Complete characterization of KADCYLA using a new Orbitrap Excedion Pro BioPharma hybrid mass spectrometer with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation

Significance of the Topic

Antibody-drug conjugates (ADCs) represent a major advance in targeted cancer therapy. Comprehensive structural analysis of ADCs such as KADCYLA is critical to ensure defined drug-to-antibody ratio (DAR), confirm conjugation sites, detect post-translational modifications and glycoforms, and support quality control and regulatory compliance.

Objectives and Overview of the Study

This study demonstrates the capabilities of a new Orbitrap Excedion Pro BioPharma hybrid mass spectrometer with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation for in-depth characterization of the lysine-linked ADC KADCYLA. Key goals include accurate DAR determination, subunit and intact mass analysis, detailed peptide mapping, and localization of conjugation sites and structural variants.

Methodology and Used Instrumentation

The experimental workflow comprised:

• Native intact mass analysis: KADCYLA diluted to 10 mg/mL and analyzed by UHPLC coupled to native-mode MS to resolve DAR variants.
• Subunit analysis under denaturing conditions: IdeS digestion and DTT reduction followed by RPLC separation and intact MS at 240,000 resolution.
• Peptide mapping: Trypsin and AspN digests on denatured ADC, separated by reversed-phase UHPLC and fragmented using both HCD and EThcD for complementary information.

Major instrumentation:

• Thermo Scientific Vanquish Horizon UHPLC system
• Thermo Scientific MAbPac SEC-1 and MAbPac RP columns
• Thermo Scientific Orbitrap Excedion Pro BioPharma hybrid mass spectrometer
• BioPharma Finder and Proteome Discoverer software

Main Results and Discussion

Native MS resolved DAR species from 0 to 8 and yielded an average DAR of 3.47. Low-abundance high DAR species (DAR 8) were detected. Subunit analysis separated 20 distinct payload-bearing fragments. Peptide mapping achieved 100 percent sequence coverage with both enzymes. EThcD fragmentation provided abundant c/z and b/y ions, enabling confident localization of 43 out of 46 potential conjugation sites and confirmation of payload stereochemistry. Post-translational modifications including asparagine deamidation, oxidation, succinimidation and major N-glycoforms (dominant A2G0F at 51 percent) were identified and quantified. Aspartic acid isomerization was confirmed by characteristic c+57/z-57 ion pairs.

Benefits and Practical Applications of the Method

• High-resolution native MS for accurate DAR measurement and intact mass determination
• Denaturing subunit analysis for detailed heterogeneity profiling
• EThcD-enabled peptide mapping for site-specific conjugation and PTM localization
• Streamlined workflow supports biopharmaceutical development, quality control, and regulatory submissions

Future Trends and Possibilities of Use

Emerging directions include integration of advanced fragmentation strategies with multi-omics data, implementation of real-time ADC release testing, and application of machine learning for automated data interpretation. Ongoing enhancements in instrument speed, sensitivity and software algorithms will further support comprehensive characterization of next-generation biotherapeutics.

Conclusion

The Orbitrap Excedion Pro BioPharma hybrid mass spectrometer with EThcD fragmentation delivers robust, high-resolution analysis of complex ADCs, enabling precise DAR quantitation, detailed subunit and peptide-level insights, and confident localization of conjugation sites and modifications. This platform offers a powerful toolset for the biopharmaceutical industry to accelerate ADC development and ensure product quality.

Reference

1. Pharmaceuticals 2020, 13, 245
2. Analytical Chemistry 2010, 82(17), 7485–7491