Multi-day biologics stability experiments with Uncle

Importance of the topic

Protein stability assessment is a cornerstone of biologics development, guiding formulation optimization and candidate selection. Traditional thermal ramp methods—measuring melting temperature (Tm) and aggregation onset (Tagg)—offer rapid ranking but may fail to resolve subtle differences when values are similar. Isothermal stability experiments extend the analytical window, capturing unfolding and aggregation kinetics over hours or days, and provide deeper insight into long-term behavior under stress.

Objectives and study overview

This study evaluates the performance of the Uncle stability platform for multi-day isothermal monitoring of a human polyclonal IgG in phosphate-buffered saline (PBS) with increasing arginine concentrations. Key aims include:

• Determining how arginine affects Tm and Tagg under thermal ramp conditions.
• Quantifying unfolding and aggregation kinetics during a 36-hour isothermal hold at 58 °C.
• Comparing intrinsic fluorescence, static light scattering (SLS), and dynamic light scattering (DLS) readouts to characterize stability differences.

Methods and used instrumention

The Uncle platform integrates multiple detection modes and precise temperature control (15–95 °C) to run 48 low-volume sealed samples concurrently. Key features:

• Intrinsic fluorescence (350/330 nm ratio) for conformational changes.
• Static light scattering at 266 nm and 473 nm for aggregate detection.
• Dynamic light scattering for hydrodynamic diameter and polydispersity.

Study design:

• Thermal ramps (15–95 °C at 0.5 °C/min) measured Tm and Tagg of IgG in PBS or PBS plus 25, 50, 100, or 250 mM arginine.
• Isothermal experiments loaded triplicates of each formulation at 58 °C for 36 h, collecting fluorescence/SLS spectra every 10 min and DLS acquisitions every 13 min.

Main results and discussion

Thermal ramp analysis revealed a progressive decrease in Tm (66.5 °C to 59 °C) and increase in Tagg (57 °C to 65 °C) as arginine concentration rose; aggregation was undetectable at 250 mM arginine. During the 36-h isothermal hold:

• In PBS alone, intrinsic fluorescence ratio increased over time, while SLS signals at 266 nm and 473 nm rose sharply, indicating aggregation onset, peak formation, and eventual aggregate precipitation after ~25 h.
• With 250 mM arginine, spectral traces remained nearly constant, showing strong aggregation suppression.
• DLS data confirmed a >10-fold hydrodynamic diameter increase in PBS (20 nm to >250 nm), whereas high-arginine samples exhibited <2-fold size change, demonstrating concentration-dependent stabilization.

Continuous readouts provided granular insight into kinetic phases that endpoint measurements cannot capture.

Benefits and practical applications

The Uncle platform enables comprehensive stability profiling—thermal melting, aggregation onset, and size evolution—in one versatile instrument. Benefits include:

• High-throughput screening of formulations and excipients with minimal sample volume.
• Integrated, hands-off isothermal assays to differentiate closely matched constructs.
• Actionable kinetic data supporting formulation decisions and shelf-life predictions.

Future trends and possibilities

Emerging directions include coupling isothermal stability data with machine-learning models for predictive formulation design, expanding isothermal protocols to additional stress conditions (pH, oxidative stress), miniaturized microfluidic implementations for ultra-high throughput, and integrating multi-parameter readouts to construct comprehensive stability fingerprints for biologics.

Conclusion

Uncle’s integrated fluorescence, scattering, and sizing measurements deliver high-resolution, multi-day stability and aggregation profiles. This capability accelerates biologics formulation screening and provides confidence in candidate selection by revealing kinetic behaviors beyond traditional thermal ramp assays.

References

• Arakawa T, Ejima D, Tsumoto K, Obeyama N, Tanaka Y, Kita Y, Timasheff SN. Suppression of protein interactions by arginine: A proposed mechanism of the arginine effects. Biophysical Chemistry. 2007;127(1–2):1–8.
• Ishibashi M, Tsumoto K, Tokunaga M, Ejima D, Kita Y, Arakawa T. Is arginine a protein-denaturant? Protein Expression and Purification. 2005;42(1):1–6.