Applying cross-correlation for thermal stability analysis of protein therapeutics

The thermal transition temperature (Tm) method of differential scanning calorimetry (DSC) and nano differential scanning fluorimetry (nanoDSF) has conventionally been used in the biopharmaceutical industry to characterize the protein thermal stability attribute. While the Tm method has demonstrated its feasibility for thermal stability analysis, the limitations of the Tm method have also been observed. To overcome these limitations, a cross-correlation method has been applied to thermal stability analysis as an enhanced analytical approach to thermal stability characterization. The cross-correlation method uses every data point in the nanoDSF and DSC thermograms to assess the overall profiles, while the conventional Tm method only compares a few data points corresponding to the Tm's. Applying the cross-correlation method enables differentiation of subtle variances between dissimilar thermograms that the Tm method may overlook. In this study we have analyzed several monoclonal antibody (mAb) samples before and after forced stress mainly inducing H₂O₂-induced stress and exposure to UV light, which causes tryptophan, methionine, and histidine oxidation. The results demonstrate that the cross-correlation method can distinguish significantly more stressed samples compared to the conventional Tm method and therefore enhances the differentiation capability of thermal stability analysis and can be used as a complementary method in research and candidate selection studies.