Figure out aggregation early: simultaneous, independent measurements of B22 and kD on Uncle

Significance of the Topic

High-concentration protein therapeutics face an increased risk of aggregation, which can compromise efficacy, safety and shelf life. Measuring colloidal stability early in development helps select optimal candidates and formulations before consuming large amounts of material or time.

Objectives and Study Overview

This study demonstrates a rapid, sample-efficient approach to simultaneously determine two key interaction parameters—the diffusion interaction parameter (kD) and the second virial coefficient (B22)—using a single experiment. The model protein lysozyme was examined under low (100 mM NaCl) and high (400 mM NaCl) salt conditions to illustrate the method’s ability to detect repulsive versus attractive interactions.

Methods and Instrumentation

Samples of lysozyme in 10 mM sodium acetate buffer (pH 5.2) with either 100 mM or 400 mM NaCl were prepared at 5, 10, 15 and 20 mg/mL. After filtration and centrifugation, triplicate aliquots were loaded into a 48-well plate. Four dynamic light scattering (DLS) acquisitions were collected per well to derive hydrodynamic diameters and diffusion coefficients. Static light scattering (SLS) measurements were calibrated against toluene as a reference to determine scattering intensities and calculate B22 values.

Instrumentation:

• Uncle stability platform (Unchained Labs)
• DLS and SLS detectors with temperature control (15–95 °C)

Main Results and Discussion

Under low-salt conditions, lysozyme exhibited positive kD (3.8 mL/g) and B22 (1.7 × 10^-4 mol·mL/g^2), indicating net repulsive interactions. High-salt conditions reversed these values (kD = –6.0 mL/g; B22 = –1.6 × 10^-4 mol·mL/g^2), consistent with attractive self-association. The measured parameters aligned closely with literature values, confirming method accuracy. Visually, samples in 400 mM NaCl turned cloudy at 4 °C but clarified upon warming or dialysis, indicating reversible native‐state aggregation rather than irreversible denaturation.

Benefits and Practical Applications

• Simultaneous measurement of kD and B22 in under 10 minutes
• Low protein consumption enabled by 48-well parallel processing
• Early formulation screening to de-risk development
• Alignment with published data supports reliability

Future Trends and Opportunities

Integration of simultaneous kD/B22 screening into high-throughput formulation pipelines can accelerate candidate selection. Combining light scattering data with advanced predictive models or machine learning could further refine aggregation risk assessment. Expanding this approach to diverse protein classes and stress conditions will broaden its applicability in biologics development.

Conclusion

This application note highlights a streamlined workflow to obtain independent kD and B22 values in a single run using the Uncle platform. The method reliably distinguishes between repulsive and attractive protein interactions, enabling rapid, early-stage assessment of aggregation propensity and supporting informed formulation decisions.

References

1. Shi S, et al. Method qualification and application of diffusion interaction parameter and virial coefficient. International Journal of Biological Macromolecules 62 (2013): 487–493.