Structural Signatures for Quality Control of Proteins

描述（由申请人提供）：生物分子的高阶结构信息是重组蛋白质量控制活动的重要组成部分。不幸的是，详细的结构信息通常需要高度复杂，耗时的实验技术（例如，核磁共振或x射线晶体学），不能被认为适合于质量控制应用。本提案旨在开发和验证一套适用于生物制药产品快速质量控制研究的技术。具体来说，我们的目标是开发可用于检测碱基状态（例如，错误折叠与正确折叠）结构变化的技术，并对特定蛋白质的某些变化进行分类（例如，脱酰胺，糖基化改变，突变等）。此外，我们的目标是自动化分析方法，以提高其吞吐量和一致性，并准备纳入生物分析质量控制服务。结构特征的概念依赖于将从对小结构修改敏感的单独的水两相分配实验中获得的数据与使特征高度特异性的数学分析技术相结合。签名包含的信息比底层结构本身少得多，因此是专门为每个应用程序构建的。第一阶段的研究工作集中在提供可行性数据，以证明一种或多种生物制药产品在不同结构修饰方面的特征的普遍性，这些结构修饰对于质量控制是典型的和重要的。第一阶段还将提供有关适当设计实验技术和优选的数学和统计方法的初步数据，以便在日常基础上构建签名。第二阶段的工作将在此基础上进一步推广和验证具有多种潜在降解途径的多种蛋白质的方法，并将该技术纳入自动化签名工作站，以快速低成本确定和破译生产样品的结构信息。这项建议进一步扩展了以前与生物恐怖主义相关疫苗产品质量控制有关的工作，以便为工业界适应提供必要的深度和广度分析和数据。

DESCRIPTION (provided by applicant): Higher-order structural information for biomolecules is an important component of quality control activities for recombinant proteins. Unfortunately, detailed structural information typically require highly complex, time-consuming experimental techniques (e.g., NMR or X-ray crystallography) that can not be considered as suitable for quality control applications. This proposal aims at developing and validating a set of techniques suitable for rapid quality control studies of biopharmaceutical products. Specifically, we aim to develop techniques that could be used to detect changes in the structure from a base state (e.g., misfolded vs. properly folded) and classify certain changes for specific protein (e.g., deamidation, altered glycosylation, mutations, etc.). Furthermore, we aim to automate the analysis methodology to increase its throughput and consistency and for ready incorporation into bioanalytical quality control services. The concept of a signature of a structure relies on combining data obtained from separate aqueous two-phase partitioning experiments that are sensitive to small structural modifications, with mathematical analysis techniques that make the signature highly specific. Signatures contain much less information than the underlying structure itself and are thus constructed specifically for each application. Phase I research work is centered on providing feasibility data to demonstrate the generality of signatures with for one or more biopharmaceutical products with respect to different structure modifications typical of and important for quality control. Phase I would also provide initial data concerning the proper design of experimental techniques and preferred mathematical and statistical methods for constructing signatures on a routine basis. Phase II work would expand upon this work to further generalize and validate the methodology for multiple proteins with multiple potential degradation pathways, and on incorporating the techniques into an automated signature workstation for rapid low-cost determining and deciphering of structural information of production samples. This proposal further extends previous work related to quality control of bioterrorism-related vaccine products to provide the depth and breadth of analysis and data necessary for adaptation by industry.