Charge-Variant Analysis of Antibody-Drug Conjugates with Cation-Exchange Chromatography

Importance of the Topic

Antibody‐drug conjugates (ADCs) represent a cutting‐edge class of biotherapeutics that combine the targeting specificity of monoclonal antibodies with potent cytotoxic payloads. By selectively delivering drugs to tumor cells, ADCs improve therapeutic efficacy while minimizing systemic side effects. Comprehensive charge‐variant profiling is essential for assessing ADC heterogeneity, drug‐to‐antibody ratio, and overall quality in both research and quality‐control environments.

Aims and Overview

This study evaluates salt‐ and pH‐gradient cation‐exchange chromatography methods on BioResolve SCX mAb Columns for charge‐variant analysis of two model ADCs: the lysine‐linked ado‐trastuzumab emtansine and a cysteine‐linked ADC. The goal is to establish robust, generic workflows suitable for routine characterization and potential multidimensional separations.

Methodology and Instrumentation

Two elution strategies were optimized:

• Salt Gradient: 20 mM MES buffer at pH 6.6, linear NaCl increase from 30 to 110 mM over 30 min, flow 0.80 mL/min on a BioResolve SCX mAb 4.6 × 100 mm Column.
• pH Gradient: BioResolve CX pH Concentrates, linear 0–100% mobile phase B over 30 min, flow 1.00 mL/min on a BioResolve SCX mAb 4.6 × 50 mm Column.

UV detection was performed at 280 nm. Organic co‐solvent effects were assessed by adding 5% and 10% acetonitrile via a quaternary solvent manager.

Used Instrumentation

• BioResolve SCX mAb Columns (4.6 × 100 mm and 4.6 × 50 mm)
• BioResolve CX pH Concentrates mobile‐phase system
• Quaternary solvent manager for gradient blending
• Reversed‐phase UPLC system with UV detection at 280 nm

Key Results and Discussion

Both ADCs yielded well‐resolved charge‐variant profiles under optimized salt and pH gradients. The pH gradient offered marginally better separation of acidic species, especially for the cysteine‐linked ADC. Addition of 5% acetonitrile had minimal impact on retention or peak area, indicating negligible secondary hydrophobic interactions. However, 10% acetonitrile caused light/heavy chain dissociation, underscoring a limit for organic modifier use.

Benefits and Practical Applications

• Generic pH‐gradient method reduces development time and resources.
• Robust separation suitable for batch‐to‐batch comparability and QA/QC workflows.
• Minimal secondary interactions allow accurate heterogeneity assessment.
• Methods are directly applicable as the first dimension in multidimensional LC approaches.

Future Trends and Opportunities

Advancements may include coupling these separation modes with mass spectrometry for detailed glycoform and drug‐load mapping, integration into automated multidimensional platforms for high throughput, and extension to emerging ADC formats and novel conjugation chemistries.

Conclusion

BioResolve SCX mAb Columns combined with BioResolve CX pH Concentrates provide versatile, high‐resolution charge‐variant analysis for both lysine‐ and cysteine‐linked ADCs. Optimized salt and pH gradient protocols deliver reliable profiling with minimal secondary interactions, supporting robust characterization and future multidimensional workflows.

References

1. Bobaly B.; et al. Current possibilities of liquid chromatography for the characterization of antibody–drug conjugates. J. Pharm. Biomed. Anal. 2018, 147, 493–505.
2. Gandhi A. V.; Randolph T. W.; Carpenter J. F. Conjugation of emtansine onto trastuzumab promotes aggregation of the antibody–drug conjugate by reducing repulsive electrostatic interactions and increasing hydrophobic interactions. J. Pharm. Sci. 2019.
3. Wang Q.; Rzewuski S. C.; Lauber M. A. Development of pH Gradient Mobile Phase Concentrates for Robust, High Resolution mAb Charge Variant Analysis. Waters Application Note 720006491EN (2019).
4. Yang H.; Warren B.; Koza S. M. Development of Monoclonal Antibody Charge Variant Analysis Methods using Waters BioResolve SCX mAb Column. Waters Application Note 720006477EN (2019).