Mechanisms of Damage to Pharmaceutical Proteins at Oil-Water Interfaces

油水界面药物蛋白的损伤机制

• 批准号: 1133871
• 负责人: Theodore Randolph
• 金额: $ 33.9万
• 依托单位: University of Colorado at Boulder
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1133871&HistoricalAwards=false
• 关键词: Mechanisms; Damage; Pharmaceutical; Proteins; Oil

1133871RandolphIntroduction: During their production, processing, storage and delivery to patients, therapeutic proteins are exposed to various interfaces, such as the interface between the silicone oils that are used to lubricate glass syringes and aqueous solutions in which the proteins are formulated. Proteins may adsorb to these interfaces, which in turn can result in aggregation of the protein. Protein aggregation in pharmaceutical formulations is associated with changes in potency, risks of increased immunogenicity, and shortened shelf life, and hence is a major contributor to the estimated $1.2 billion required for development of a new protein-based therapeutic. Interfacial damage of proteins is a particular problem at fluid-fluid interfaces, where the dynamic nature of the interface (e.g., in response to shear forces experienced during shipping and handling of a protein formulation) may offer increased exposure of interfaces to proteins. This project examines the behavior of therapeutic proteins as they interact with silicone oil/water interfaces. The mechanisms of such interactions are probed with advanced spectroscopic and physical techniques, with a goal of developing rational design strategies to prevent interfacial protein damage and reduce associated costs and health risks. Intellectual Merit: Protein adsorption and aggregation at interfaces is ubiquitous, but the fundamental mechanisms leading to protein adsorption at interfaces and consequent generation of aggregates remain poorly understood. This project, a collaboration between two research groups with expertise in protein interfacial science, protein conformational thermodynamics and aggregation kinetics will address both the microscale mechanisms that lead to protein adsorption and unfolding at interfaces, and the kinetic processes that result in macroscopically observable protein aggregation. To characterize the kinetics of adsorption and interfacial aggregation at oil-water interfaces, a combination of several state-of-the-art experimental techniques will be developed and applied. These include single-molecule tracking micro-rheology (using fluorescence microscopy), emulsion adsorption, fluorescence-activated cell sorting (FACS) and front-face fluorescence quenching of adsorbed protein. Molecular conformation of proteins at silicone oil-water interfaces will be measured using Forster resonant energy transfer (FRET) in order such as to establish direct connections between protein conformation and dynamic processes such adsorption, desorption, interfacial mobility, aggregation. Likewise, protein adsorption at the air-water interface will be measured using dynamic pendant bubble tensiometry, emulsion depletion experiments and FACS, and the resulting protein aggregation monitored by flow microscopy, chromatography and FACS. The links between interfacially-induced protein damage and formulation conditions will be explored by determining the effects of interfacial area change, protein concentration, thermodynamic conditions, and excipients. By manipulating the thermodynamic stability of the protein's native state structure (e.g., with stabilizing excipients), microscopic protein unfolding processes will be linked to interfacial phenomena and macroscopic measurements of agitation-induced-aggregation kinetics.Broader Impacts: The project will have several broad impacts. First, a detailed understanding of protein adsorption at oil-water interfaces will aid the design of formulations that provide protection against interfacially-induced protein aggregation, reduce development costs and offer increased patient safety. Second, the many new techniques that will be tested and developed will be of use to a wide variety of academic and industrial scientists. Furthermore, protein adsorption and aggregation at interfaces is of critical importance to several other scientific endeavors, such as vaccinology, applications of microfluidics and nanotechnology in the diagnostics arena and the development of implantable medical devices. By linking two research groups with diverse expertise in interfacial science and protein formulation, the graduate and undergraduate students who participate in this research will receive a broad, cross-disciplinary training. In addition, because of the groups close ties with the biopharmaceutical industry, the results of this research will be rapidly disseminated so as to afford maximum impact in practical applications of the proposed new, fundamental scientific studies.

1133871Randolph简介：在生产、加工、储存和交付给患者的过程中，治疗性蛋白质会暴露于各种界面，例如用于润滑玻璃注射器的硅油与配制蛋白质的水溶液之间的界面。 蛋白质可能吸附到这些界面上，进而导致蛋白质聚集。药物制剂中的蛋白质聚集与效力变化、免疫原性增加的风险和保质期缩短有关，因此是开发新的蛋白质疗法所需的约 12 亿美元的主要贡献者。 蛋白质的界面损伤是流体-流体界面的一个特殊问题，其中界面的动态性质（例如，响应蛋白质制剂运输和处理过程中经历的剪切力）可能会增加界面与蛋白质的接触。 该项目研究了治疗性蛋白质与硅油/水界面相互作用时的行为。 利用先进的光谱和物理技术探讨这种相互作用的机制，目的是制定合理的设计策略，以防止界面蛋白损伤并降低相关成本和健康风险。 智力优点：蛋白质在界面处的吸附和聚集是普遍存在的，但导致蛋白质在界面处吸附并随后产生聚集体的基本机制仍然知之甚少。该项目由两个在蛋白质界面科学、蛋白质构象热力学和聚集动力学方面拥有专业知识的研究小组合作，将解决导致蛋白质在界面吸附和展开的微观机制，以及导致宏观可观察到的蛋白质聚集的动力学过程。为了表征油水界面吸附和界面聚集的动力学，将开发和应用几种最先进的实验技术的组合。其中包括单分子跟踪微流变学（使用荧光显微镜）、乳液吸附、荧光激活细胞分选 (FACS) 和吸附蛋白质的正面荧光猝灭。将使用福斯特共振能量转移（FRET）测量硅油-水界面处蛋白质的分子构象，以便在蛋白质构象与吸附、解吸、界面迁移、聚集等动态过程之间建立直接联系。同样，将使用动态悬垂气泡张力测定法、乳液消耗实验和 FACS 来测量空气-水界面处的蛋白质吸附，并通过流式显微镜、色谱法和 FACS 监测所得的蛋白质聚集。通过确定界面面积变化、蛋白质浓度、热力学条件和赋形剂的影响，将探索界面诱导的蛋白质损伤与配方条件之间的联系。通过操纵蛋白质天然状态结构的热力学稳定性（例如，使用稳定赋形剂），微观蛋白质展开过程将与界面现象和搅拌诱导聚集动力学的宏观测量联系起来。 更广泛的影响：该项目将产生几个广泛的影响。首先，对油水界面蛋白质吸附的详细了解将有助于设计配方，防止界面诱导的蛋白质聚集，降低开发成本并提高患者安全性。其次，将要测试和开发的许多新技术将供广泛的学术和工业科学家使用。此外，界面上的蛋白质吸附和聚集对于其他一些科学事业至关重要，例如疫苗学、微流体和纳米技术在诊断领域的应用以及植入式医疗设备的开发。通过将两个在界面科学和蛋白质配方方面具有不同专业知识的研究小组联系起来，参与这项研究的研究生和本科生将接受广泛的跨学科培训。此外，由于该小组与生物制药行业有着密切的联系，这项研究的成果将被迅速传播，以便在所提出的新的基础科学研究的实际应用中产生最大的影响。