High Throughput Differential Scanning Fluorimetry (DSF) Formulation Screening with Complementary Dyes to Assess Protein Unfolding and Aggregation in Presence of Surfactants

摘要

Purpose The purpose was to evaluate DSF for high throughput screening of protein thermal stability (unfolding/ aggregation) across a wide range of formulations. Particular focus was exploring PROTEOSTAT? - a commercially available fluorescent rotor dye - for detection of aggregation in surfactant containing formulations. Commonly used hydrophobic dyes (e.g. SYPRO? Orange) interact with surfactants, complicating DSF measurements. Methods CRM197 formulations were prepared and analyzed in standard 96-well plate rT-PCR system, using SYPRO? Orange and PROTEOSTAT? dyes. Orthogonal techniques (DLS and IPF) are employed to confirm unfolding/aggregation in selected formulations. Selected formulations are subjected to non-thermal stresses (stirring and shaking) in plate based format to characterize aggregation with PROTEOSTAT?. Results Agreement is observed between SYPRO? Orange (unfolding) and PROTEOSTAT? (aggregation) DSF melt temperatures across wide range of non-surfactant formulations. PROTEOSTAT? can clearly detect temperature induced aggregation in low concentration (0.2 mg/mL) GRM197 formulations containing surfactant. PROTEOSTAT? can be used to explore aggregation due to non-thermal stresses in plate based format amenable to high throughput screening. Conclusions DSF measurements with complementary extrinsic dyes (PROTEOSTAT?, SYPRO? Orange) are suitable for high throughput screening of antigen thermal stability, across a wide range of relevant formulation conditions - including surfactants -with standard, plate based rT-PGR instrumentation.