Analysis of Antibody-Drug Conjugates Using Size Exclusion Chromatography and Mass Spectrometry

Significance of the Topic

Antibody–drug conjugates (ADCs) combine the targeting specificity of monoclonal antibodies with the potency of cytotoxic payloads, offering precise delivery to tumor cells while minimizing damage to healthy tissues. Accurate characterization of ADCs, including drug-to-antibody ratio (DAR) and heterogeneity assessment, is essential for ensuring safety, efficacy, and regulatory compliance in biopharmaceutical development.

Objectives and Study Overview

This application note presents a streamlined workflow for analyzing trastuzumab and its ADC form, trastuzumab emtansine (T-DM1), using size exclusion chromatography coupled online with quadrupole time-of-flight mass spectrometry (SEC-MS). The goals are to demonstrate minimal method optimization, effective desalting with MS-compatible buffers, and reliable DAR determination.

Methodology and Instrumentation

• Sample Preparation: Both mAb and ADC samples were deglycosylated with N-glycanase in Tris buffer at 37 °C overnight to reduce spectral complexity.
• Mobile Phase: A volatile mixture of 50 % acetonitrile, 49 % water, and 1 % formic acid preserved native protein structure and ensured MS compatibility.
• Chromatography: Separation on a Bio SEC-3 column (7.8 × 300 mm, 3 µm) at 30 °C, 0.8 mL/min flow rate, 25 µL injection volume.
• Detection: Agilent 1290 Infinity II LC system coupled to Agilent 6530 Accurate-Mass Q-TOF MS; diode array detection for UV profiling; MassHunter Workstation with Bioconfirm and DAR Calculator modules for deconvolution and quantitation.

Main Results and Discussion

• The MS-friendly buffer produced sharp SEC peaks and excellent signal-to-noise without extensive method development, compared to nonvolatile PBS.
• Intact trastuzumab displayed multiple glycoform peaks around 145 kDa; deglycosylation simplified the spectrum, clarifying mass assignments.
• Deglycosylated T-DM1 revealed eight distinct species corresponding to DAR values from 0 to 7, with mass increments matching the combined mass of the DM1 payload and linker (~959.7 Da).
• The average DAR of 3.2, calculated via the Agilent DAR Calculator, matched literature values, confirming method accuracy.

Benefits and Practical Applications

• Rapid, robust profiling of monoclonal antibodies and ADCs without high temperatures or nonvolatile salts.
• Precise DAR determination supports quality control, batch release, and comparability studies.
• Easy integration into existing LC/MS platforms and software accelerates implementation in R&D and QC laboratories.

Future Trends and Opportunities

• Integration of ion mobility spectrometry for deeper structural and conformational analysis of ADC species.
• Automation and high-throughput workflows to support accelerated ADC screening and optimization.
• Extension to novel linker chemistries and payload classes, broadening ADC design space.
• Inline process monitoring during manufacturing for real-time quality assurance.

Conclusion

Size exclusion chromatography coupled with MS using a volatile mobile phase delivers a straightforward and reliable approach for characterizing monoclonal antibodies and ADCs. Deglycosylation enhances spectral clarity, enabling precise DAR measurement and supporting effective quality control strategies in biotherapeutic development.

Instrumentation Used

• Agilent 1290 Infinity II LC system (binary pump, multisampler, column compartment, diode array detector).
• Agilent 6530 Accurate-Mass Q-TOF LC/MS.
• Agilent 1260 Bio-Inert Infinity Quaternary LC for comparison with PBS buffer runs.
• MassHunter Workstation Software with Bioconfirm and DAR Calculator.

References

1. Rohrer T. Antibody drug conjugates: potent weapons for the oncology arsenal. Chemistry Today. 2009;27(5):56–60.
2. Panowski S. Site-specific antibody drug conjugates for cancer therapy. mAbs. 2014;6(1):34–45.
3. Krop I et al. Phase I study of Trastuzumab-DM1 in HER2-positive metastatic breast cancer. ASCO 2010;28(16):2689–2704.
4. Wakankar A et al. Analytical methods for physiochemical characterization of antibody drug conjugates. mAbs. 2011;3(2):161–172.
5. Marcoux J. Native mass spectrometry and ion mobility characterization of trastuzumab emtansine. Protein Science. 2015;24:1210–1223.
6. Lazar AC et al. Analysis of immunoconjugate composition using SEC-MS. Rapid Commun Mass Spectrom. 2005;19:1806–1814.
7. Kim MT et al. Statistical modeling of drug load distribution on trastuzumab emtansine. Bioconjugate Chemistry. 2014;25(6):1223–1232.