title: 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.
abstract: Aggregation propensity is a critical attribute of protein therapeutics that can influence production, manufacturing, delivery, and potential activity and safety (immunogenicity). 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. Although many biophysical methods have been developed to characterize protein aggregation, most established methods are low-throughput, requiring large quantities of protein, lengthy assay 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, versatility and low protein material requirements. The results suggest that kosmotrope-based solubility assessment has broad applicability to selecting protein therapeutic candidates with low aggregation propensity and high "developability" to progress into development.
authors.affiliation: Bristol-Myers Squibb, Princeton, New Jersey 08540, USA. aaron.yamniuk@bms.com
authors.forename: Aaron P
authors.initials: AP
authors.lastname: Yamniuk
authors.forename: Noah
authors.initials: N
authors.lastname: Ditto
authors.forename: Mehul
authors.initials: M
authors.lastname: Patel
authors.forename: Jun
authors.initials: J
authors.lastname: Dai
authors.forename: Preeti
authors.initials: P
authors.lastname: Sejwal
authors.forename: Paul
authors.initials: P
authors.lastname: Stetsko
authors.forename: Michael L
authors.initials: ML
authors.lastname: Doyle
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry*; D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D002460:Cell Line; D055808:Drug Discovery; D006801:Humans; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012995:Solubility
chemical_list: D000906:Antibodies; D011993:Recombinant Fusion Proteins; D000645:Ammonium Sulfate
keywords: ammonium sulfate; antibody; biologics; formulation; high throughput technologies; kosmotrope; protein aggregation; protein therapeutic; solubility; stability