Mechanisms of Damage to Pharmaceutical Proteins at Oil-Water Interfaces

1133871 Randolph简介：在其生产、加工、储存和输送给患者的过程中，治疗性蛋白质暴露于各种界面，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。 蛋白质可以吸附到这些界面上，这又可以导致蛋白质的聚集。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货架期缩短相关，因此是开发新的基于蛋白质的治疗剂所需的估计12亿美元的主要贡献者。 蛋白质的界面损伤是流体-流体界面处的特定问题，其中界面的动态性质（例如，响应于在蛋白质制剂的运输和处理过程中所经历的剪切力）可以增加界面对蛋白质的暴露。 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。 这种相互作用的机制与先进的光谱和物理技术进行了探讨，其目标是制定合理的设计策略，以防止界面蛋白质损伤，并降低相关的成本和健康风险。 智力优势：蛋白质在界面上的吸附和聚集是普遍存在的，但导致蛋白质在界面上吸附和随之产生的聚集体的基本机制仍然知之甚少。该项目是两个具有蛋白质界面科学，蛋白质构象热力学和聚集动力学专业知识的研究小组之间的合作，将解决导致蛋白质吸附和界面展开的微观机制，以及导致宏观上可观察到的蛋白质聚集的动力学过程。为了表征在油-水界面处的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。这些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）和正面荧光淬灭吸附的蛋白质。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动态过程（如吸附、解吸、界面迁移率、聚集）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测量空气-水界面处的蛋白质吸附，并通过流动显微镜、色谱法和FACS监测所得蛋白质聚集。将通过确定界面面积变化、蛋白质浓度、热力学条件和辅料的影响来探索界面诱导的蛋白质损伤和制剂条件之间的联系。通过操纵蛋白质天然状态结构的热力学稳定性（例如，与稳定赋形剂），微观蛋白质展开过程将与界面现象和宏观测量搅拌诱导聚集动力学。更广泛的影响：该项目将有几个广泛的影响。首先，详细了解蛋白质在油-水界面的吸附将有助于设计配方，以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。第二，将被测试和开发的许多新技术将对各种各样的学术和工业科学家有用。此外，蛋白质在界面处的吸附和聚集对于其他几种科学努力是至关重要的，例如疫苗学、微流体和纳米技术在诊断竞技场中的应用以及可植入医疗装置的开发。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，由于该集团与生物制药行业的密切联系，这项研究的结果将迅速传播，以便在拟议的新的基础科学研究的实际应用中产生最大的影响。

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.