(692g) Accurate Determination of B22 Data – Multivariate Analysis to Improve B22 As a Predictive Tool

Protein-protein molecular interactions are known to be involved in protein solution aggregation behaviour and are a common issue for the manufacturing of therapeutic proteins such as mAbs. Much effort has been employed to gain a better understanding of aggregation, however the mechanisms leading to protein aggregation are still not fully understood. The osmotic second virial coefficient (B₂₂) is a fundamental physiochemical property that describes protein-protein interactions in solution, which can be a useful tool for not only predicting the aggregation propensity of proteins but also protein solubility, crystallisation and phase behaviour.

A rapid and high-throughput way of determining B₂₂ is self-interaction chromatography (SIC). However, the method is still critiqued to be less accurate than its predecessors such as static light scattering. This paper is evaluating the impact of the different experimental strategies and theoretical estimations on the parameters involved in the second virial coefficient determined by SIC. This has been done to help focus on the small variations and combinations that have the largest impact on B₂₂ and can directly determine a critically different value.

A number of journal papers recently published describe important experimental considerations for more accurate B₂₂ estimations. Many of these small differences in the experimental procedure could result in wrongly estimated B₂₂ values and the interpretation that the protein-protein interactions are repulsive when they are actually attractive and an instable condition for the protein. To easily identify what initial parameters that have the greatest impact an extensive multivariate and sensitivity analysis has been performed to report the impact of a single or multiple parameters on B₂₂. The values that were found to be the most accurate representations of B₂₂ were compared to aggregation data to identity that B₂₂ was a useful and powerful predictor of aggregation propensity.

Apart from proving to be an accurate predictor for stability in pre-formulation and formulation studies, this tool has also shown to be a good indicator for suitable conditions that can be used for purification and manufacturing of mAbs to enable better product stability while not compromising recovery and quality.

In summary, this work is providing a good insight into important considerations when determining protein-protein interactions and virial coefficients to obtain accurate values that can be used as a powerful tool for biopharmaceutical development.

REFERENCES

Hedberg, S. H. M., Heng, J. Y. Y., Williams, D. R. & Liddell, J. M. (2015). Self-Interaction Chromatography of MAbs: Accurate Measurement of Dead Volumes. Pharmaceutical Research, 32, 3975-3985.

Quigley, A., Heng, J., Liddell, J. & Williams, D. (2013). The Accurate Measurement of Second Virial Coefficients Using Self Interaction Chromatography: Experimental Considerations. European Journal of Pharmaceutics and Biopharmaceutics, 85, 1103-1111.