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）：药物蛋白质构成了药物市场的一个巨大且快速增长的部分。为了安全有效，药用蛋白质不仅必须拥有正确的共价结构，还必须拥有正确的非共价三维折叠结构——并且没有现有的方法能够在质量保证（QA）的基础上保证这种构象完整性。一种称为羟自由基蛋白足迹 (HRFP) 的技术已被描述用于对重组药物蛋白与 FDA 批准的配方进行严格的结构比较。 HRFP 使用大量短命羟基 (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与质谱联用 (LCMS) 识别的修饰。对于 QA，蛋白质测试样品的 HRFP 结果必须与蛋白质标准品的 HRFP 结果匹配。否则，测试样品的结构就会受到损害。如果观察到标准品和样品之间存在差异，则可以确定这些异常的大致位置，从而有助于排除故障。虽然该方法作为分析和 QA 工具展现出强大的潜力，但 HRFP 需要大量高度专业化的专业知识才能生成足够可靠的数据以用作 QA 方法。我们提出了一个用于执行整个 HRFP 流程的自动化平台。在实践和哲学上，该平台将被插入到现有的自下而上的质谱蛋白质分析自动化工作流程中。我们建议生产自动化样品处理和闪光光解系统、闪光光解生成羟基自由基的替代方案、HRFP 每个步骤的 QA 监视器以及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质结构异常的能力。由此产生的系统将是一个单一供应商自动化 HRFP 平台，可插入现有的 LCMS 系统中，从而允许具有蛋白质质谱专业知识的传统水平的研究人员应用该技术。