Characterization of C-terminal and Disulfide Bond Peptides of Monoclonal Antibody (mAb) on Q-TOF Mass Spectrometer

Significance of the Topic

Monoclonal antibody therapeutics are critical for treating chronic diseases, and ensuring their structural integrity is essential for biosimilar quality control. Peptide mapping of C-terminal sequences and disulfide bond linkages provides insight into primary structure and post-translational modifications.

Objectives and Study Overview

This study aimed to characterize a bevacizumab biosimilar by mapping and sequencing its C-terminal and cysteine-containing peptides using a Shimadzu LCMS-9030 Q-TOF mass spectrometer. Key goals included detecting lysine variants, disulfide-linked peptides, and confirming sequence integrity.

Methods and Instrumentation

• Sample preparation: Reduction with dithiothreitol, alkylation with iodoacetamide, tryptic digestion at 37 °C, quenching with trifluoroacetic acid and centrifugation.
• LC conditions: Shim-pack GISS C18 column (250 × 4.6 mm, 5 μm), 0.1 % formic acid in water and acetonitrile, gradient from 0 % to 50 % B over 85 min, flow rate 0.3 mL/min, column temperature 40 °C.
• MS settings: Heated electrospray ionization in positive mode, MS scan range m/z 250–2500, MS/MS fragmentation, nitrogen nebulizing and drying gases.

Main Results and Discussion

• TIC and extract ion chromatograms enabled detection of fifteen cysteine-containing peptides and three C-terminal peptides with mass accuracy < 2 ppm.
• Heavy chain C-terminal peptides exhibited lysine truncation, with the clipped form dominating (> 99 %) over the lysine-retained variant.
• Carbamidomethyl modifications on cysteines were confirmed, and de novo sequencing of selected peptides showed comprehensive b- and y-ion coverage, validating sequence assignments.

Benefits and Practical Applications

The described workflow delivers a fast and reliable approach for monitoring critical quality attributes of mAb biosimilars. The EIC-based method with high mass accuracy simplifies peptide identification and supports routine QC environments.

Future Trends and Opportunities

Advancements may include automated data processing, deeper coverage of complex post-translational modifications, integration of ion mobility for conformational analysis, and machine-learning-driven de novo sequencing to further streamline biosimilar characterization.

Conclusion

The LCMS-9030 Q-TOF system effectively characterized C-terminal and disulfide bond-linked peptides of a bevacizumab biosimilar. The study confirms the instrument’s capability for accurate peptide mapping and sequence confirmation in biosimilar development.

Used Instrumentation

• Shimadzu LCMS-9030 Q-TOF mass spectrometer
• Shim-pack GISS C18 column, 5 μm, 250 × 4.6 mm
• Heated ESI interface and nitrogen gas supplies

References

• Dick LW Jr, Qiu D, Mahon D, Adamo M, Cheng K. C-terminal lysine variants in fully human monoclonal antibodies: Investigation of test methods and possible causes. Biotechnol Bioeng. 2008;100:1132-1143.
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• Shimadzu Application News AD-0176. Peptide Mapping of Monoclonal Antibody (mAb) Using Nexera Bio with Q-TOF Mass Spectrometer for Full Sequence Confirmation. 2019.