Spot aggregation early with B22 and kD on Stunner

Significance of the Topic

The early detection of protein aggregation is essential for the development and manufacturing of high-concentration biopharmaceuticals. Aggregation can compromise drug efficacy, safety and shelf life. Quantitative parameters such as the diffusion interaction parameter (kD) and the second virial coefficient (B22) provide insight into colloidal stability and aggregation propensity, guiding formulation optimization.

Objectives and Study Overview

This study evaluates the ability of the Stunner platform to rapidly and simultaneously measure kD and B22 for two model proteins. Hen egg-white lysozyme (HEWL) formulations at low and high salt conditions were compared, and a NIST monoclonal antibody reference material (RM 8671) was assessed in a standard histidine buffer. The aim is to demonstrate high-throughput aggregation screening using just micro-volumes.

Used Methodology and Instrumentation

The Stunner system integrates micro-volume UV/Vis spectral quantification with dynamic light scattering (DLS) and static light scattering (SLS) in a single cuvette platform. Samples (2 µL) are loaded in microfluidic plates in triplicate or higher replicates. DLS acquisitions (4 × 5 s) yield diffusion coefficients and scattering intensities across a dilution series, while fixed path-length absorbance reads protein concentrations without dilution errors. The Homebrew toolkit was used to develop a custom viscosity assay employing 30 nm polystyrene beads, enabling accurate correction of diffusion calculations.

Main Results and Discussion

HEWL in 10 mM acetate, pH 4.6 showed a positive kD under 100 mM NaCl (net repulsion) and a negative kD under 400 mM NaCl (net attraction), consistent with clouding behavior at low temperature and clarification upon warming. B22 values mirrored these trends. The NIST mAb in 25 mM histidine, pH 6 exhibited positive kD and B22, indicating a formulation resistant to aggregation. Viscosities measured by Homebrew increased with salt concentration (1.16 cP at 100 mM NaCl to 1.25 cP at 400 mM NaCl) and were 1.49 cP for the antibody buffer, matching literature values.

Benefits and Practical Applications

• Simultaneous quantification of protein concentration, kD and B22 accelerates formulation screening.
• Micro-volume, dilution-free workflow conserves precious samples.
• Automated outlier detection and advanced DLS view enhance data confidence.
• Customizable Homebrew assays permit tailored viscosity and quality assessments.

Future Trends and Applications

The integration of high-throughput light scattering with automated microfluidics is poised to evolve toward real-time stability monitoring, AI-driven formulation prediction and ultra-miniaturized screening. Expanding Homebrew libraries will support novel assays for viscosity, aggregation kinetics and excipient interactions.

Conclusion

Stunner offers a unified platform for rapid, accurate assessment of protein self-interactions via kD and B22, validated against published benchmarks. Its micro-volume, high-throughput design and flexible Homebrew toolkit enable comprehensive formulation development and aggregation risk mitigation in biopharmaceutical research.

References

• Volkin DB, et al. Protein-excipient interactions: mechanisms and biophysical characterization. Adv Drug Deliv Rev. 2011;63:1118–1159.
• Saluja A, et al. Diffusion and sedimentation interaction parameters as predictors of protein aggregation. Biophys J. 2010;99(8):2657–2665.
• Schiel J, et al. NISTmAb case study: technical comparison. HOS 2017 Workshop.