilent 1290 Infinity Thermostat Column Compartment
• Agilent 6550 iFunnel Q-TOF LC/MS System with JetStream source

Main Results and Discussion

Both innovator and biosimilar achieved >95% sequence coverage of the heavy chain and 100% for the light chain. Mirror plots of total ion chromatograms revealed minor differences in specific peptide abundances, such as the C-terminal peptide (SLSLSPGK). Oxidation at methionine in the DLTMISR peptide was higher in the biosimilar. Deamidation at Asn388 in the GFYPSDIAVEWESNGQPENNYK peptide was similar (~8%) in both products. PTMs were confirmed by MS/MS fragment ion shifts matching theoretical mass changes.

Benefits and Practical Applications

This workflow offers high resolution, accuracy, and sensitivity for detailed comparability studies. Automated data extraction and BioConfirm software streamline identification of sequence variants and PTMs, facilitating quality control and regulatory submissions for biosimilar development.

Future Trends and Potential Applications

Advancements in high-resolution MS and data analysis software will enable deeper characterization of low-abundance variants. Integration of intact mass analysis and multi-enzymatic digestions may improve PTM mapping. Real-time data interpretation with AI-driven algorithms could accelerate biosimilar comparability assessments.

Conclusion

Agilent’s UHPLC-QTOF MS platform coupled with peptide mapping provides a robust approach to assess molecular similarity between innovator and biosimilar mAbs. The methodology achieves comprehensive sequence coverage and reliable PTM analysis, supporting biosimilar development and regulatory compliance.

Reference

• FDA. Biosimilars: FDA-Approved Products. 2017.
• Agilent Technologies. Application Note 5991-5557EN.
• Agilent Technologies. Application Note 5990-8769EN.
• Chelius D, Rehder DS, Bondarenko PV. Anal Chem. 2005, 77, 6004–6011.