Mechanisms of Damage to Pharmaceutical Proteins at Oil-Water Interfaces

在生产，加工，储存和交付给患者的过程中，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和用于配制蛋白质的水溶液之间的界面。蛋白质可以吸附在这些界面上，这反过来又会导致蛋白质的聚集。药物配方中的蛋白质聚集与效力的变化、免疫原性增加的风险和保质期缩短有关，因此是开发一种新的基于蛋白质的治疗方法所需的估计12亿美元的主要贡献者。蛋白质的界面损伤是流体-流体界面的一个特殊问题，其中界面的动态性质（例如，在运输和处理蛋白质配方过程中所经历的剪切力的响应）可能会增加界面对蛋白质的暴露。该项目研究了治疗蛋白与硅油/水界面相互作用时的行为。利用先进的光谱和物理技术探索这种相互作用的机制，目的是制定合理的设计策略，以防止界面蛋白质损伤，降低相关成本和健康风险。知识价值：蛋白质在界面上的吸附和聚集是普遍存在的，但是导致蛋白质在界面上吸附和随之产生的聚集的基本机制仍然知之甚少。这个项目是两个研究小组在蛋白质界面科学、蛋白质构象热力学和聚集动力学方面的专业知识的合作，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。为了表征油水界面的吸附和界面聚集动力学，将开发和应用几种最先进的实验技术。这些包括单分子跟踪微流变学（使用荧光显微镜），乳液吸附，荧光活化细胞分选（FACS）和吸附蛋白质的正面荧光猝灭。为了建立蛋白质构象与吸附、解吸、界面迁移、聚集等动态过程之间的直接联系，将利用福斯特共振能量转移（FRET）技术测量硅油-水界面上蛋白质的分子构象。同样，蛋白质在空气-水界面的吸附将使用动态垂坠气泡张力测定法、乳液耗尽实验和FACS来测量，并通过流动显微镜、色谱法和FACS来监测所产生的蛋白质聚集。通过确定界面面积变化、蛋白质浓度、热力学条件和辅料的影响，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，对油水界面上蛋白质吸附的详细了解将有助于配方的设计，从而防止界面诱导的蛋白质聚集，降低开发成本并提高患者的安全性。其次，许多将被测试和开发的新技术将被各种各样的学术和工业科学家使用。此外，蛋白质在界面上的吸附和聚集对其他一些科学研究至关重要，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获得广泛的跨学科培训。此外，由于该小组与生物制药工业关系密切，这项研究的结果将迅速传播，以便对拟议的新的基础科学研究的实际应用产生最大的影响。

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.