LC-MS Analysis of Intact Lysine-Conjugated ADCs using the ACQUITY™ Premier UPLC™ and Xevo™ G3 QTof Mass Spectrometer

Significance of the Topic

Antibody–Drug Conjugates (ADCs) have emerged as a critical class of targeted therapeutics in oncology, combining the specificity of monoclonal antibodies with potent cytotoxic drugs. Accurate measurement of the drug-to-antibody ratio (DAR) is essential for ensuring batch-to-batch consistency, safety, and therapeutic efficacy.

Objectives and Study Overview

This application note illustrates the deployment of the ACQUITY Premier UPLC system coupled with the Xevo G3 QTof mass spectrometer to analyze intact lysine-conjugated ADCs. Using ado-trastuzumab emtansine (Kadcyla) as a model, the study aims to:

• Demonstrate high-resolution intact protein analysis of deglycosylated ADCs.
• Automate DAR calculation via the waters_connect UNIFI App.
• Evaluate detection of conjugation heterogeneity and impurities.

Methods and Instrumentation

Samples of trastuzumab and its ADC form were treated with PNGaseF to remove N-glycans. Intact reversed-phase LC-MS analysis employed:

• ACQUITY Premier UPLC with high-performance surfaces for consistent recovery.
• Xevo G3 QTof mass spectrometer for sensitive, high-resolution biomolecule detection.
• waters_connect UNIFI Intact Mass Workflow for data acquisition and processing.
• Intelligent Data Capture (IDC) for real-time noise reduction.
• MaxEnt1 deconvolution: m/z range 2200–3400; mass range 140–155 kDa; resolution 1 Da; 25 iterations; peak width FWHM 0.8 m/z; mass matching tolerance 50 ppm.

Main Results and Discussion

Mirror plots of raw spectra highlighted increased complexity in deglycosylated ADCs compared to unconjugated antibodies. MaxEnt1 deconvolution revealed a distribution of 0–9 linker-drug attachments and minor linker-only impurities. Automated DAR calculation yielded a value of 3.53, aligning with published data and demonstrating the workflow’s accuracy in profiling conjugation heterogeneity.

Benefits and Practical Applications

• Streamlined ADC analysis with automated DAR metrics reduces manual processing errors.
• High-performance UPLC surfaces enhance uptime and recovery.
• Benchtop QTof MS delivers robust detection of complex intact proteins.
• Compliance-ready informatics platform ensures traceable reporting.

Future Trends and Possibilities

• Integration of AI-driven data analytics for deeper structural insights.
• Advancement in site-specific conjugation techniques to reduce ADC heterogeneity.
• Real-time process monitoring to support bioprocess control.
• Extended workflows for subunit and peptide mapping to complement intact analysis.

Conclusion

The combined ACQUITY Premier UPLC–Xevo G3 QTof platform, with the UNIFI Intact Mass Workflow, offers a rapid, sensitive, and automated solution for intact ADC characterization. The approach enables reliable DAR determination and impurity profiling within a compliance-ready environment.

References

• Gogia P, Ashraf H, Bhasin S, Xu Y. Antibody-Drug Conjugates: A Review of Approved Drugs and Their Clinical Level of Evidence. Cancers (Basel). 2023;15(15):3886.
• Chen L, Wang L, Shion H, Yu C, Yu YQ, Zhu L, Li M, Chen W, Gao K. In-Depth Structural Characterization of Kadcyla (ado-trastuzumab emtansine) and Its Biosimilar Candidate. mAbs. 2016;8(7):1210–1223.
• Ippoliti S, Cornwell O, Reid L, Yu YQ, Harry E, Towers M. Comprehensive Biosimilar Comparability Assessment via Intact and Subunit RP-MS and IEX-UV-MS Using the Xevo G3 QTof System. Waters Appl Note. 2022;720007635.