Lysozyme-Sucrose Interactions in the Solid State: Glass Transition, Denaturation, and the Effect of Residual Water

The freeze-dryingof proteins, along with excipients, offers asolution for increasing the shelf-life of protein pharmaceuticals.Using differential scanning calorimetry, thermogravimetric analysis,sorption calorimetry, and synchrotron small-angle X-ray scattering(SAXS), we have characterized the properties at low (re)hydrationlevels of the protein lysozyme, which was freeze-dried together withthe excipient sucrose. We observe that the residual moisture contentin these samples increases with the addition of lysozyme. This resultsfrom an increase in equilibrium water content with lysozyme concentrationat constant water activity. Furthermore, we also observed an increasein the glass transition temperature (T (g)) of the mixtures with increasing lysozyme concentration. Analysisof the heat capacity step of the mixtures indicates that lysozymedoes not participate in the glass transition of the sucrose matrix;as a result, the observed increase in the T (g) of the mixtures is the consequence of the confinement of the amorphoussucrose domains in the interstitial space between the lysozyme molecules.Sorption calorimetry experiments demonstrate that the hydration behaviorof this formulation is similar to that of the pure amorphous sucrose,while the presence of lysozyme only shifts the sucrose transitions.SAXS analysis of amorphous lysozyme-sucrose mixtures and unfoldingof lysozyme in this environment show that prior to unfolding, thesize and shape of lysozyme in a solid sucrose matrix are consistentwith its native state in an aqueous solution. The results obtainedfrom our study will provide a better understanding of the low hydrationbehavior of protein-excipient mixtures and support the improvedformulation of biologics.