New approach to stability assessment of protein solution formulations by differential scanning calorimetry.

A central composite rotate second order design was used to evaluate chicken egg-white lysozyme (lysozyme) thermal stability at different pH, and lysozyme, sucrose and 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD) concentrations, by means of differential scanning calorimetry (DSC). Four measurements were used to characterize the thermogram: the calorimetric enthalpy (DeltaH(cal)), the temperature at maximum heat flux (T(m)), the ratio of maximum heat flux over thermogram area (C(pT(m))/area), and the ratio of calorimetric enthalpies from the second heating cycle to the first enthalpy (R(DeltaH(cal))). These parameters were interpreted using the three step equilibrium model for protein degradation (irreversible degradation following reversible unfolding). In addition to degradation, increased lysozyme concentration leads to a sizeable decrease in DeltaH(cal) and area ratio, showing how it causes protein aggregation; which in turn promotes protein degradation. DeltaH(cal) and T(m) reach maxima at pH 5, R(DeltaH(cal)) at pH 4.19, while C(pT(m))/area increases linearly with pH, revealing a specific base catalysis of the irreversible degradation step. The role of sucrose concentration in lysozyme stabilization is linked to the stabilization of the unfolded moiety; it neither affects DeltaH(cal) nor C(pT(m))/area, but increases both T(m) and R(DeltaH(cal)). No influence of HPbetaCD on the stability of lyzozyme was observed, probably due to low concentrations employed.