Spot aggregation early with B22 and kD on Stunner

Importance of the Topic

High-concentration protein therapeutics are increasingly used in biopharmaceutical applications but carry a heightened risk of aggregation, which can compromise safety, efficacy and shelf life. Early identification of formulation conditions that minimize aggregation is therefore critical for accelerating development and ensuring product quality.

Objectives and Study Overview

This study demonstrates how the Stunner platform simultaneously quantifies two colloidal stability parameters—the diffusion interaction parameter (kD) and the second virial coefficient (B22)—using a single dilution series for rapid formulation screening. Model proteins, hen egg-white lysozyme (HEWL) and the NIST monoclonal antibody reference material (RM 8671), were used to validate the approach under different buffer conditions.

Methodology and Instrumentation

Samples were prepared and filtered before analysis to remove particulates. HEWL was studied in 10 mM acetate buffer (pH 4.6) with either 100 mM or 400 mM NaCl at 1–11 mg/mL, and NIST mAb in 25 mM histidine/histidine-HCl buffer (pH 6) at 0.25–6 mg/mL. The Stunner platform integrates micro-volume UV/Vis absorbance with dynamic light scattering (DLS) and static light scattering (SLS) in a 96-well microfluidic plate, consuming just 2 µL per sample. Four DLS acquisitions of 5 s each were recorded per sample, with buffer blanks and PEG40 as a scattering reference. A Homebrew application measured buffer viscosities via 30 nm reference beads to refine diffusion coefficient calculations.

Main Results and Discussion

Stunner analysis revealed a positive kD for HEWL in low-salt buffer (repulsive interactions) and a negative kD under high-salt conditions (net attraction), consistent with cloudiness observed at 4 °C in high salt. B22 values mirrored these trends, confirming increased aggregation propensity when electrostatic shielding by NaCl reduces repulsion. For NIST mAb, both kD and B22 were positive in its optimized histidine buffer, aligning closely with published reference values. Measured buffer viscosities (1.16–1.49 cP) were incorporated into data processing to improve accuracy.

Benefits and Practical Applications

• High-throughput, low-volume screening of formulations without serial dilutions
• Simultaneous determination of protein concentration, kD and B22 from a single run
• Automated outlier detection and advanced DLS views for data confidence
• Compatibility with automation systems and 21 CFR Part 11 compliance

Future Trends and Potential Applications

Expanding this approach to larger protein libraries and integrating machine-learning for pattern recognition could further streamline formulation development. Coupling early aggregation prediction with orthogonal biophysical techniques (e.g., thermal shift or capillary electrophoresis) will enhance understanding of stability under real-world storage and delivery conditions.

Conclusion

The Stunner platform enables rapid, reproducible assessment of protein aggregation risk by measuring kD and B22 independently yet simultaneously. Its minimal sample consumption and high-throughput workflow make it a valuable tool for early formulation screening in biopharmaceutical development.

References

• DB Volkin et al., Advanced Drug Delivery Reviews, 2011;63:1118–1159
• A Saluja et al., Biophysical Journal, 2010;99(8):2657–2665
• J Schiel et al., NISTmAb technical case study, HOS 2017 workshop