Evaluation of SEC Columns for Analysis of ADC Aggregates and Fragments

Significance of the Topic

Antibody–drug conjugates (ADCs) combine the targeting precision of monoclonal antibodies with potent cytotoxic agents, offering highly specific treatment for various diseases. Accurate characterization of ADC aggregates and fragments is critical for ensuring safety and efficacy, yet their increased hydrophobicity compared to naked antibodies poses challenges in size exclusion chromatography (SEC) due to nonspecific interactions.

Objectives and Study Overview

This study evaluated the performance of three commercially available SEC columns in analyzing two classes of ADCs (Lys-linked trastuzumab emtansine and Cys-linked brentuximab vedotin). The goal was to identify stationary phases and mobile phase conditions that minimize secondary interactions and optimize aggregate resolution.

Methodology

• SEC columns: Agilent AdvanceBio SEC 300 Å, 2.7 µm; Vendor 1 SEC 200 Å, 1.7 µm; Vendor 2 SEC 300 Å, 1.8 µm.
• Mobile phase: phosphate-buffered saline (PBS), pH 7.4, 0.35 mL/min; injection volumes: 5 µL for trastuzumab emtansine, 10 µL for brentuximab vedotin.
• Experimental variables: pH adjusted from 6.8 to 7.4; NaCl concentrations at 100, 200, and 300 mM.

Instrumentation

Analysis employed a UHPLC system with UV detection, maintaining column temperature at 30 °C and a 20-minute run time.

Key Findings and Discussion

• The Agilent AdvanceBio SEC column exhibited the sharpest peaks, lowest tailing factors (USP ≤1.47), and highest resolution for both ADCs, enabling reliable monomer/dimer separation.
• Vendor 1 columns showed pronounced peak tailing and multiple unresolved components, indicating significant nonspecific interactions.
• Vendor 2 columns improved peak width but still underperformed in resolution compared to AdvanceBio.
• Optimal dimer/monomer resolution was achieved at pH 7.4; lower pH reduced aggregate detection accuracy.
• Increasing NaCl concentration up to 300 mM had negligible effect on peak shape and resolution, suggesting ionic strength is less critical than stationary phase hydrophilicity.

Benefits and Practical Applications of the Method

By selecting a hydrophilic polymer-coated SEC column and optimizing mobile phase pH, laboratories can enhance the accuracy and reproducibility of ADC aggregate quantification without resorting to organic modifiers, streamlining quality control in biopharmaceutical development.

Future Trends and Potential Applications

• Design of next-generation stationary phases combining hydrophilic shielding with mixed-mode interactions to further suppress nonspecific binding.
• Integration of SEC with mass spectrometry for simultaneous molecular weight and structural analysis of ADC species.
• Automation and high-throughput SEC platforms for accelerated screening during ADC formulation and stability studies.

Conclusion

The choice of SEC stationary phase and mobile phase parameters critically influences the resolution and reliability of ADC aggregate analysis. The Agilent AdvanceBio SEC 300 Å, 2.7 µm column at pH 7.4 consistently outperformed alternative columns, minimizing secondary interactions and delivering robust separation of monomeric and high-molecular-weight species.

Reference

• Agilent Technologies, Application Note 5994-3276EN, 2021.