2.8. Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry (DSC) is one of the most common techniques for characterization of freeze-dried protein drugs [121]. It is commonly used as a complementary method to spectroscopic and chromatographic characterization of proteins in the solid state [122]. The method consists of heating and cooling the sample together with a reference, measuring the difference in the amount of heat required to raise the temperature of the sample and reference. The difference is measured as a function of temperature, maintaining similar temperatures for the sample and reference [123]. DSC is a widely used technique for studying the unfolding of protein secondary structures and for characterizing the conformational stability of proteins under various conditions. DSC can be used in both solid and liquid states. Nano differential scanning calorimetry (nanoDSC) is used to analyze liquid samples of protein formulations. Lyophilized proteins are usually dissolved in buffers with pH around neutral, but water can also be used. From the heat capacity curve, the melting temperature (T_m), as well as the calorimetric enthalpy (ΔH_m) and entropy (ΔS_m) of the unfolding process can be accessed. For example, nanoDSC has been used to determine the melting temperature of proteins with FTIR spectroscopy [124]. On the other hand, DSC is more commonly used to study solid samples of protein formulations. While the unfolding of proteins produces endothermic peaks, their aggregation is shown to be an exothermic event. Moreover, in the study by Pikal et al. aggregation could be correlated with the loss of secondary structure and a decrease in the area under the denaturation endotherm. This process was reversible with trehalose as an excipient in the formulations [122]. By comparing the melting temperatures (T_m) of different protein lyophilizates, insights can be gained into their different secondary structures. DSC can also be used to optimize lyophilization parameters, ultimately resulting in a better appearance of the lyophilized protein cake. For example, Han et al. investigated the effect of sucrose and mannitol. It was found that the addition of sucrose to the formulations resulted in an upward shift in melting points, thus providing protection, while the addition of mannitol did not show the same benefit [125]. Ihnat et al. also investigated the effects of various excipients on the thermal stability of lyophilized protein samples [126].

Interestingly, protein stability does not always correlate well with glass transition temperature (T_g) values [127]. On the other hand, formulations stored above their T_g are generally less stable than those stored below. While storage below the T_g is necessary, it is not always sufficient to ensure stability. Therefore, when evaluating protein stability in lyophilized formulations, DSC characterization should be accompanied by other analytical methods [6,128,129]. In addition, modulated DSC (mDSC) can resolve total heat flow into thermodynamic (reversal) heat flow and kinetic (non-reversal) heat flow. For example, the enthalpy relaxation endotherm (kinetic) can be separated from the glass transition event (thermodynamic) [130]. Therefore, mDSC is particularly useful for detecting T_g present with other overlapping thermal events (glass transition) [131]. Therefore, mDSC is particularly useful for detecting T_g present with other overlapping thermal events (glass transition) [132], to correlate eutectic temperature (lowest possible melting temperature over all of the mixing ratios for the involved component species) events leading to cake collapse [133] and to study the effects of annealing on the thermal properties of frozen sucrose solutions [134,135,136]. It was also used as an additional method to ssHDX-MS for the analysis of the conformation of lyophilized IgG1 (Table 2) [137].

The effect of sucrose and histidine as excipients on T_g is presented in the table. The moisture content was measured by Karl Fischer titration [137].