GOALI: Aggregation of Protein Therapeutics in Aqueous Solutions

治疗性蛋白质为人类疾病（如糖尿病和癌症）提供了许多独特的治疗方法。然而，由于其精细的三维结构，蛋白质很难在药品所需的18-24个月的保质期内保持稳定。此外，目前还不可能预测哪种解决条件会产生足够的稳定性。首席研究员（pi）建议对蛋白质的关键物理性质进行基础研究，以开发预测长期稳定的最佳解决条件的方法。这个多学科项目结合了科罗拉多大学化学工程和制药科学系与工业合作伙伴安进公司的努力。pi建议确定临界溶液条件（pH，离子强度和稳定辅料的存在）对几种模型蛋白的聚集速率和途径的影响。将测量可溶性低聚物和不溶性沉淀物的形成和损失（如适用）的水平和动力学，并使用红外和衍生紫外光谱确定聚集体中的蛋白质结构。为了了解这些影响，pi将确定pH、离子强度和稳定辅料对蛋白质结构和热力学性质的影响。蛋白质的二级和三级结构将研究与圆二色，红外，荧光和衍生紫外光谱。将测量展开的自由能和氢-氘交换速率，以验证增加天然态的热力学稳定性和将天然态集合向最紧凑的物种移动是抑制蛋白质聚集的重要因素的假设。第二渗透维里系数将通过光散射来测量，以评估溶液条件对蛋白质体系胶体稳定性的影响。高压光谱技术将用于补充这些测量。最后，溶液条件对模型蛋白化学稳定性的影响（例如，脱胺、氧化和非天然二硫形成）将由安进的工业合作者确定，模型蛋白的物理和化学稳定性之间的联系将被阐明。

Therapeutic proteins provide numerous unique treatments for human diseases (e.g., diabetes, and cancer). However, due to their delicate three-dimensional structures, proteins are very difficult to keep stable during the required 18-24 month shelf life of a pharmaceutical product. Furthermore, it is currently not possible to predict which solution conditions will result in sufficient stability. The Principal Investigators (PIs) propose to conduct fundamental studies of critical physical properties of proteins to develop methods for predicting optimal solution conditions for long-term stability. This multidisciplinary project combines efforts from Chemical Engineering and Pharmaceutical Sciences Departments at the University of Colorado with those of Amgen, an industrial partner. The PIs propose to determine the effects of critical solution conditions (pH, ionic strength and presence of stabilizing excipients) on rate and pathway for aggregation of several model proteins. Levels, and kinetics for formation and loss (where applicable) of soluble oligomeric species and insoluble precipitates will be measured, and structure of proteins in aggregates will be determined with infrared and derivative UV spectroscopies. To understand these effects, the PIs will determine the effects of pH, ionic strength and stabilizing excipients on protein structure and thermodynamic properties. Protein secondary and tertiary structures will be studied with circular dichroism, infrared, fluorescence and derivative-UV spectroscopies. The free energy of unfolding and hydrogen-deuterium exchange rates will be measured to test the hypothesis that increasing the thermodynamic stability of the native state and shifting the native state ensemble toward most compact species are important factors for inhibiting protein aggregation. The second osmotic virial coefficient will be measured by light scattering to assess the effects of solution conditions on the colloidal stability of the protein systems. High-pressure spectroscopic techniques will be used to complement these measurements. Finally, the effect of solution conditions on chemical stability (e.g., deamidation, oxidation and non-native disulfide formation) of the model proteins will be determined by the industrial collaborators at Amgen, and linkages between physical and chemical stability of the model proteins will be elucidated.