The stability of biologically produced
pharmaceuticals is the limiting
factor to various applications, which can be improved by formulation
in solid-state forms, mostly via lyophilization. Knowledge about the
protein structure at the molecular level in the solid state and its
transition upon rehydration is however scarce, and yet it most likely
affects the physical and chemical stability of the biological drug.
In this work, synchrotron small- and wide-angle X-ray scattering (SWAXS)
are used to characterize the structure of a model protein, lysozyme,
in the solid state and its structural transition upon rehydration
to the liquid state. The results show that the protein undergoes distortion
upon drying to adopt structures that can continuously fill the space
to remove the protein–air interface that may be formed upon
dehydration. Above a hydration threshold of 35 wt %, the native structure
of the protein is recovered. The evolution of SWAXS peaks as a function
of water content in a broad range of concentrations is discussed in
relation to the structural changes in the protein. The findings presented
here can be used for the design and optimization of solid-state formulations
of proteins with improved stability.
Although unfolding of protein in the liquid state is relatively well studied, its mechanisms in the solid state, are much less understood. We evaluated the reversibility of thermal unfolding of lysozyme with respect to the water content using a combination of thermodynamic and structural techniques such as differential scanning calorimetry, synchrotron small and wide-angle X-ray scattering (SWAXS) and Raman spectroscopy. Analysis of the endothermic thermal transition obtained by DSC scans showed three distinct unfolding behaviors at different water contents. Using SWAXS and Raman spectroscopy, we investigated reversibility of the unfolding for each hydration regime for various structural levels including overall molecular shape, secondary structure, hydrophobic and hydrogen bonding interactions. In the substantially dehydrated state below 37 wt% of water the unfolding is an irreversible process and can be described by a kinetic approach; above 60 wt% the process is reversible, and the thermodynamic equilibrium approach is applied. In the intermediate range of water contents between 37 wt% and 60 wt%, the system is phase separated and the thermal denaturation involves two processes: melting of protein crystals and unfolding of protein molecules. A phase diagram of thermal unfolding/denaturation in lysozyme - water system was constructed based on the experimental data.
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