PEPTIDE CHARACTERIZATION AND MONITORING WORKFLOW FOR BIOSIMILAR mAb DRUG PRODUCTS USING A COMPLIANCE READY LC-MS AND INFORMATICS PLATFORM

Significance of the Topic

Monoclonal antibody biosimilars require rigorous analytical characterization to ensure safety, efficacy and regulatory compliance.
Peptide mapping and multi-attribute method (MAM) enable detailed profiling of post-translational modifications and degradation products.
Such workflows support faster batch release and comparability studies between innovator and biosimilar products.

Objectives and Study Overview

This study presents a dual-phase workflow:

• Peptide map generation and attribute definition using a Xevo G3 QTof mass spectrometer and UNIFI App.
• Transition to MAM-based monitoring on a BioAccord LC-MS System with Peptide MAM App.

Four infliximab products (Remicade and three biosimilars) were evaluated under stress conditions to assess PTM profiles and new peak formation.

Methodology and Instrumentation

Sample Preparation:

• Infliximab samples incubated at 37 °C for 0, 1, and 2 weeks.
• Reduction, alkylation, desalting, trypsin digestion, acidification and dilution to 0.2 µg/µL.

LC-MS Analysis:

• Xevo G3 QTof with ACQUITY Premier Peptide CSH Column on ACQUITY Premier UPLC, 1–35% B over 50 minutes.
• Data-independent acquisition (MSE) for peptide mapping in UNIFI App.
• BioAccord LC-MS System and Peptide MAM App for targeted attribute monitoring and new peak detection using the same chromatographic conditions.

Instrumentation:

• Xevo G3 QTof mass spectrometer
• ACQUITY Premier UPLC System with CSH Peptide Column
• BioAccord LC-MS System
• UNIFI App and waters_connect Informatics Platform
• Peptide MAM App for attribute monitoring

Main Results and Discussion

The peptide mapping workflow achieved >95% sequence coverage and localized PTMs (oxidation, deamidation, glycosylation, lysine clipping).
Stress studies revealed consistent increases in deamidation over time, while oxidation trends varied across products.
Comparative analysis showed similar C-terminal lysine levels for Remicade, Inflectra, and Avsola, but lower levels in Renflexis.
New peak detection identified missed cleavages and low-abundance species, enabling user confirmation via extracted ion chromatograms and MS spectra.

Benefits and Practical Applications of the Method

• High-confidence, compliance-ready workflow for biosimilarity assessment.
• Seamless method transfer between instruments on a unified informatics platform.
• Quantitative monitoring of critical quality attributes across large sample sets.
• Simultaneous detection of known and novel product-related impurities.

Future Trends and Potential Applications

• Integration of higher-resolution instruments and extended dynamic range for broader attribute coverage.
• Automation of data analysis and AI-driven peak annotation to accelerate decision making.
• Expansion of MAM workflows to other biologics, including fusion proteins and bispecific antibodies.
• Cloud-based informatics for real-time monitoring in global manufacturing networks.

Conclusion

The combined Xevo G3 QTof peptide mapping and BioAccord MAM workflow delivers robust, high-throughput characterization and monitoring of mAb biosimilars.
This approach ensures regulatory compliance, enhances process control, and supports efficient biosimilar development and quality assessment.

References

• Rogers RS, Nightlinger NS, Livingston B et al. Development of a Quantitative Mass Spectrometry Multi-Attribute Method for Characterization, Quality Control Testing, and Disposition of Biologics. mAbs. 2015;7(5):881-890.
• Ranbaduge N, Yu YQ. A Streamlined Compliant Ready Workflow for Peptide-Based Multi-Attribute Method (MAM). Waters Application Note. 2020;720007094.
• DeLaney K, Ippoliti S, Reid L et al. Applying Peptide Mapping and Multi-Attribute Method (MAM) Workflow for Biosimilar mAb Drug Products Comparison on the Xevo G3 QTof Platform. Waters Application Note. 2022;720007632.