Meet Our Uncle: 12 Stability Applications on One Platform

Significance of the topic

The development and formulation of protein-based therapeutics depend heavily on comprehensive stability assessment. Understanding unfolding, aggregation, and intermolecular interactions under various stresses guides early-stage screening, reduces failure rates, and accelerates time to clinic.

Objectives and overview of the application note

This technical note introduces Uncle, an integrated stability platform capable of performing twelve distinct analyses on a single instrument. By combining intrinsic fluorescence, static light scattering (SLS), and dynamic light scattering (DLS) detection modes, Uncle delivers high-content profiling of protein stability across multiple assays without changing samples or instruments.

Used instrumentation

• Uncle stability platform by Unchained Labs
• Temperature control from 15 °C to 95 °C with sealed quartz multi-well cuvette chambers (Unis)
• Detection modules: intrinsic fluorescence (tryptophan/tyrosine), SLS at multiple wavelengths, DLS
• Software for simultaneous data acquisition and automated analysis

Methodology

All assays use nine microliters of sample per well in 48-well quartz Unis. Thermal ramps (typically 15–95 °C, 0.3–0.5 °C/min) and isothermal holds are combined with fluorescence, SLS, and/or DLS measurements. Chemical denaturation curves, viscosity assays, and diffusion/self-interaction studies employ the same workflow with minimal sample preparation.

Main results and discussion

• Thermal melting (Tm): intrinsic fluorescence barycentric mean reveals multiple unfolding transitions (e.g., 57.8, 71.5, 88.1 °C) and formulation improvements (Tm shift to 73.6 °C).
• Thermal aggregation (Tagg): SLS at 266 nm detects aggregation onset (e.g., 67 °C) and formulation differences.
• DSF with SYPRO Orange: extrinsic dye assay yields two melting transitions at 62.5 °C and 76.3 °C for a monoclonal antibody.
• Chemical denaturation (ΔG): urea denaturation curve fits a three-state model, calculating free energies of unfolding (ΔG ≈ 9.9 and 10.9 kcal/mol).
• Isothermal aggregation: extended holds at sub-Tm temperatures chart aggregate formation and polydispersity increases over time.
• Thermal recovery: iterative heating/cooling ramps monitor reversible unfolding below Tm and irreversible aggregation above Tm.
• Sizing and polydispersity by DLS: hydrodynamic diameter and polydispersity track conformational changes under thermal stress (e.g., rapid size increase above 70 °C).
• Diffusion interaction (kD): DLS concentration series yields kD values that shift from repulsive to attractive interactions with pH changes.
• Second virial coefficient (B22): light scattering intensity series provides B22 values, confirming colloidal stability trends in line with kD data.
• Kirkwood–Buff integral (G22): analysis at high protein concentration differentiates net attractive or repulsive self-interactions.
• Viscosity measurements: nanoparticle tracking in protein and glycerol solutions correlates with standard viscometer data and quantifies viscosity up to 300 mg/mL protein (≈4 cP).

Benefits and practical applications

• Single-instrument workflow reduces sample volume and handling errors.
• Simultaneous multi-modal detection enhances confidence in stability profiles.
• Automated software accelerates data analysis and decision making.
• Early screening of formulations, buffers, and excipients for optimal stability.
• Applicable to research labs, QA/QC, and industrial biologics development.

Future trends and possible applications

Integration of high-throughput screening, machine learning for predictive formulation optimization, coupling with mass spectrometry or chromatography modules, and real-time process monitoring will further expand the utility of all-in-one stability platforms.

Conclusion

Uncle offers a versatile, low-volume approach to twelve key protein stability assays, streamlining workflows and delivering comprehensive stability data from a single instrument. This integrated solution empowers researchers to identify optimal constructs and formulations earlier in development, minimizing risk and accelerating timelines.

Reference

Technical Note: Meet Our Uncle: 12 Stability Applications on One Platform; Unchained Labs, Rev E, 2018.