A central composite design to investigate the thermal stabilization of lysozyme

Purpose. The formulation and processing of protein drugs requires the stabi lization of the native, biologically active structure. Our aim was to inves tigate the thermal stability of a model protein, lysozyme, in the presence of two model excipients, sucrose and hydroxypropyl-beta-cyclodextrin (HP-be ta-CD). Methods. We used high sensitivity differential scanning calorimetry (HSDSC) in combination with a central composite design (CCD). As indicators of pro tein thermal stability, the measured responses were the unfolding transitio n temperature (T-m), the onset temperature of the denaturation (T-o), and t he extrapolated onset temperature (T-o,T-c). Results. A highly significant (F probability <0.001) statistical model resu lted From analysis of the data. The largest effect was due to pH lover the range 3.2-7.2), and the pH value that maximized T-m was 4.8. Several minor but significant effects were detected that were useful for mechanistic unde rstanding. In particular, the effects of protein concentration and cyclodex trin concentration on T-m and T-o,T-c were found to be pH-dependent This wa s indicative of the partially hydrophilic nature of protein-protein interac tions and protein-cyclodextrin interactions, respectively. Conclusions. Response surface methodology (RSM) proved efficient for the mo deling and optimization of lysozyme thermal stability as well as for the ph ysical understanding of the protein-sugar-cyclodextrin system in aqueous so lution.