WADC: High Resolution Mass Spectrometry of Antibody Drug Conjugates Using the Orbitrap Mass Analyzer

Significance of the Topic

The characterization of antibody–drug conjugates (ADCs) is critical for the development and quality control of biotherapeutic agents.
High complexity, heterogeneity of glycoforms and drug loads demand high-resolution mass spectrometry to ensure accurate determination of drug-to-antibody ratios (DAR) and structural integrity.
The Orbitrap platform with BioPharma workflows offers unified denaturing and native analysis to capture both subunit- and intact-molecule information.

Objectives and Study Overview

This study aimed to demonstrate a comprehensive workflow for intact ADC analysis using Orbitrap Q Exactive Plus and Q Exactive HF instruments equipped with the BioPharma Option.
Two clinically relevant ADCs were selected: Brentuximab vedotin and Trastuzumab emtansine.
The goals included:

• Evaluating three operating modes (Normal, Protein, High Mass Range) for intact mass detection up to m/z 8000.
• Comparing denaturing reversed-phase LC-MS and native SEC-MS for ADC structural analysis.
• Determining DAR values using ReSpect deconvolution and the Sliding Window feature in BioPharma Finder software.

Methodology and Instrumentation

Chromatography and Sample Preparation:

• Denaturing LC: MAbPac RP column, 10 min gradient of 10–90% ACN with 0.1% FA at 250 µL/min.
• Native LC: Size‐exclusion SEC column, isocratic 50 mM ammonium acetate at 300 µL/min.
• Intact samples prepared by dissolving dried ADCs according to manufacturer protocols.

Mass Spectrometry:

• Instruments: Thermo Scientific Q Exactive Plus and Q Exactive HF with BioPharma Option.
• Operating Modes: Normal (m/z ≤ 6000), Protein (enhanced resolution, cooling in HCD cell), HMR (m/z ≤ 8000).
• Source: Electrospray ionization; S-Lens RF increased to 200 for HMR.

Data Analysis:

• Software: BioPharma Finder 2.0 with ReSpect deconvolution and Sliding Window integration.
• Database: ADC-specific FASTA entries; mass tolerance 50 ppm; static Gln→Pyro-Glu modification.

Results and Discussion

Denaturing RP‐LC-MS of Brentuximab vedotin revealed nine distinct mass species corresponding to DAR 0–8 glycoforms.
ReSpect deconvolution and Sliding Window integration yielded an average DAR of 4.07, matching published values.
Native SEC-MS produced lower charge state envelopes and improved mass separation, facilitating detection of intact ADC variants under near-physiological conditions.

Similarly, Trastuzumab emtansine analysis under both denaturing and native conditions provided consistent deconvolution results.
The average DAR of 3.71 obtained by native intact MS aligned with expected values, confirming method robustness.

Comparison of operating modes:

• Protein Mode enhanced signal for subunit ions compared to Normal Mode.
• HMR Mode extended detection to m/z 8000, essential for large intact ADC species.

Benefits and Practical Applications

Unified platform enables both denaturing and native intact MS in a single instrument.
High mass range detection and advanced deconvolution workflows allow:

• Accurate DAR determination for quality control.
• Glycoform profiling and low‐abundance species detection.
• Preservation of non-covalent interactions to study ADC structural integrity.

Future Trends and Potential Applications

Integration of ion mobility separations to enhance conformational analysis.
Automation of multi-attribute method workflows for high-throughput QC.
Extended software capabilities for real-time deconvolution and reporting.
Coupling online SEC-MS with peptide mapping for comprehensive ADC characterization.
Expanding native MS to study higher-order assemblies and ADC stability in formulation studies.

Conclusion

The Orbitrap BioPharma Option effectively extends intact mass analysis capabilities up to m/z 8000, enabling complete characterization of ADCs under both denaturing and native conditions.
ReSpect deconvolution and Sliding Window integration deliver reliable DAR values consistent with literature.
This unified workflow supports robust structural and quantitative assessment of complex biotherapeutics.

References

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• [4] Dabaene F. et al. Innovative native MS methodologies for antibody drug conjugate characterization: High resolution native MS and IM-MS for average DAR and DAR distribution assessment. Anal Chem. 2014;86(21):10674–10683.
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