Uncle stays hot for proteins with isothermal apps

Importance of the topic

Early detection of protein stability failures under thermal stress is essential in biopharmaceutical development to rank formulation candidates and predict long-term behavior. High-throughput isothermal assays combined with thermal ramp analysis enable efficient screening of protein unfolding and aggregation using minimal sample volumes.

Objectives and overview of the study

This application note demonstrates how the Uncle platform integrates full-spectrum fluorescence, static light scattering (SLS), and dynamic light scattering (DLS) to evaluate thermal and isothermal stability of a monoclonal antibody (mAb 1) and RNase A across varying salt conditions. The note illustrates three workflows: Tm & Tagg, Tm using SYPRO Orange, and extended stability assays.

Methods and instrumentation

The Uncle instrument provides:

• Temperature control from 15 to 95 °C for thermal ramps and isothermal holds
• Full-spectrum fluorescence detection (250–720 nm)
• 473 nm and 266 nm excitation lasers for intrinsic and dye-based assays
• Static and dynamic light scattering for aggregation and size distribution

Sample protocols:

• Tm & Tagg: 1 °C/min ramp from 15–95 °C, intrinsic fluorescence BCM analysis (300–430 nm) and SLS at 266 nm
• Tm using SYPRO Orange: 10× dye concentration, fluorescence emission 510–680 nm and SLS at 473 nm
• Isothermal: 16 h hold at 60 °C with minute-by-minute monitoring
• Out-of-the-box stability: daily DLS size measurements over 3 days

Main results and discussion

• The mAb 1 thermal ramp showed Tm values around 70 °C and Tagg values sensitive to NaCl concentration, with highest aggregation onset in 50 mM NaCl.
• SYPRO Orange assays revealed two melting transitions (Tm1 ~64–67 °C and Tm2 ~80–82 °C) and confirmed dye-induced Tm shifts.
• Isothermal holds at 60 °C identified faster aggregation in salt-containing buffers, while the salt-free formulation delayed aggregate formation.
• RNase A exhibited a single Tm at ~61 °C by SYPRO Orange; thermal ramps alone did not resolve stability differences between salt conditions.
• Isothermal and 3-day DLS experiments ranked high-salt RNase A formulation as most stable under prolonged heat stress.

Benefits and practical applications

The integrated Uncle workflows enable:

• Rapid ranking of formulation stability using both unfolding and aggregation metrics
• Reduced sample consumption with 9 µL quartz cuvettes
• Flexible design for short-term thermal ramps, isothermal holds, and long-term incubation
• Comprehensive data to guide candidate selection and minimize downstream storage failures

Future trends and potential applications

Ongoing developments may include:

• Expanding multi-parameter screening to a broader range of biologics and buffer systems
• Automated integration with high-throughput formulation libraries and robotic handlers
• Data analytics pipelines leveraging machine learning for predictive stability modeling
• Remote monitoring and cloud-based data management for decentralized laboratories

Conclusion

The Uncle platform offers a versatile solution for early-stage characterization of protein stability by combining intrinsic and extrinsic fluorescence with light scattering under thermal and isothermal conditions. Its high-throughput format and multi-method detection accelerate formulation ranking and reduce the risk of long-term storage failures.

Reference

No external references were provided in the original application note.