Routine analysis of drug to antibody ratio and drug distribution with maXis II

Significance of the Topic

Antibody drug conjugates represent a frontier in targeted cancer therapy by coupling the high specificity of monoclonal antibodies with the potent cytotoxic effects of small molecule drugs. Precise characterization of intact mass and drug-to-antibody ratio is essential to control efficacy, stability and safety profiles of these complex biotherapeutics.

Objectives and Study Overview

This study focuses on the intact mass analysis and average drug-to-antibody ratio (DAR) determination for Kadcyla (ado-trastuzumab emtansine, T-DM1) using the Bruker maXis II ultrahigh resolution QTOF mass spectrometer. The goal is to validate experimental DAR values against published benchmarks and demonstrate workflow efficiency for routine ADC analysis.

Methodology and Instrumentation

• Sample Preparation: Kadcyla diluted to 1 mg/mL, with 1 µL injected per analysis.
• Liquid Chromatography: Bruker Elute UHPLC system equipped with a BEH 300 C4 column (2.1×100 mm) operated at 60 °C. A 12 min gradient from 5 % to 95 % mobile phase B (0.1 % acetonitrile) at 200 µL/min was applied.
• Mass Spectrometry: Bruker maXis II ETD in high-mass mode, resolution up to 80 000. Source conditions: 4500 V spray voltage, nebulizer gas 1.5 bar, dry gas 8 L/min at 200 °C, 120 eV ISCID.
• Data Processing: Automated deconvolution and DAR calculation performed in BioPharma Compass 2021 using maximum entropy algorithm over m/z 2500–3500 and Mr 140 000–180 000.

Main Results and Discussion

The deconvoluted mass spectrum revealed:

• Minor peak corresponding to unconjugated trastuzumab G0F/G0F glycoform.
• Series of conjugated ADC species with mass increments of ~958.5 Da, matching SMCC-DM1 linker additions.
• Average DAR values of 3.56 (main glycoform) and 3.54 (including major glycoforms plus free linker), in close agreement with literature.
• Clear resolution of DAR species from 0 to 8, enabled by high resolution and intra-scan dynamic range.

Benefits and Practical Applications

• Accelerated process development through rapid intact mass DAR assessment, serving as an orthogonal method to HIC.
• Reduction of analysis time to under 1 minute per dataset via automated BioPharma Compass workflows.
• Robust reference profile creation and batch-to-batch comparability for routine QC of mAb and ADC products.

Future Trends and Opportunities

• Integration of native mass spectrometry and size-exclusion chromatography-MS for comprehensive ADC characterization.
• Advancement of AI-driven deconvolution and predictive profiling to enhance routine release testing.
• Expansion to multi-linker chemotypes and higher DAR constructs with high-throughput platforms.
• Application in biosimilar comparability studies and pharmacokinetic-targeted profiling.

Conclusion

The combination of Bruker maXis II ultrahigh resolution QTOF-MS with BioPharma Compass delivers a precise, automated and rapid workflow for intact mass and DAR analysis of ADCs. Experimental DAR values for Kadcyla align with published data, affirming the method’s reliability for routine characterization.

Reference

• Rinnerthaler G et al (2019) International Journal of Molecular Sciences 20(5):1115
• Jones J et al (2020) mAbs 12(1):1682895