Comprehensive Characterization of Cysteine-conjugated Antibody Drug Conjugate (ADC) on a Hybrid Quadrupole-Orbitrap Mass Spectrometer

Significance of the Topic

Antibody–drug conjugates (ADCs) represent a rapidly growing class of targeted biotherapeutics for cancer treatment. Cysteine-linked ADCs are particularly attractive due to controlled conjugation chemistry and well-defined drug-to-antibody ratios (DAR). Comprehensive analytical characterization of these bioconjugates is essential to ensure product quality, safety and efficacy during development and manufacturing.

Objectives and Study Overview

This study aimed to perform an in-depth characterization of a cysteine-fluorophore ADC standard (Sigma ADC Mimic) using a hybrid quadrupole-Orbitrap mass spectrometer. Key goals included determination of intact mass profiles under native and denatured conditions, measurement of DAR distribution, identification of conjugation sites and glycoform analysis, and detailed peptide mapping to confirm sequence coverage and drug-linker attachment patterns.

Methodology and Instrumentation

Sample Preparation:

• Reduction of interchain disulfides with DTT, alkylation with iodoacetic acid.
• Tryptic digestion for peptide mapping; dilution to 1 mg/mL for intact mass analysis.

Chromatography and MS Conditions:

• Native intact mass: size exclusion LC (MAbPac SEC-1, 5 µm, 2.1 × 150 mm) in 50 mM ammonium acetate.
• Denatured intact mass: reversed-phase LC (MAbPac RP, 4 µm, 2.1 × 100 mm) with formic acid/acetonitrile gradient.
• Peptide mapping: Acclaim Vanquish C18 column (2.2 µm, 2.1 × 150 mm), 0.1% formic acid in water/acetonitrile.
• MS detection: Thermo Scientific™ Orbitrap Exploris™ 240 with BioPharma Option, resolution up to 60 000; acquisition of MS1 and HCD-MS2 spectra.

Main Results and Discussion

Denatured Intact Mass Analysis:

• Detection of multiple ADC species carrying 0–8 SMCC linker-drug attachments.
• High-accuracy deconvoluted masses (sub-5 ppm) enabled quantification of each DAR species.

Native Intact Mass Analysis:

• Size exclusion LC-MS revealed a narrower DAR distribution, with an average DAR calculated by software (~3.45).

Peptide Mapping:

• Sequence coverage exceeded 93%, confirming primary structure integrity.
• Hinge peptide THTCPPCPAPELLGGPSVFLFPPKPK exhibited four distinct conjugated forms (linker-only, drug-linker, carboxymethylated, combinations).
• Diagnostic MS2 ions from drug fragmentation and peptide b/y series facilitated unambiguous site assignment.
• Most expected conjugated peptides were identified; two small carboxymethylated hinge fragments were not detected, likely due to low MS2 trigger efficiency.

Benefits and Practical Applications

The combined native and denatured intact mass analyses, together with detailed peptide mapping on an Orbitrap Exploris 240 platform, enable comprehensive assessment of critical quality attributes (CQAs) for cysteine-linked ADCs. This approach supports:

• Reliable DAR measurement and distribution profiling.
• Accurate glycoform quantitation and mass assignment.
• Precise localization of conjugation sites and occupancy levels.
• High-confidence peptide identification and post-translational modification mapping.

Future Trends and Possibilities

Emerging trends in ADC characterization include integration of top-down proteomics, ion mobility separation to resolve isobaric species, and application of machine learning for automated spectral interpretation. Advances in higher-resolution MS, faster acquisition speeds and improved bioinformatics tools will further enhance throughput and depth of characterization for next-generation ADCs.

Conclusion

This work demonstrates that a hybrid quadrupole-Orbitrap mass spectrometer coupled with tailored LC methods provides robust, high-resolution intact mass and peptide-level analyses of cysteine-conjugated ADCs. The workflow delivers detailed insights into DAR distribution, glycoform composition and conjugation site mapping, reinforcing its value for ADC development, quality control and regulatory compliance.

Used Instrumentation

• Thermo Scientific™ Orbitrap Exploris™ 240 mass spectrometer (BioPharma Option).
• Thermo Scientific™ Vanquish™ UHPLC system.
• MAbPac™ SEC-1 and MAbPac™ RP columns.
• Acclaim™ Vanquish™ C18 column.