High-throughput protein stability screens with Aunty

Significance of the Topic

High-throughput assessment of protein stability is essential in biopharmaceutical development and quality control. Rapid, sensitive screening of multiple formulations accelerates lead selection, optimizes excipient conditions, and reduces time to clinic.

Objectives and Study Overview

This study demonstrates Aunty, an integrated platform combining full-spectrum fluorescence with static and dynamic light scattering in a quartz 96-well format. The goal is to showcase its throughput, sensitivity, reproducibility, and utility for protein melting and aggregation analysis under various buffer and excipient conditions.

Methodology and Instrumentation

Aunty leverages an SBS-format quartz 96-well plate requiring just 8 µL per sample.

• Full-spectrum fluorescence: intrinsic or dye-based detection with a robust 280 nm excitation LED and high-sensitivity spectrophotometer (0.025–300 mg/mL dynamic range).
• Static light scattering (SLS): average intensity to detect aggregation onset down to 50 µg/mL IgG.
• Dynamic light scattering (DLS): hydrodynamic diameter and polydispersity analysis with detection limit of 0.1 mg/mL lysozyme.
• Thermal control: programmable ramps from 15–95 °C at 0.1–10 °C/min, temperature accuracy ±0.1 °C.
• Automation readiness: plate input ports, sealing, and API support.

Key Results and Discussion

Parallel monitoring identified melting temperature (Tm), aggregation onset (Tagg), and size increase temperature (Tsize) in one run. Bovine IgG in PBS showed Tm 70.8 °C (CV 0.8 %) and Tagg 67.0 °C (CV 1.1 %) across 96 replicates. Addition of 500 mM arginine lowered Tm by 3.5 °C but raised Tagg by ~4.9 °C, indicating delayed aggregation but accelerated unfolding. Comparative screens of therapeutic antibodies under various excipients revealed distinct formulation preferences: NaCl stabilized bovine IgG (Tagg +2.8 °C) but destabilized trastuzumab (Tm –2.6 °C).

Benefits and Practical Applications

Aunty delivers:

• Ultra-high throughput screening with minimal sample volume.
• Simultaneous conformational and colloidal stability metrics.
• High sensitivity and reproducibility for rigorous formulation comparisons.
• Flexible detection modes for intrinsic or dye-based assays.

Future Trends and Opportunities

Integration with machine learning for predictive stability modeling, expansion to additional detection modalities (e.g., Raman scattering), and adoption of microfluidic-enabled plates for single-cell or membrane proteins will further empower rapid developability assessments.

Conclusion

Aunty’s combination of quartz 96-well format, full-spectrum fluorescence, SLS, DLS, precise thermal control, and automation readiness makes it a powerful tool for accelerated, comprehensive protein stability screening.

References

• Kamerzell TJ, Esfandiary R, Joshi SB, Noe D, Middaugh CR. Protein-excipient interactions: Mechanisms and biophysical characterization applied to protein formulation development. Advanced Drug Delivery Reviews. 2011;63(13):1118–1159.