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

摘要 Immuto Science Inc.正在开发一种坚固的自动化台式仪器，以执行高通量 蛋白质生物疗法的结构表征在传统结构的一小部分时间内 X射线结晶学和低温电子显微镜等表征技术。基于蛋白质的生物疗法(如 如抗体、胰岛素、重组疫苗等)是现代医学中最有效的类别之一 用于治疗多种疾病，包括癌症、自身免疫/炎症、遗传性疾病、 以及新冠肺炎等传染病。细胞因子的生物学功能和理化性质 生物治疗是由其高级结构(HOS)决定的--折叠和三维 在很大程度上决定功能和稳定性的构象。因此，分析这三个问题势在必行-- 药物开发过程中几个阶段的蛋白质疗法的高维结构 以确保药物的安全性和有效性。生物制药制造商被要求展示 蛋白质HOS构象的一致性符合调控机构的要求。此外，识别绑定 治疗性蛋白(如单抗)与其相应抗原(称为表位)的结合部位 对新的抗体疗法、疫苗和诊断的发展至关重要。食物和 药品监督管理局(FDA)指南要求药物与其靶点之间的特定结合部位信息 监管申报文件。目前用于蛋白质HOS表征和表位映射的技术是复杂的， 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多家公司收到的反馈 客户制药客户，我们开发了一种名为等离子体诱导修饰的技术 生物分子(PLIMB)，满足行业对常规结构、基于质谱学的需求 蛋白质HOS分析。PLIMB从水中产生亚微秒级的羟基(OH)自由基以标记 溶液中的蛋白质。OH自由基共价标记蛋白质的溶剂可及区域和 随后的质谱分析揭示了单一氨基酸水平的结构信息。有了PLIMB， HOS分析和表位映射可以在48小时内完成，目前的技术，如x- 射线结晶学和冷冻-EM需要几个月的时间才能完成。在第二阶段，我们会先把 羟基自由基检测系统在第一阶段设计为全自动、可制造的PLIMB 仪器，然后验证PLIMB系统的商业使用。最终，PLIMB将是一场革命性的 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 蛋白质生物疗法和加快药物发现的时间表。

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.