Analytical Scale Native SEC-MS for Antibody-Drug Conjugates (ADCs) Characterization

Significance of the Topic

Antibody–drug conjugates (ADCs) represent a transformative modality in targeted therapy by combining the selectivity of monoclonal antibodies with potent cytotoxic payloads. Accurate determination of the drug-to-antibody ratio (DAR) and drug load distribution is critical for ensuring consistent efficacy, safety, and regulatory compliance in ADC development and manufacturing.

Objectives and Study Overview

This study presents an analytical-scale native SEC-MS method for characterizing both cysteine- and lysine-conjugated ADCs. The goals were to:

• Develop a robust, high-sensitivity workflow that preserves non-covalent complexes.
• Automate DAR and drug load distribution calculations within the UNIFI Scientific Information System.
• Compare results to hydrophobic interaction chromatography (HIC) and previous native SEC-MS approaches.

Methodology and Instrumentation

Samples of cysteine- and lysine-linked ADCs were stored at –80 °C, then thawed and diluted to 2 μg/μL in 50 mmol/L ammonium acetate. Analyses were performed on an ACQUITY UPLC H-Class Bio system with a Protein BEH SEC column (2.1 × 150 mm, 1.7 μm) under isocratic conditions (50 mmol/L ammonium acetate, 0.065 mL/min, 10 min). A tunable UV detector (280 nm) monitored elution, and eluted species were introduced to a Vion IMS QTof mass spectrometer operating in ESI+ mode (m/z 500–8,000) with Glu1-Fibrinopeptide B as lockmass. Data acquisition and automated intact-protein analysis workflows, including DAR calculations, were executed in UNIFI.

Main Results and Discussion

The smaller-ID SEC column provided over fivefold sensitivity improvement compared to previous 4.6 mm columns, enabling milder source conditions and enhanced ionization. The QuanTof 2-enabled Vion IMS QTof delivered improved raw-spectrum glycoform resolution, obviating the need for PNGase F deglycosylation in cysteine-linked ADCs. Average DARs and drug distribution profiles from native SEC-MS closely matched HIC results and earlier SEC-MS data across low, moderate, and high conjugation levels. For lysine-linked ADC (Kadcyla), native SEC-MS without deglycosylation yielded a DAR of 3.56 versus the reported 3.50.

Benefits and Practical Applications

This analytical-scale native SEC-MS method offers:

• Streamlined sample prep without deglycosylation.
• High throughput with a 10-minute isocratic run.
• Automated data processing for critical quality attributes.
• Enhanced sensitivity and retention reproducibility for batch-to-batch comparisons.

Future Trends and Potential Applications

Advances in ion mobility separation, higher-resolution MS, and expanded UNIFI workflows will further enhance in-depth ADC characterization. Prospective developments include site-specific conjugation profiling, multi-attribute monitoring of biotherapeutics, and application to emerging payloads and linkers in early-stage research and routine quality control.

Conclusion

The analytical-scale native SEC-MS platform described here delivers reliable, high-sensitivity DAR and drug load distribution measurements for cysteine- and lysine-conjugated ADCs. Its concordance with orthogonal HIC data and legacy methods, combined with simplified sample handling and automated processing, makes it well suited for routine ADC characterization and critical quality attribute assessment.

Instrumental Setup

• Vion IMS QTof Mass Spectrometer (Waters)
• ACQUITY UPLC H-Class Bio System (Waters)
• ACQUITY UPLC Tunable UV Detector (280 nm)
• Protein BEH SEC Column, 200 Å, 1.7 μm, 2.1 × 150 mm
• UNIFI Scientific Information System, version 1.8.2

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