Fang Rui, Grobelny Pawel J, Bogner Robin H, Pikal Michael J
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269.
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269; Institute of Materials Science, University of Connecticut, Storrs, Connecticut 06269.
J Pharm Sci. 2016 Nov;105(11):3290-3295. doi: 10.1016/j.xphs.2016.07.028. Epub 2016 Sep 16.
Lyophilized proteins are generally stored below their glass transition temperature (T) 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-T endotherm. The pre-T 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-T 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-T 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.
冻干蛋白质通常储存在其玻璃化转变温度（T）以下，以保持长期稳定性。一些处于（纯）固态的蛋白质在远低于玻璃化转变温度的某个温度下出现明显的吸热峰，称为预T吸热峰。预T吸热事件与蛋白质内部流动性的转变有关。本研究的目的是研究两种蛋白质，胰岛素和人生长激素（hGH）的内部动力学，这两种蛋白质都表现出在50°C - 60°C开始的预T吸热事件。通过傅里叶变换红外光谱在30°C至80°C的温度范围内对两种蛋白质的固态氢/氘（H/D）交换进行了表征。H/D交换程度的明显S形转变，胰岛素的中点为56.1±1.2°C，hGH的中点为61.7±0.9°C，表明在这些温度下蛋白质分子向更大的流动性转变。数据支持这样的假设，即预T事件与与蛋白质动态温度相关的蛋白质内部流动性转变有关。超过蛋白质动态温度预计会激活蛋白质内部运动，因此可能会对稳定性产生影响。
