Multi-day biologics stability experiments with Uncle

Significance of the Topic

Assessing protein stability over extended periods is a key requirement in biologics development. Traditional thermal ramp measurements (melting temperature, Tm, and aggregation onset temperature, Tagg) provide rapid ranking of candidate formulations but can lack resolution when values are closely clustered. Isothermal stability experiments conducted just below the melting point offer a detailed view of unfolding and aggregation kinetics over hours or days, enabling more discriminating comparison of similar protein constructs or formulations.

Objectives and Study Overview

This application note describes a series of multi-day isothermal stability experiments conducted with the Uncle platform. The study aims to evaluate how increasing concentrations of arginine influence the thermal unfolding, aggregation behavior, and hydrodynamic size of a human polyclonal IgG at 58 °C over 36 hours. Preliminary thermal ramp experiments establish Tm and Tagg values to guide selection of the isothermal temperature.

Methodology and Instrumentation

All experiments were performed on Uncle (Unchained Labs), an integrated stability analyzer combining intrinsic fluorescence, static light scattering (SLS), and dynamic light scattering (DLS) with precise temperature control (15–95 °C) and sealed low-volume sample handling.

• Thermal Ramp Measurements: IgG at 5 mg/mL in PBS or PBS with 25, 50, 100, or 250 mM arginine was ramped from 15 to 95 °C at 0.5 °C/min. Tm was derived from the first derivative of the 350:330 nm fluorescence ratio; Tagg from the onset of SLS at 266 nm.
• Fluorescence + SLS Isothermal Study: IgG at 1 mg/mL in the same formulations was held at 58 °C for 36 h with spectra (250–720 nm) recorded every 10 minutes.
• DLS Isothermal Study: Parallel DLS data were acquired every 13 minutes (four 5 s acquisitions) over 36 h at 58 °C.

Key Results and Discussion

• Thermal Ramp Findings: Tm decreased from 66.5 °C (PBS) to 59 °C (250 mM Arg), while Tagg increased from 57 °C to 65 °C with higher arginine, indicating a stabilization of the aggregate-prone state despite a modest decrease in conformational stability.
• Isothermal Fluorescence + SLS: IgG in PBS exhibited large SLS peaks at 266 nm and 473 nm after 36 h, signifying extensive aggregation. In contrast, the 250 mM arginine sample showed almost unchanged spectra, with minimal scattering increase and slightly altered initial fluorescence, reflecting suppressed aggregation and altered baseline conformation.
• Continuous SLS Monitoring: Scattering intensity at 473 nm rose sharply in PBS, peaking around 25 h before decreasing due to precipitate removal. Increasing arginine concentrations progressively reduced maximum scattering, with 250 mM preventing detectable aggregate formation over 36 h.
• Intrinsic Fluorescence Ratio: All samples showed gradual increases in the 350:330 nm ratio over time, consistent with unfolding. Higher arginine shifted the starting ratio upward, implying a slightly destabilized native state in the presence of arginine.
• DLS Data: Correlation functions and calculated hydrodynamic diameter revealed a >10-fold size increase (20 nm to >250 nm) for IgG in PBS, whereas 250 mM arginine limited growth to less than two-fold. Triplicate measurements confirmed high reproducibility.

Benefits and Practical Applications

The Uncle platform enables comprehensive stability profiling—thermal unfolding, aggregation onset, and size distribution—on a single instrument with minimal hands-on effort and low sample consumption. Continuous isothermal monitoring captures kinetic phases that endpoint assays miss, improving formulation screening and optimization in biologics development and QC workflows.

Future Trends and Potential Applications

As biologics pipelines expand, automated high-throughput isothermal stability screening will become increasingly important. Integrating additional detection modes (e.g., Raman, FTIR) and linking kinetic datasets with machine learning models may further accelerate formulation design. Real-time aggregation monitoring could also be applied to novel modalities such as antibody–drug conjugates or nanoparticle-based therapies.

Conclusion

This study demonstrates that arginine effectively suppresses antibody aggregation and size growth during extended isothermal incubation, despite modest destabilization of the folded state. The integrated fluorescence, SLS, and DLS measurements on Uncle provide a robust, reproducible approach for detailed multi-day stability assessment, enabling informed formulation decisions early in development.

References

1. 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.
2. 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.