Differential scanning calorimetry and fluorimetry measurements of monoclonal antibodies and reference proteins: Effect of scanning rate and dye selection



Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) were used to measure the transition temperatures of four proteins: RNase A, invertase, rituximab, and the NISTmAb (NIST Reference Material, RM 8671). The proteins were combined with several different fluorescent dyes for the DSF measurements. This study compares the results of DSC and DSF measurements of transition temperatures with different types of proteins, dye combinations, and thermal scan rates. As protein unfolding is often influenced by kinetic effects, we measured the transition temperatures of the proteins using DSC over a range of temperature scan rates and compared them to the data obtained from DSF over comparable temperature scan rates. The results when the proteins were combined with Sypro Orange® and bis-ANS for the DSF measurements had the best correlations with the transition temperatures determined by calorimetry. The scan rate was found to be an important variable when comparing results between DSC and DSF. The van't Hoff enthalpy changes for the transitions were calculated from the DSC data by using a non-two-state model and from the DSF values using a two-state model. The calculated van't Hoff enthalpy changes did not show a good correlation between the two methods. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:677–686, 2017