Method Development for Separating Charge Variants of Antibody-Drug Conjugates by Ion- Exchange Chromatography

Importance of the Topic

Monoclonal antibodies and antibody–drug conjugates (ADCs) exhibit structural heterogeneity that can generate charge variants affecting product stability and efficacy.
Quality control of these biotherapeutics requires robust separation and detection of charge variants to ensure consistency, safety, and regulatory compliance.

Objectives and Study Overview

This application note demonstrates an efficient workflow for developing an ion‐exchange chromatography method to separate charge variants of trastuzumab deruxtecan (T-DXd) using pH gradient conditions.
The study leverages LabSolutions MD software to automate method development steps and identify optimal mobile phase composition and organic solvent ratios.

Methodology and Instrumentation

Screening design:

• Analyte: T-DXd at 5 mg/mL in ultrapure water.
• Mobile phase salt concentration: MES-HEPES-sodium acetate from 10 to 100 mmol/L at pH 5.0 and pH 10.0.
• Organic modifier ratio: 0, 5, 10, 15 % acetonitrile or methanol.
• Gradient: 2 % to 100 % pH 10.0 buffer over 10 min, hold, then return.

Automated workflow with LabSolutions MD:

1. Define mobile phase components and concentration levels.
2. Select column and set temperature.
3. Enter sample details.
4. Specify gradient profile, flow rate (1.0 mL/min), and detection at 280 nm.
5. Generate analysis schedule for all combinations.

Instrumentation Used

• Chromatography system: Nexera lite inert method scouting system.
• Column: Shim-pack Bio IEX SP-NP (50 × 4.6 mm I.D., 5 µm).
• Detector: UV at 280 nm using SPD-40 UHPLC inert cell.
• Software: LabSolutions MD for automated method development and peak evaluation.

Main Results and Discussion

Salt concentration effects:
Screening chromatograms revealed that a mobile phase salt concentration of 30 mmol/L provided the highest resolution and number of charge variant peaks.
Organic modifier effects:
Varying acetonitrile and methanol ratios showed 5 % acetonitrile yielded the highest peak‐to‐valley ratios without baseline disturbances, while higher organic content induced ADC denaturation.
Quantitative ranking:
LabSolutions MD computed an evaluation value (number of peaks × sum of resolutions) to rank conditions, streamlining selection of the optimal method.

Benefits and Practical Applications

• Significantly reduces manual workload and human error in mobile phase preparation and schedule creation.
• Enables rapid generation and evaluation of large data sets for method optimization.
• Provides objective, quantitative criteria for selecting separation conditions.

Future Trends and Applications

Integration of machine learning algorithms to predict optimal conditions from historical data.
Extension of automated method development to other therapeutic proteins and complex biologics.
Enhanced remote and cloud‐based workflows for global QC laboratories.

Conclusion

This study demonstrates that automated method development with LabSolutions MD and pH gradient ion‐exchange chromatography can efficiently identify optimal conditions for separating ADC charge variants.
The combination of systematic screening, quantitative evaluation, and automated workflows streamlines biopharmaceutical method development and supports robust quality control.

Reference

• Application News No. 01-00259-EN: Analyses of Antibody Drugs Using Ultra High Performance Liquid Chromatography.
• Application News No. 01-00473A-EN: Efficient Method Development of Monoclonal Antibody Size Variants by Size Exclusion Chromatography.
• Application News No. 01-00986-EN: Efficient Method Development for Separation of Antibody Charge Variants by Ion-Exchange Chromatography.