Intrinsic Differential Scanning Fluorimetry for Protein Stability Assessment in Microwell Plates

Intrinsic differential scanning fluorimetry (DSF) is essential for analyzing protein thermal stability. Until now, intrinsic DSF was characterized by medium throughput and high consumable costs. Here, we present a microplate-based intrinsic DSF approach that enables the measurement of up to 384 samples in parallel by consuming only 10 μL per sample. We systematically test and benchmark the new intrinsic DSF against gold-standard methods such as differential scanning microcalorimetry and circular dichroism. Using a range of model proteins and sample conditions, we demonstrate the robustness and versatility of the intrinsic DSF method for characterizing protein stability and ranking protein drug candidates. In addition, we demonstrate modulated scanning fluorimetry (MSF) capabilities on the intrinsic DSF hardware that enable simultaneous MSF measurements in 384-microwell plates. Overall, the presented technology is a powerful tool for the early stability analysis of various protein samples and drug candidates.