High-throughput protein stability screens with Aunty

Importance of the Topic

A robust, high-throughput platform for protein stability screening is essential in biopharmaceutical development to assess conformational and colloidal stability rapidly. By monitoring unfolding and aggregation in parallel, researchers can optimize formulations, improve developability assessments, and reduce timelines for candidate selection.

Objectives and Study Overview

This technical note demonstrates how the Aunty platform integrates intrinsic fluorescence, static light scattering (SLS), and dynamic light scattering (DLS) in a quartz SBS-format 96-well plate to achieve fast, sensitive, and automated thermal stability screening. Key goals include:

• Determining protein melting temperatures (Tm) via full-spectrum fluorescence
• Detecting aggregation onset (Tagg) and particle size changes (Tsize)
• Validating precision, reproducibility, and low sample consumption

Methodology

Samples (8 µL per well) are loaded into etched quartz 96-well plates and sealed to prevent evaporation. Temperature ramps from 15 °C to 95 °C at rates of 0.1–10 °C/min are applied. Intrinsic fluorescence changes are tracked to identify Tm by monitoring shifts and intensity changes in the emission spectrum. SLS measures average scattering intensity to detect aggregation, while DLS analyzes fluctuation rates to calculate hydrodynamic diameters and size distributions.

Instrumentation

The study employs the Aunty stability platform featuring:

• Quartz SBS-format 96-well plates with 8 µL sample volume
• 280 nm excitation LED and high-sensitivity spectrophotometer for full-spectrum fluorescence
• Parallel SLS and DLS detection modules
• Precise thermal control with ±0.1 °C accuracy
• Automation-ready design with SBS-format input ports and API integration

Main Results and Discussion

Intrinsic fluorescence and light scattering data reveal both unfolding and aggregation behavior in real time:

• Bovine IgG (1 mg/mL) in PBS exhibited a Tm of 70.8 °C (CV 0.8 %) and Tagg of 67 °C (CV 1.1 %) across 96 replicates, demonstrating excellent precision.
• Adding 500 mM arginine lowered Tm by 3.5 °C but shifted Tagg from 68.5 °C to 73.4 °C, indicating delayed aggregation despite accelerated unfolding.
• Excipients screening of seven antibody formulations (trastuzumab and pembrolizumab biosimilars) in a single run yielded Tm and Tagg CVs below 2 %, illustrating the platform’s throughput and reproducibility.

Benefits and Practical Applications

The Aunty platform offers:

• High data resolution and sensitivity for low-volume samples
• Simultaneous monitoring of unfolding and aggregation in a single run
• Automation compatibility for large-scale formulation screens
• Rapid identification of optimal excipient conditions and protein constructs

Future Trends and Applications

Advances may include integration with automated liquid-handling systems, expanded dye-based assays for diverse protein classes, AI-driven data analysis for predictive stability modeling, and extension to multi-parametric screening in early developability studies.

Conclusion

The Aunty platform uniquely combines full-spectrum fluorescence, SLS, and DLS in a quartz 96-well format to deliver fast, precise, and high-throughput protein stability data. Its minimal sample requirements, precise thermal control, and automation readiness accelerate formulation optimization and developability assessments, supporting more efficient biologics development.

References

1. Kamerzell TJ, et al. Protein-excipient interactions: mechanisms and biophysical characterization applied to protein formulation development. Adv Drug Deliv Rev. 2011;63(13):1118–1159.