RESEARCH ARTICLES Drug Discovery-Development Interface Application of a Kosmotrope-Based Solubility Assay to Multiple Protein Therapeutic Classes Indicates Broad Use as a High-Throughput Screen for Protein Therapeutic Aggregation Propensity

Aggregation propensity is a critical attribute of protein therapeutics that can influence production, manufacturing, delivery, and potential activity and safety (immunogenic- ity). It is therefore imperative to select molecules with low aggregation propensity in the early stages of drug discovery to mitigate the risk of delays or failure in clinical development. Al- though many biophysical methods have been developed to characterize protein aggregation, most established methods are low-throughput, requiring large quantities of protein, lengthy as- say times, and/or significant upstream sample preparation, which can limit application in early candidate screening. To avoid these limitations, we developed a reliable method to characterize aggregation propensity, by measuring the relative solubility of protein therapeutic candidates in the presence of the kosmotropic salt ammonium sulfate. Manual bench-scale and automated plate-based methods were applied to different protein therapeutic formats including Adnectins, domain antibodies, PEGylated Adnectins, Fc fusion proteins, and monoclonal antibodies. The kosmotrope solubility data agreed well with the aggregation propensity observed by established methods, while being amenable to high-throughput screening because of speed, simplicity, ver- satility and low protein material requirements. The results suggest that kosmotrope-based sol- ubility assessment has broad applicability to selecting protein therapeutic candidates with low aggregation propensity and high "developability" to progress into development. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2424-2439, 2013 Keywords: protein aggregation; solubility; protein therapeutic; biologics; antibody; stability; kosmotrope; ammonium sulfate; high throughput technologies; formulation