Protein Internal Dynamics Associated With Pre-System Glass Transition Temperature Endothermic Events: Investigation of Insulin and Human Growth Hormone by Solid State Hydrogen/Deuterium Exchange.

Lyophilized proteins are generally stored below their glass transition temperature (Tg) to maintain long-term stability. Some proteins in the (pure) solid state showed a distinct endotherm at a temperature well below the glass transition, designated as a pre-Tg endotherm. The pre-Tg endothermic event has been linked with a transition in protein internal mobility. The aim of this study was to investigate the internal dynamics of 2 proteins, insulin and human growth hormone (hGH), both of which exhibit the pre-Tg endothermic event with onsets at 50°C-60°C. Solid state hydrogen/deuterium (H/D) exchange of both proteins was characterized by Fourier transform infrared spectroscopy over a temperature range from 30°C to 80°C. A distinct sigmoidal transition in the extent of H/D exchange had a midpoint of 56.1 ± 1.2°C for insulin and 61.7 ± 0.9°C for hGH, suggesting a transition to greater mobility in the protein molecules at these temperatures. The data support the hypothesis that the pre-Tg event is related to a transition in internal protein mobility associated with the protein dynamical temperature. Exceeding the protein dynamical temperature is expected to activate protein internal motion and therefore may have stability consequences.

Using the fluorescence red edge effect to assess the long-term stability of lyophilized protein formulations.

Nanosecond relaxation processes in sugar matrices are causally linked through diffusional processes to protein stability in lyophilized formulations. Long-term protein degradation rates track mean-squared displacement (⟨u(2)⟩) of hydrogen atoms in sugar glasses, a parameter describing dynamics on a time scale of picoseconds to nanoseconds. However, measurements of ⟨u(2)⟩ are usually performed by neutron scattering, which is not conducive to rapid formulation screening in early development. Here, we present a benchtop technique to derive a ⟨u(2)⟩ surrogate based on the fluorescence red edge effect. Glycerol, lyophilized trehalose, and lyophilized sucrose were used as model systems. Samples containing 10(-6) mole fraction of rhodamine 6G, a fluorophore, were excited at either 532 nm (main peak) or 566 nm (red edge), and the ⟨u(2)⟩ surrogate was determined based the corresponding Stokes shifts. Results showed reasonable agreement between ⟨u(2)⟩ from neutron scattering and the surrogate from fluorescence, although deviations were observed at very low temperatures. We discuss the sources of the deviations and suggest technique improvements to ameliorate these. We expect that this method will be a valuable tool to evaluate lyophilized sugar matrices with respect to their ability to protect proteins from diffusion-limited degradation processes during long-term storage. Additionally, the method may have broader applications in amorphous pharmaceutical solids.