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）：药物蛋白包括制药市场的大型且快速增长的细分市场。为了安全有效，药物蛋白不仅必须具有正确的共价结构，而且还必须具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）基础上保证这种构象完整性。已经描述了一种称为羟基自由基蛋白足迹（HRFP）的技术，用于对具有FDA批准配方的重组药物的严格结构比较。 HRFP使用短寿命的羟基（OH。）自由基在蛋白质表面上与氨基酸反应，从而产生了通过液相色谱与质谱法（LCMS）耦合的液相色谱很容易识别的修饰。对于QA，来自蛋白质测试样本的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损害。在观察到标准和样本之间的差异的情况下，可以确定这些异常的一般位置，以帮助进行故障排除。尽管该方法作为分析和质量检查工具具有强大的潜力，但HRFP需要大量高度专业的专业知识，以生成足够可靠的数据以用作QA方法。我们提出了一个自动化平台，用于执行整个HRFP流程。实际上，从哲学上讲，该平台将通过质谱插入自下而上蛋白质分析的现有自动化工作流程中。我们建议生产一个自动样品处理和闪光分解系统，替代方案，用于闪光灯的羟基自由基，HRFP的每个步骤的QA监视以及统计质量质量保证计算机程序。我们将展示综合系统使用当前药物蛋白检测结构异常的能力。最终的系统将是一个单供应商自动化的HRFP平台，用于插入已经存在的LCMS系统，允许在蛋白质质谱学方面具有常规专业知识的研究人员应用该技术。