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

Mol Pharm. 2023 Sep 4;20(9):4664-4675. doi: 10.1021/acs.molpharmaceut.3c00403. Epub 2023 Aug 9.


The freeze-drying of proteins, along with excipients, offers a solution 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)hydration levels of the protein lysozyme, which was freeze-dried together with the excipient sucrose. We observe that the residual moisture content in these samples increases with the addition of lysozyme. This results from an increase in equilibrium water content with lysozyme concentration at constant water activity. Furthermore, we also observed an increase in the glass transition temperature (Tg) of the mixtures with increasing lysozyme concentration. Analysis of the heat capacity step of the mixtures indicates that lysozyme does not participate in the glass transition of the sucrose matrix; as a result, the observed increase in the Tg of the mixtures is the consequence of the confinement of the amorphous sucrose domains in the interstitial space between the lysozyme molecules. Sorption calorimetry experiments demonstrate that the hydration behavior of 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 unfolding of lysozyme in this environment show that prior to unfolding, the size and shape of lysozyme in a solid sucrose matrix are consistent with its native state in an aqueous solution. The results obtained from our study will provide a better understanding of the low hydration behavior of protein-excipient mixtures and support the improved formulation of biologics.

Keywords: differential scanning calorimetry; glass transition; hydration; proteins; small-angle X-ray scattering; solid-state formulations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calorimetry, Differential Scanning
  • Excipients / chemistry
  • Freeze Drying / methods
  • Muramidase* / chemistry
  • Proteins / chemistry
  • Scattering, Small Angle
  • Sucrose / chemistry
  • Vitrification*
  • Water / chemistry
  • X-Ray Diffraction


  • Muramidase
  • Sucrose
  • Water
  • Excipients
  • Proteins