Uncle stays hot for proteins with isothermal apps

Importance of the Topic

Protein stability assessments are essential in biologics development to ensure candidates maintain structure and function under storage and stress conditions. Thermal unfolding and aggregation temperatures (Tm and Tagg) combined with isothermal stability profiles guide formulation screening and minimize long-term trial failures by enabling rapid, high-throughput analysis of low-volume samples.

Objectives and Study Overview

This study demonstrates the application of a multi-detector stability platform to evaluate the thermal and isothermal stability of a monoclonal antibody (mAb1) and ribonuclease A (RNase A) across three buffer formulations. Four experimental modes—thermal ramp with intrinsic fluorescence and light scattering (Tm & Tagg), dye-based differential scanning fluorescence (Tm using SYPRO), isothermal hold, and long-term stability—are used to rank and compare formulation performance.

Methodology and Instrumentation

Samples were prepared in 25 mM His-HCl pH 7.4 with 0, 50 or 250 mM NaCl. mAb1 was diluted to 1 mg/mL and RNase A to 1 mg/mL. Measurements were performed on the Uncle platform, featuring:

• Full-spectrum intrinsic fluorescence excitation at 266 nm (300–430 nm emission)
• Extrinsic SYPRO Orange fluorescence excitation at 473 nm (510–680 nm emission)
• Static light scattering (SLS) at both wavelengths to detect aggregation
• Dynamic light scattering (DLS) for hydrodynamic sizing
• Precise thermal control from 15–95 °C and isothermal holds

This setup enables 9 µL sample analysis in quartz cuvettes with minimal evaporation and throughput of up to 48 samples per run.

Results and Discussion

mAb1 stability:

• Intrinsic fluorescence ramps (Tm & Tagg) revealed minor Tm shifts (70.4 °C → 69.5 °C) and Tagg variations with increasing NaCl, indicating salt-dependent aggregation behavior.
• Dye-based DSF with SYPRO Orange showed two unfolding transitions, with the higher-temperature event correlating to aggregation (Tagg).
• Isothermal incubation at 60 °C highlighted faster aggregate formation in 50 mM and 250 mM NaCl versus salt-free buffer.

RNase A stability:

• Intrinsic fluorescence was not applicable due to low tryptophan content, so SYPRO-based ramps yielded a single Tm (~61 °C) for all formulations without immediate aggregation.
• Isothermal holds at 60 °C over 16 h identified significant aggregation in low-salt conditions, while 250 mM NaCl maintained stability.
• Long-term DLS monitoring confirmed the protective effect of high salt over a 3-day incubation.

This combinatorial approach enables clear differentiation between unfolding and aggregation and robust ranking of formulation performance.

Benefits and Practical Applications

The integrated platform offers:

• Rapid Tm and Tagg determination within 2 h using small volumes.
• Simultaneous intrinsic and extrinsic fluorescence with SLS/DLS for comprehensive stability profiling.
• Isothermal and long-term assays to forecast aggregation over relevant time scales.
• High sample throughput facilitating early-stage formulation screening and candidate prioritization.

Future Trends and Applications

Advancements may include:

• Integration with automated formulation libraries and machine learning for predictive stability optimization.
• Miniaturized microfluidic formats for multiplexed protein screening.
• Real-time monitoring of stability in bioprocess streams and containerized storage studies.

Conclusion

The multi-detector isothermal platform streamlines protein stability characterization by combining thermal ramp, isothermal, and long-term assays in a single instrument. This approach accelerates formulation development, reveals nuanced stability behaviors, and reduces resource demands in biologics research.

References

No external references were provided.