Conquer ADC stability characterization with Aunty

Importance of Topic

Antibody–drug conjugates (ADCs) integrate potent cytotoxic agents with monoclonal antibodies to target cancer cells with high specificity. However, the addition of hydrophobic small-molecule payloads compromises the native stability of antibodies, promoting unfolding and aggregation. Optimizing ADC formulations to mitigate these liabilities is critical for manufacturability, shelf life, and clinical performance.

Study Objectives and Overview

This application note demonstrates the use of the Aunty platform to perform high-throughput conformational and colloidal stability screening of four therapeutics: the monoclonal antibodies Herceptin and its biosimilar Kanjinti, and the ADCs Kadcyla and Enhertu. Two excipient conditions—10 mM histidine, pH 6, with either 80 mg/mL sucrose or 0.9% NaCl—were assessed to identify stabilizing formulation components.

Methodology and Instrumentation

Samples were buffer-exchanged into histidine buffer and characterized on a Stunner instrument for protein concentration, size, and polydispersity. Stability assays were conducted on the Aunty quartz-plate thermal stability platform, integrating static light scattering (SLS), dynamic light scattering (DLS), and full-spectrum fluorescence. Colloidal stability parameters (diffusion interaction parameter kD and second virial coefficient B22) were calculated from DLS and SLS dilution series. Thermal unfolding and aggregation were monitored by intrinsic fluorescence barycentric mean shifts (Tm) and SLS intensity increases (Tagg and Tsize). SYPRO Orange reporter dye assays were used for ADCs with payload fluorescence interference.

Results and Discussion

In histidine buffer with sucrose, all four proteins exhibited positive kD and B22 values, indicative of repulsive interactions and low aggregation propensity. In contrast, formulations with NaCl produced negative slopes, reflecting attractive interactions, particularly pronounced for the ADCs. Thermal assays revealed two melting transitions (Tm1, Tm2) for the mAbs, with Tagg temperatures closely aligned. Replacing sucrose with NaCl reduced both Tm1 and Tagg across all samples, with Enhertu showing the greatest destabilization. ADCs consistently displayed lower thermal stability and higher aggregation propensity than their parent antibodies.

Benefits and Practical Applications

Aunty enables parallel, automation-ready screening of up to 96 formulations using minimal sample volume (8 µL per well). By combining SLS, DLS, and fluorescence in a single run, it accelerates excipient selection and formulation optimization for ADC development, reducing material consumption and experimental complexity.

Future Trends and Opportunities

Advances in assay multiplexing and robotic integration will enhance throughput and reproducibility in ADC stability studies. Customizable full-spectrum fluorescence assays can address payload interference, while integrating stability data with machine learning may predict developability and guide early formulation design for next-generation bioconjugates.

Conclusion

The Aunty platform successfully characterized the colloidal and conformational stability differences between mAbs and ADCs, confirming that a histidine buffer with sucrose markedly improves stability over NaCl for trastuzumab-based products. ADCs remain inherently less stable than their antibody scaffolds, underscoring the need for tailored formulation strategies. Aunty’s comprehensive, high-throughput capabilities support more efficient ADC development workflows.

References

1. Physical and Chemical Stability of Antibody Drug Conjugates: Current Status. PL Ross et al. Journal of Pharmaceutical Sciences. 2016;105(2):391–397.
2. A Single Molecular Descriptor to Predict Solution Behavior of Therapeutic Antibodies. JS Kingsbury et al. Science Advances. 2020;6(32).
3. Accelerated Formulation Development of Monoclonal Antibodies (mAbs) and mAb-Based Modalities. VI Razinkov et al. Journal of Biomolecular Screening. 2015;20(4):468–483.
4. The Use of Fluorescence Methods to Monitor Unfolding Transitions in Proteins. MR Eftink. Biophysical Journal. 1994;66(2 Pt 1):482–501.