Automated Conformational Analysis of Pharmaceutical Proteins

药物蛋白质的自动构象分析

• 批准号: 8646687
• 负责人: PETER Justin TODD
• 金额: $ 34.96万
• 依托单位: PHOTOCHEM TECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8646687
• 关键词: Amino Acids; Autoimmune Responses; Chemical Structure; Chemicals; Chemistry; Circular Dichroism; Coupled; Data; Data Set; Detection; Development; Digestion; Drug Formulations; Exhibits; FDA approved; Generations; Generic Drugs; Hydroxyl Radical; Laboratories; Lasers; Legal patent; Life; Liquid Chromatography; Liquid substance; Location; Manuals; Marketing; Mass Spectrum Analysis; Membrane Proteins; Methods; Modification; Molecular Conformation; Monitor; Personal Computers; Pharmaceutical Preparations; Pharmacologic Substance; Process; Production; Protein Analysis; Protein Conformation; Protein Footprinting; Proteins; Recombinants; Research Personnel; Robotics; Safety; Sampling; Scheme; Shapes; Site; Solutions; Structure; Surface; System; Techniques; Technology; Testing; Time; Translations; Variant; Vendor; Xenon; analytical method; base; computer program; data reduction; design; experience; flash photolysis; liquid chromatography mass spectrometry; meetings; nanosecond; operation; oxidation; protein folding; prototype; public health relevance; quality assurance; screening; small molecule; tool

DESCRIPTION (provided by applicant): Pharmaceutical proteins comprise a large and rapidly growing segment of the pharmaceutical market. To be safe and effective, pharmaceutical proteins must possess not only the correct covalent structure, but also the correct non-covalent three-dimensional folded structure - and there is no existing method capable of guaranteeing this conformational integrity on a quality assurance (QA) basis. A technology known as hydroxyl radical protein footprinting (HRFP) has been described for the rigorous structural comparison of recombinant pharmaceutical proteins with FDA-approved formulations. HRFP uses a burst of short-lived hydroxyl (OH.) radicals to react with amino acids on the protein surface, creating modifications easily recognized by liquid chromatography coupled to mass spectrometry (LCMS). For QA, results of HRFP from a test sample of a protein must match HRFP results from the protein standard. Otherwise, the structure of the test sample has been compromised. In cases where differences between standard and sample are observed, the general location of these abnormalities can be determined, aiding in troubleshooting. While the method exhibits strong potential as an analytical and QA tool, HRFP requires a substantial degree of highly specialized expertise to generate data sufficiently reliable for use as a QA method. We propose an automated platform for executing the entire HRFP process. Practically, and philosophically, the platform will be inserted into the existing automated workflow of bottom-up protein analysis by mass spectrometry. We propose to produce an automated sample handling and flash photolysis system, alternate schemes for flash photolytic generation of hydroxyl radicals, QA monitors for each step of HRFP, and statistical quality assurance computer programs. We will demonstrate the ability of the integrated system to detect structural abnormalities with current pharmaceutical proteins. The resulting system will be a single-vendor automated HRFP platform for insertion into already-existing LCMS systems allowing application of this technology by researchers with a conventional level of expertise in protein mass spectrometry.

描述（由申请人提供）：药用蛋白质是医药市场中一个巨大且快速增长的部分。为了安全有效，药用蛋白不仅必须具有正确的共价结构，而且必须具有正确的非共价三维折叠结构，而目前还没有能够在质量保证（QA）的基础上保证这种构象完整性的方法。一种被称为羟基自由基蛋白足迹（HRFP）的技术被描述为重组药物蛋白与fda批准的配方的严格结构比较。HRFP利用短寿命羟基（OH）自由基与蛋白质表面的氨基酸发生反应，产生易于被液相色谱-质谱联用（LCMS）识别的修饰。对于质量保证，蛋白质测试样品的HRFP结果必须与蛋白质标准的HRFP结果相匹配。否则，测试样品的结构已被破坏。在观察到标准和样品之间差异的情况下，可以确定这些异常的一般位置，帮助排除故障。虽然该方法显示出作为分析和QA工具的强大潜力，但HRFP需要大量高度专业化的专业知识来生成足够可靠的数据，以便作为QA方法使用。我们提出了一个执行整个HRFP过程的自动化平台。从实践和理念上讲，该平台将被插入到现有的自下而上的蛋白质质谱分析自动化工作流程中。我们建议建立一个自动样品处理和闪光光解系统，闪光光解生成羟基自由基的备选方案，HRFP每个步骤的QA监视器和统计质量保证计算机程序。我们将展示集成系统检测当前药物蛋白结构异常的能力。最终的系统将是一个单一供应商的自动化HRFP平台，可插入到现有的LCMS系统中，允许具有传统蛋白质质谱专业水平的研究人员应用该技术。