Mechanisms of Damage to Pharmaceutical Proteins at Oil-Water Interfaces

油水界面药物蛋白的损伤机制

基本信息

  • 批准号:
    1133871
  • 负责人:
  • 金额:
    $ 33.9万
  • 依托单位:
  • 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

项目摘要

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.
1133871Randolphintruction:在其生产,加工,储存和分娩期间,治疗蛋白会暴露于各种界面,例如用于润滑玻璃注射器的硅油和蛋白质蛋白质的水溶液之间的接口。 蛋白质可能会吸附到这些界面上,进而导致蛋白质聚集。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缩短的保质期有关,因此是开发新的基于蛋白质的治疗性的12亿美元所需的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上的一个特殊问题,其中界面的动态性质(例如,响应于在运输和处理蛋白质配方期间经历的剪切力)可能会增加接口对蛋白质的接触。 该项目研究了治疗蛋白与硅油/水界面相互作用时的行为。 通过先进的光谱和物理技术探测了这种相互作用的机制,其目的是制定理性设计策略,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:界面上的蛋白质吸附和聚集无处不在,但是导致界面上蛋白质吸附的基本机制以及随之而来的聚集体的产生仍然很少了解。该项目是两个在蛋白质界面科学方面具有专业知识的研究小组之间的合作,蛋白质构象的热力学和聚集动力学将解决导致蛋白质吸附和界面上蛋白质吸附和展开的微观机制,以及在宏观上可观察的蛋白质聚集中的动力学机制。为了表征油水界面上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侵蚀学(使用荧光显微镜),乳液吸附,荧光激活的细胞分选(FACS)和吸附蛋白的前面荧光猝灭。将使用Forster谐振能量转移(FRET)测量蛋白质在硅油水界面处的分子构象,例如在蛋白质构象和动态过程之间建立直接连接,例如吸附,解吸,互化,互化迁移率,聚合。同样,将使用动态吊坠张力仪,乳液消耗实验和FACS以及通过流动显微镜,色谱和FACS监测的蛋白质聚集来测量空气水接口处的蛋白质吸附。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,将探索界面引起的蛋白质损伤与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,具有稳定赋形剂),显微镜蛋白展开过程将与界面现象和抗激态测量的抗激发诱导的 - 聚集动力学的测量相关。首先,对油水界面上蛋白质吸附的详细了解将有助于设计配方,以防止互动诱导的蛋白质聚集,降低发育成本并提供提高患者的安全性。其次,将要测试和开发的许多新技术将用于各种学术和工业科学家。此外,界面上的蛋白质吸附和聚集对于其他几项科学努力,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耐植物医疗设备的发展至关重要。通过将两个研究小组与界面科学和蛋白质制定方面的专业知识联系起来,参加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,由于这些团体与生物制药行业有着密切的联系,因此这项研究的结果将迅速传播,以便在拟议的新的,基本的科学研究的实际应用中给予最大的影响。

项目成果

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Theodore Randolph其他文献

Theodore Randolph的其他文献

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{{ truncateString('Theodore Randolph', 18)}}的其他基金

GOALI: Aggregation of Protein Therapeutics in Aqueous Solutions
目标:蛋白质治疗药物在水溶液中的聚集
  • 批准号:
    0138595
  • 财政年份:
    2002
  • 资助金额:
    $ 33.9万
  • 项目类别:
    Continuing Grant
Acquisition of a Nanoparticle Analysis Ensemble
获得纳米颗粒分析套件
  • 批准号:
    0079612
  • 财政年份:
    2000
  • 资助金额:
    $ 33.9万
  • 项目类别:
    Standard Grant
Mechanisms for Success or Failure of Excipients and Protein-Stabilizers
赋形剂和蛋白质稳定剂成功或失败的机制
  • 批准号:
    9816975
  • 财政年份:
    1999
  • 资助金额:
    $ 33.9万
  • 项目类别:
    Standard Grant
Thermodynamic and Molecular Mechanisms of Protein Stabilization by Polymers During Freezing, Drying, and Rehydration: EPR and FTIR Studies
冷冻、干燥和再水合过程中聚合物稳定蛋白质的热力学和分子机制:EPR 和 FTIR 研究
  • 批准号:
    9505301
  • 财政年份:
    1995
  • 资助金额:
    $ 33.9万
  • 项目类别:
    Continuing Grant
High Pressure Effects on Protein Crystallization (Collaborative Research)
高压对蛋白质结晶的影响(合作研究)
  • 批准号:
    9529288
  • 财政年份:
    1995
  • 资助金额:
    $ 33.9万
  • 项目类别:
    Continuing Grant
Reactions in Supercritical Fluids: Experimental and Simulation Studies of Microscopic Phenomena
超临界流体中的反应:微观现象的实验和模拟研究
  • 批准号:
    9414759
  • 财政年份:
    1994
  • 资助金额:
    $ 33.9万
  • 项目类别:
    Standard Grant
Presidential Young Investigators Award: Spectroscopic Studies of Proteins in Engineering Environments
总统青年研究员奖:工程环境中蛋白质的光谱研究
  • 批准号:
    9496042
  • 财政年份:
    1993
  • 资助金额:
    $ 33.9万
  • 项目类别:
    Continuing Grant
Presidential Young Investigators Award: Spectroscopic Studies of Proteins in Engineering Environments
总统青年研究员奖:工程环境中蛋白质的光谱研究
  • 批准号:
    9157318
  • 财政年份:
    1991
  • 资助金额:
    $ 33.9万
  • 项目类别:
    Continuing Grant

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