Take a snapshot of your protein quality with Uncle

Importance of the Topic

Assessing protein sample integrity before downstream analysis is essential for reliable characterization outcomes. Early identification of unfolding or aggregation events helps optimize purification protocols, storage conditions and experimental designs, saving time and resources.

Objectives and Study Overview

This work illustrates how the Uncle platform performs rapid quality checks of monoclonal antibodies under various stress conditions. Thermal stress, freeze/thaw cycling and storage temperature effects on mAb1, mAb2 and the NIST monoclonal antibody reference material (NISTmAb) were evaluated to demonstrate the platform’s sensitivity to conformational changes, aggregation and concentration loss.

Methodology and Instrumentation

Protein samples were prepared in sodium acetate or histidine buffers and diluted to 0.5 mg/mL. Thermal stress (75 °C, 1 h), six freeze/thaw cycles and 7-day storage at 4 °C or 25 °C were applied. The Uncle platform measured intrinsic fluorescence spectra (250–720 nm), static light scattering at 266 and 473 nm, and dynamic light scattering (DLS) to determine BCM fluorescence shifts, aggregate formation and hydrodynamic size. Protein concentrations were quantified on the Lunatic UV/Vis system using the A280 Classic application.

Instrumentation Used

• Uncle protein stability platform (Unchained Labs) for fluorescence, SLS and DLS
• Lunatic spectrophotometer (Unchained Labs) for A280 absorbance
• Uncle Analysis software for BCM and sizing analysis

Main Results and Discussion

Thermal stress induced a significant BCM fluorescence increase and elevated 266 nm SLS signal for mAb1, indicating unfolding and small aggregate formation, while NISTmAb remained stable with transitions matching DSC benchmarks. Concentration measurements revealed partial precipitation of mAb1 (recovery ratio 0.64) versus minor loss for NISTmAb (0.91). Repeated freeze/thaw cycles caused native-state aggregation of mAb1, detected by a size increase up to ~20 nm without unfolding signatures. Storage at 25 °C for 7 days led to modest unfolding of mAb2, whereas samples maintained at 4 °C showed stable profiles in fluorescence and DLS.

Benefits and Practical Applications

The Uncle platform delivers comprehensive protein quality data—including conformational stability, early aggregation detection and concentration loss—in minutes using only 9 μL per sample. High-throughput capability (up to 48 samples) supports batch comparisons, formulation screening and quality control workflows.

Future Trends and Potential Applications

Integration of multi-mode stability profiling into automated formulation development and predictive stability modeling is anticipated. Combining rapid screening with machine learning could further accelerate biologic optimization and ensure robust product quality for therapeutic proteins.

Conclusion

Uncle combines intrinsic fluorescence, static and dynamic light scattering into a single workflow for efficient protein quality control. The platform accurately detects unfolding, aggregation and concentration changes under diverse stress conditions, supporting faster decision-making in biologics research and development.

References

1. State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume 2. Edited by Schiel J.E., Davis D.L., Borisov O.V., American Chemical Society, 2015.