Aggregation of Antibody-Drug Conjugates: The Light Scattering Toolbox for Screening and Characterization

Significance of the Topic

Antibody–drug conjugates (ADCs) represent a growing class of targeted therapeutics for oncology and other diseases. Their complex structure, featuring antibodies linked to cytotoxic drugs, enhances efficacy but also increases the risk of aggregation. Aggregation can impair safety, efficacy and shelf-life. Light scattering techniques offer versatile, sensitive and label-free methods to screen formulation conditions, monitor stability and characterize aggregate species, supporting robust ADC development.

Objectives and Study Overview

This work presents a comprehensive light scattering toolkit to assess ADC aggregation propensity and characterize aggregates at multiple levels. Key goals include:

• Comparing aggregation onset across different linker chemistries.
• Demonstrating high-throughput screening for thermal and colloidal stability.
• Profiling size and molar mass distributions of native and stressed ADC samples.

Methodology and Instrumentation

Dynamic light scattering (DLS) and multi-angle light scattering (MALS) were integrated with fractionation and plate-based screening:

• High-throughput DLS using the DynaPro® Plate Reader II to measure hydrodynamic radius, polydispersity and aggregation onset temperature (Tonset) via thermal ramps.
• Asymmetric-flow field-flow fractionation (AF-FFF) coupled to Wyatt DAWN® HELEOS® II for size-based separation without stationary phase artifacts.
• Static MALS detection to derive absolute molar mass (Mw) and size distributions from first principles, independent of calibration standards.
• Additional techniques mentioned include composition-gradient MALS (CG-MALS) and phase-analysis light scattering (MP-PALS) for charge and interaction studies.

Main Results and Discussion

DLS screening revealed distinct thermal stability profiles for two ADC variants:

• ADC1 exhibited a higher aggregation onset (Tonset ≈ 63.9 °C) compared to ADC2 (Tonset ≈ 48.6 °C), indicating that linker chemistry critically influences stability.
• ADC2 unfolded and aggregated at lower temperatures, highlighting its increased aggregation propensity.

AF-FFF-MALS fractograms documented:

• Monomeric ADC populations with Mw ~149 kDa in both unstressed and stressed samples.
• Emergence of higher-order oligomeric species (~339 kDa) upon stress, representing a small but detectable fraction of total mass.

Complementary MALS-UV-RI analysis determined drug-antibody ratio (DAR) while quantifying fragment and aggregate fractions. The combination of techniques enabled high-resolution mapping of size, mass and aggregation pathways.

Benefits and Practical Applications of the Method

The integrated light scattering approach offers:

• Rapid, high-throughput screening of formulation and linker chemistry to minimize aggregation risk.
• Absolute molar mass determination without column calibration, enhancing reproducibility.
• Sensitive detection of low-abundance aggregates, critical for early stability assessment.
• Actionable data for formulation scientists to optimize ADC design and storage conditions.

Future Trends and Applications

Advances likely to enhance ADC characterization include:

• Integration of AI-driven data analysis for real-time aggregation risk prediction.
• Expansion of CG-MALS and MP-PALS for detailed interaction and charge profiling.
• Automation of fractionation and plate-based assays for end-to-end ADC screening pipelines.
• Miniaturization and multiplexing to accelerate early-stage linker and payload screening.

Conclusion

The light scattering toolbox, combining high-throughput DLS, AF-FFF-MALS and complementary detectors, provides a powerful platform for ADC aggregation screening and aggregate characterization. By elucidating the impact of linker chemistry on stability and enabling rapid, absolute measurements of size and mass, these methods streamline ADC development and support robust, reliable therapeutics.

Reference

• Broomell C., Some D. Aggregation of Antibody-Drug Conjugates: The Light Scattering Toolbox for Screening and Characterization. Wyatt Technology Corporation; 2014.