Take a snapshot of your protein quality with Uncle

Importance of the Topic

Rapid and reliable evaluation of protein sample quality is essential in analytical chemistry and biopharmaceutical development. Early detection of unfolding, aggregation, or concentration loss helps prevent wasted resources and inconsistent results in downstream assays or formulations.

Objectives and Overview

This study demonstrates a snapshot approach using the Uncle platform to assess protein stability by combining intrinsic fluorescence, static light scattering (SLS), and dynamic light scattering (DLS). The aim is to:

• Compare the effects of thermal and freeze–thaw stress on monoclonal antibodies (mAb1 and NISTmAb)
• Monitor long-term storage impact on mAb2 at different temperatures
• Showcase the throughput and sample efficiency of the method

Methodology and Instrumentation

Proteins were prepared in appropriate buffers (sodium acetate pH 5.0 or histidine pH 6.0) at 0.5 mg/mL. Stress conditions included heating at 75 °C for 1 hour, six freeze–thaw cycles at –80 °C, and storage at 4 °C or 25 °C for 7 days. Measurements employed:

• Uncle platform: full-spectrum intrinsic fluorescence (250–720 nm) with barycentric mean (BCM) analysis, SLS at 266 and 473 nm, and DLS for hydrodynamic diameter and polydispersity
• Lunatic UV/Vis (2 µL sample) for protein concentration by A280

Main Results and Discussion

Thermal Stress

• mAb1 showed a pronounced increase in BCM fluorescence and elevated SLS at 266 nm, indicating unfolding and small aggregate formation
• NISTmAb remained stable, with minimal changes in fluorescence, SLS, and DLS; thermal ramp transitions aligned with reported DSC values at 69, 83, and 94 °C, confirming integrity
• DLS confirmed mAb1 aggregates ~70 nm with increased polydispersity, whereas NISTmAb only exhibited a trace population of large particles (<0.1% mass)
• Protein concentration measurements revealed significant loss for heat-treated mAb1 (ratio 0.64) and minor loss for NISTmAb (ratio 0.91), suggesting precipitation of unfolded species

Freeze–Thaw Stress

• Repeated freeze–thaw cycles caused an increase in mAb1 hydrodynamic diameter up to ~20 nm, consistent with native-state aggregation
• BCM fluorescence remained constant, indicating no significant unfolding despite size increases

Long-Term Storage

• mAb2 stored at 25 °C exhibited a gradual rise in BCM fluorescence over 7 days, signaling partial unfolding
• Samples at 4 °C remained stable in fluorescence, SLS, and DLS, with hydrodynamic diameter ~10 nm

Benefits and Practical Applications

The snapshot approach provides:

• Comprehensive stability profiles in one experiment
• High throughput: up to 48 samples in parallel
• Low sample consumption: 9 µL per sample
• Rapid results: ~7.5 minutes for fluorescence/SLS and 23 minutes for DLS
• Simultaneous detection of unfolding, aggregation, and concentration changes

Future Trends and Applications

Emerging directions include:

• Integration with predictive modeling and machine learning for advanced stability forecasting
• Automated high-throughput screening of formulation buffers or excipients
• Extended application to other biologics such as enzymes, vaccines, or protein–ligand complexes
• Miniaturization and multiplexing for point-of-care quality checks

Conclusion

The Uncle platform offers a fast, reliable, and sample-efficient solution for protein quality control by combining full-spectrum fluorescence, static, and dynamic light scattering. It enables laboratories to make informed decisions on sample integrity before proceeding to critical downstream applications.

Instrumentation Used

• Uncle protein stability platform (fluorescence, SLS, DLS)
• Lunatic UV/Vis spectrophotometer for A280 concentration measurements

References

1. Schiel JE, Davis DL, Borisov OV, editors. State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume 2: The NISTmAb Case Study. American Chemical Society; 2015.