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

Significance of the Topic

High concentration biologic formulations require robust assessment of aggregation propensity to ensure safety, efficacy, and manufacturability

Objectives and Study Overview

This work illustrates how simultaneous measurement of the diffusion interaction parameter kD and second virial coefficient B22 on the Uncle platform can predict protein colloidal stability. Lysozyme was used as a model to compare formulation performance in low and high salt conditions

Methodology

Lysozyme was prepared at 25 mg/mL in 10 mM sodium acetate buffer pH 5.2 with either 100 mM or 400 mM NaCl
Serial dilutions to 20, 15, 10, and 5 mg/mL were filtered and centrifuged
Dynamic light scattering (DLS) data were collected with four acquisitions of 5 seconds per sample to determine hydrodynamic diameters
Debye plots from diffusion coefficient versus concentration and scattering intensity versus concentration were generated to calculate kD and B22, respectively

Used Instrumentation

Uncle stability platform equipped with dynamic light scattering and static light scattering detectors
Temperature control from 15 to 95 C and sealed sample handling for up to 48 samples in parallel

Main Results and Discussion

• At 400 mM NaCl negative kD (–6.0 mL/g) and B22 (–1.6E-4 mol·mL/g2) values indicate attractive interactions leading to reversible aggregation under cold storage
• At 100 mM NaCl positive kD (3.8 mL/g) and B22 (1.7E-4 mol·mL/g2) values reflect repulsive interactions that promote solubility
• Results closely align with published literature, confirming method reliability
• Reversible solution cloudiness at high salt demonstrates non denaturing protein associations

Benefits and Practical Applications

• Fast, simultaneous acquisition of kD and B22 using minimal sample volumes and low concentration series
• Early screening of aggregation risk to accelerate formulation development and reduce costs
• High sample throughput enables profiling of multiple molecules and formulations in parallel
• Supports decision making in biologic candidate selection and quality control workflows

Future Trends and Potential Applications

• Integration into high throughput screening of extensive biologic libraries
• Combining dual parameter data with machine learning models for predictive stability assessment
• Expansion to next generation modalities and broader colloidal systems
• Unified platforms integrating kD, B22, thermal stability, and aggregation onset measurements

Conclusion

The Uncle platform provides an efficient, single experiment approach for independent, simultaneous measurement of kD and B22. Results correlate with established literature, enabling informed formulation decisions to mitigate aggregation risk in high concentration biologics.

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