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-pTm/area), and the ratio of calorimetric enthalpies from the second heating cycle to the first enthalpy (R-DeltaHcal). 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-DeltaHcal at pH 4.19, while C-pTm/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-pTm/area, but increases both T-m and R-DeltaHcal. No influence of HPPCD on the stability of lyzozyme was observed, probably due to low concentrations employed. (C) 2002 Elsevier Science B.V. All rights reserved.