Characterization of Monoclonal Antibody Biosimilar through C-terminal and Disulfide Bond Peptides Sequencing Analysis on Q-TOF Mass Spectrometer

Significance of the Topic

The rapid expansion of biopharmaceuticals underscores the need to verify primary structure fidelity in biosimilars. Characterizing C-terminal variants and disulfide bond peptides is essential for quality assessment and ensuring clinical performance.

Objectives and Study Overview

• Characterize C-terminal peptides with and without lysine truncation in a bevacizumab biosimilar.
• Detect and sequence cysteine-containing peptides released from disulfide bonds.
• Validate an EIC-based rapid screening approach with mass accuracy below 2 ppm.

Methodology and Instrumentation

• Sample preparation: Reduction with DTT, alkylation with IAM and tryptic digestion of bevacizumab in Tris buffer.
• Liquid chromatography: Shim-pack GISS C18 column (250×4.6 mm, 5 µm), 0.3 mL/min flow, gradient elution with 0.1% formic acid in water (A) and acetonitrile (B).
• Mass spectrometry: LCMS-9030 Q-TOF with heated ESI (300 °C), MS/MS via CID with argon (230 kPa), mass accuracy <2 ppm.

Results and Discussion

• TIC analysis revealed distinct peaks for tryptic peptides, enabling extraction of C-terminal and cysteine-linked ions.
• Three C-terminal peptides and fifteen cysteine-containing peptides were detected and confirmed using extracted ion chromatograms with <2 ppm error.
• Quantitative data showed predominant removal of C-terminal lysine from the heavy chain.
• MS/MS de novo sequencing provided complete y-ion series for key peptides such as SLSLSPG and VYACEVTHQGLSSPVTK, including carbamidomethyl modifications.

Benefits and Practical Applications

• The workflow delivers a fast and reliable method for monitoring C-terminal variants and disulfide mapping in mAb biosimilars.
• High mass accuracy and EIC-based screening streamline quality control in biopharmaceutical production.
• The approach supports regulatory compliance and can be adopted for routine batch release testing.

Future Trends and Possibilities

• Automation of data processing and high-throughput LC-MS workflows for comprehensive mAb characterization.
• Use of advanced fragmentation techniques and higher-resolution mass analyzers for deeper post-translational modification analysis.
• Extension of the methodology to other biotherapeutic formats, including Fc-fusion proteins and antibody–drug conjugates.

Conclusion

The combination of targeted tryptic digestion, precise LC-Q-TOF analysis and EIC-based peptide identification offers an efficient, accurate platform for characterizing C-terminal and disulfide bond peptides in bevacizumab biosimilars, reinforcing critical quality attribute assessment.

References

• L.W. Dick Jr, D. Qiu, D. Mahon, M. Adamo, K. Cheng. C-terminal lysine variants in fully human monoclonal antibodies: Investigation of test methods and possible causes. Biotechnology and Bioengineering, 100:1132–1143 (2008).