Plasma-Generated Hydroxyl Radicals for Analysis of Three-Dimensional Structures in Protein Therapeutics

等离子体产生的羟基自由基用于蛋白质治疗中三维结构的分析

• 批准号: 10547178
• 负责人: Faraz Choudhury
• 金额: $ 103.78万
• 依托单位: IMMUTO SCIENTIFIC, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10547178
• 关键词: 3-Dimensional; Address; Adopted; Amino Acids; Amplifiers; Antibodies; Antigens; Autoimmune Diseases; Autoimmunity; Binding Sites; Biological Process; Biological Products; Biological Response Modifier Therapy; Biotechnology; COVID-19; Collaborations; Communicable Diseases; Complex; Computer software; Consumption; Coupling; Cryoelectron Microscopy; Crystallization; Data; Data Analyses; Development; Diagnostic; Disease; Dose; Double-Blind Method; Drug Industry; Engineering; Ensure; Epitope Mapping; Equilibrium; Exposure to; Feedback; Fiber Optics; Genetic Diseases; Goals; Guidelines; Health; Higher Order Chromatin Structure; Hour; Human; Hydroxyl Radical; Industry; Industry Standard; Inflammation; Insulin; Knowledge; Label; Lasers; Life; Malignant Neoplasms; Manufacturer Name; Mass Spectrum Analysis; Measurement; Measures; Medicine; Methionine; Methods; Modern Medicine; Modification; Molecular Conformation; Monoclonal Antibodies; Outcome; Performance; Peroxides; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Plasma; Preparation; Process; Property; Protein Engineering; Protein Footprinting; Protein Region; Proteins; Quality Control; Recombinant Vaccines; Reproducibility; Research Personnel; Resolution; Resources; Safety; Sampling; Signal Transduction; Small Business Innovation Research Grant; Solvents; Source; Speed; Structure; Surface; System; Techniques; Technology; Testing; Therapeutic antibodies; Three-dimensional analysis; Time; TimeLine; Tube; United States Food and Drug Administration; Universities; Vaccines; Water; Wisconsin; X-Ray Crystallography; antigen antibody binding; base; commercial application; design; detection platform; drug development; drug discovery; drug efficacy; improved; instrument; methionine sulfoxide; oxidation; performance tests; phase 1 study; photomultiplier; protein structure; prototype; rapid growth; response; therapeutic protein; three dimensional structure; tool; voltage

Abstract Immuto Scientific Inc. is developing a robust, automated benchtop instrument to perform high-throughput structural characterization of protein biotherapeutics in a fraction of time of traditional structural characterization techniques such as x-ray crystallography and cryo-EM. Protein-based biotherapeutics (such as antibodies, insulin, recombinant vaccines, etc.) are one of the most effective classes of modern medicines for the treatment of a wide variety of diseases including cancers, autoimmunity/inflammation, genetic disorders, and infectious diseases such as COVID-19. The biological function and physicochemical properties of biotherapeutics are determined by their higher order structures (HOS)—the folding and three-dimensional conformation that largely dictates function and stability. Therefore, it is imperative to analyze the three- dimensional higher order structure of protein therapeutics at several stages of the drug development process to ensure both safety and efficacy of the drug. Biopharmaceutical manufacturers are required to demonstrate the consistency of the protein HOS conformation to the regulatory agencies. Moreover, identifying the binding site of a therapeutic protein (such as a monoclonal antibody) to its corresponding antigen (known as epitope mapping) is critical for the development of new antibody therapeutics, vaccines and diagnostics. Food and Drug Administration (FDA) guidelines require specific binding site information between a drug and its target for the regulatory filing. Current techniques for protein HOS characterization and epitope mapping are complex, resource intensive and can take up to 6-12 months to perform. Based on feedback received from over 300 customer pharmaceutical customers, we have developed a technology called Plasma Induced Modification to Biomolecules (PLIMB) that addresses the need of the industry for routine structural, mass spectrometry-based protein HOS analysis. PLIMB generates sub microsecond bursts of hydroxyl (OH) radicals from water to label proteins in solution. The OH radicals covalently label the solvent accessible regions of the protein and subsequent mass spectrometric analysis reveals single amino acid level structural information. With PLIMB, HOS analysis and epitope mapping can be performed in under 48 hours where current techniques such as x- ray crystallography and Cryo-EM takes several months to perform. In Phase II, we will first incorporate the hydroxyl radical detection system that was designed in Phase I into a fully automated, manufacturable PLIMB instrument, and then validate the PLIMB system for commercial use. Ultimately, PLIMB will be a revolutionary new tool for pharmaceutical researchers that will provide new capabilities to better engineer highly effective protein biotherapeutics and accelerate the drug discovery timeline.

摘要 Immuto Scientific Inc.正在开发一种强大的自动化台式仪器， 蛋白质生物治疗剂结构表征， 表征技术，如X射线晶体学和cryo-EM。基于蛋白质的生物治疗剂（例如 如抗体、胰岛素、重组疫苗等）是现代药物中最有效的一类 用于治疗多种疾病，包括癌症，自身免疫/炎症，遗传性疾病， 以及COVID-19等传染病。本文介绍了其生物学功能和理化性质 生物治疗药物是由它们的高阶结构（HOS）决定的-折叠和三维结构。 构象在很大程度上决定了功能和稳定性。因此，有必要对这三个方面进行分析-- 在药物开发过程的几个阶段， 以确保药物的安全性和有效性。生物制药制造商必须证明 蛋白质HOS构象与监管机构的一致性。此外，识别绑定 治疗性蛋白质（如单克隆抗体）与其相应抗原（称为表位）结合位点 作图）对于开发新的抗体疗法、疫苗和诊断是至关重要的。食品和 药物管理局（FDA）指南要求药物与其靶标之间的特异性结合位点信息，以用于 监管备案。目前用于蛋白质HOS表征和表位作图的技术是复杂的， 资源密集型，可能需要长达6-12个月的时间来执行。根据从300多个国家收到的反馈意见， 客户制药客户，我们已经开发出一种称为等离子体诱导修饰的技术， 生物分子（Biomolecules，CIMMB）解决了行业对常规结构、基于质谱的 蛋白质HOS分析。CH 3 MB产生亚微秒的羟基（OH）自由基从水到标记的爆发 蛋白质溶液OH基团共价标记蛋白质的溶剂可及区域， 随后的质谱分析揭示了单个氨基酸水平的结构信息。有了CIMMB， HOS分析和表位作图可以在48小时内进行，其中目前的技术如x- 射线晶体学和冷冻电镜需要几个月的时间才能完成。在第二阶段，我们会首先把 羟基自由基检测系统，在第一阶段被设计成一个全自动的，可制造的CIMB 仪器，然后验证用于商业用途的CIMMB系统。最终，CIMMB将是一个革命性的 制药研究人员的新工具，将提供新的能力，以更好地设计高效的 蛋白质生物治疗和加速药物发现时间轴。