Characterization of control and stress induced samples of trastuzumab biosimilar using LCMS-9030 by bottom-up approach

Significance of the Topic

Monoclonal antibodies (mAbs) represent a rapidly expanding class of biotherapeutics used in oncology, immunology and beyond.
During production, storage and handling, mAbs can undergo chemical stress leading to modifications such as oxidation and deamidation.
Early identification of these vulnerable sites is critical for ensuring product quality, stability and therapeutic efficacy.

Objectives and Study Overview

This work aimed to apply a bottom-up peptide mapping approach using the Shimadzu LCMS-9030 to characterize control and stress-induced samples of a trastuzumab biosimilar.
The study focused on identifying oxidation and deamidation sites under forced degradation conditions.
Key goals included achieving high sequence coverage, confident post-translational modification (PTM) localization and assessing the method’s mass accuracy and sensitivity.

Methodology and Instrumentation

Control and forced degradation samples were prepared by subjecting the biosimilar to alkaline pH (pH 9, 7 days at 37 °C) for deamidation and 30 % H2O2 (1 hour at 37 °C) for oxidation.
After quenching and reduction/alkylation, proteins were digested with trypsin and peptides cleaned via SPE cartridges.
The Nexera X2 UHPLC system fitted with a Shim-pack Arata Peptide C18 column (100 mm × 2.0 mm, 2.2 µm) was employed for separation using a water/acetonitrile gradient with 0.1 % formic acid.
Mass analysis was performed on the LCMS-9030 quadrupole time-of-flight MS in data-dependent acquisition mode.
Key acquisition settings included:

• MS1 survey scan range: 200–2500 m/z
• MS/MS scan range: 100–2800 m/z
• Collision energy spread: 18–52 V
• Seven dependent MS/MS events per cycle
• External TOF calibration for stable mass measurement

Data processing was conducted using Protein Metrics software with a 6 ppm precursor and 20 ppm fragment tolerance.
Variable modifications included oxidation, deamidation and glycan database searching for N-glycosylation.

Key Results and Discussion

The bottom-up workflow achieved over 92 % sequence coverage for both heavy and light chains with a single enzyme digestion.
Mass accuracy remained below 2 ppm across peptides ranging from 4 to 63 amino acids, demonstrating exceptional stability.
Oxidation analysis of the peptide DTLMISR revealed a +16 Da shift and characteristic sulfoxide fragmentation, confirming Met oxidation.
Deamidation of ASQDVNTAVAWYQQKPGK was distinguished from natural isotopic peaks by a +0.984 Da shift in MS1 and site assignment from MS/MS fragments.
Relative quantitation highlighted specific residues prone to modification under stress:

• HC-255 showing >77 % oxidation under H2O2 stress
• Multiple Asn/Gln sites exhibiting elevated deamidation at alkaline pH

These findings underscore the method’s capability to pinpoint critical quality attribute sites.

Benefits and Practical Applications

The described LCMS-9030 peptide mapping approach offers:

• High mass accuracy and sensitivity for low-abundance PTMs
• Comprehensive MS/MS fragmentation via collision energy spread
• Robust sequence coverage facilitating CQA monitoring

This workflow supports biosimilar development, stability testing, and multi-attribute method (MAM) implementation in QC environments.

Future Trends and Opportunities

Integration of multiple proteases can further improve coverage of short or difficult regions.
Advancements in real-time data processing and AI-driven PTM identification may accelerate characterization pipelines.
Extending this platform to intact protein and middle-down approaches could enrich structural insight into higher order modifications.

Conclusion

The bottom-up LCMS-9030 method delivers reliable, in-depth peptide mapping and PTM analysis for trastuzumab biosimilar samples under control and stress conditions.
Exceptional mass accuracy, sensitivity and fragmentation fidelity enable precise localization of oxidation and deamidation sites.
This strategy enhances understanding of antibody stability, guiding formulation optimization and regulatory compliance.

References

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2. Rogstad KA, et al. Rapid assessment of oxidation via middle-down LC-MS correlates with methionine side-chain solvent-accessible surface area. MAbs. 2017;9(4):646–653.
3. Kilian K. Data-Dependent Analysis Approach in LC/HRMS: Annotation of Natural Product Components. 04-JMST-208-EN.
4. Parks BC. Monoclonal Antibody Workflows on the Shimadzu Q-TOF LCMS-9030 Using the Protein Metrics Software Suite. SSI-LCMS-103.
5. Aslanian A, et al. Structural Elucidation of Post-Translational Modifications in Monoclonal Antibodies. ACS Symposium Series. 2015;1201:119–183.