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. ? ?
