Disulfide Bond Characterization of Monoclonal Antibody (mAb) Using Q-TOF Mass Spectrometer

Significance of the Topic

Monoclonal antibodies represent a rapidly expanding class of biotherapeutics and diagnostics. The correct formation and location of disulfide bonds are critical quality attributes that influence mAb stability, folding, biological activity and immunogenicity.

Objectives and Study Overview

This study aimed to develop and validate a liquid chromatography–mass spectrometry (LC–MS) method for detailed characterization of disulfide bond linkages in a bevacizumab biosimilar. A comparative analysis under non-reduced and reduced conditions was performed to identify intact disulfide-linked peptides and free cysteine-containing peptides.

Methodology

Sample preparation involved denaturation of 5 mg/mL bevacizumab biosimilar with ProteaseMAX™ surfactant, followed by tryptic digestion both with and without reduction (DTT) and alkylation (IAM). Peptides were separated on a Shim-pack™ GISS-HP column using a gradient of water and acetonitrile (both containing 0.1% formic acid and 0.01% TFA) at 0.5 mL/min. Mass analysis was carried out in positive electrospray ionization mode with MS and data-dependent MS/MS scanning over m/z 100–2000.

Applied Instrumentation

• Liquid Chromatograph: Shimadzu Nexera series
• Column: Shim-pack GISS-HP, 3 µm, 150 × 3.0 mm
• Mass Spectrometer: Shimadzu LCMS-9030 Q-TOF
• ESI Interface: Heated ESI, 4.5 kV; capillary 250 °C; heat block 400 °C
• Gas settings: Nebulizing N₂ 3 L/min; Drying N₂ 10 L/min; Heating zero air 10 L/min

Main Results and Discussion

• Structure: Bevacizumab contains 16 disulfide bonds (12 intra-chain, 4 inter-chain).
• Detection: Under non-reduced conditions, eight intact S–S peptides were observed. After reduction, 15 free-cysteine peptides were detected with high mass accuracy (<3 ppm).
• Chromatography: Total ion chromatograms displayed distinct peaks for disulfide-linked species in non-reduced vs. reduced samples.
• Sequencing: De novo MS/MS analysis of representative peptides (e.g., non-reduced S-S-2 and reduced S-3) confirmed disulfide connectivity with annotated fragment ions.

Benefits and Practical Applications

The presented LC-MS workflow enables precise mapping of mAb disulfide bonds, supporting biosimilar comparability, structural verification, and quality control. Its robustness and sensitivity make it suitable for routine analytical laboratories in biopharmaceutical development.

Future Trends and Potential Applications

• Integration of higher-throughput LC-MS platforms and automated data analysis pipelines.
• Application to increasingly complex antibody formats (e.g., bispecifics, ADCs).
• Enhanced fragmentation techniques (e.g., ETD, UVPD) for deeper structural insights.
• Combining disulfide mapping with glycosylation and post-translational modification analyses.

Conclusion

A straightforward and reliable LCMS-9030 Q-TOF method was developed for detailed disulfide bond characterization in a bevacizumab biosimilar. The approach demonstrated high confidence in peptide identification and accurate connectivity mapping, establishing its value for mAb structural analysis.

References

No literature references were provided in the source document.