T. Wright
University of California, Los Angeles,
United States
Keywords: protein stability, thermodynamic analysis, differential scanning fluorimetry (DSF)
Summary:
Protein stability is a critical determinant of efficacy, safety, and shelf life in therapeutic and vaccine formulations. However, traditional methods used to characterize protein unfolding, such as calorimetry and circular dichroism, often require large sample volumes, high concentrations, and lengthy measurement times, which limits their utility in formulation screening. Here, we present a rapid and high-throughput differential scanning fluorimetry (DSF) platform that enables extraction of thermodynamic parameters of protein unfolding from minimal sample quantities. Operating under a two-state reversible unfolding model, DSF tracks fluorescence changes as hydrophobic dyes bind to unfolding proteins, providing quantitative measurements of Gibbs free energy (ΔG), enthalpy (ΔH), and entropy (ΔS). These parameters offer mechanistic insight into whether destabilization arises from enthalpic or entropic contributions, which are key considerations in formulation design. We demonstrate the versatility of this approach by characterizing the unfolding thermodynamics of multiple enzymes, including lysozyme, carbonic anhydrase, chymotrypsin, horseradish peroxidase, and cellulase, under varying solvent and additive conditions. Compared with conventional biophysical analyses, DSF achieves more than a twenty-four-fold reduction in experimental time while maintaining accuracy and reproducibility. This streamlined approach provides a powerful tool for rapidly screening excipients, stabilizers, and formulation conditions, advancing the rational design of stable protein-based therapeutics and biomacromolecule carriers