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

AbstractThe biological function and physicochemical properties of proteinbased therapeutics are determined by theirhigher order structuresHOSThereforeit is imperative to analyze the threedimensional higher orderstructure of protein therapeutics at several stages of the drug development process to ensure both safety andefficacy of the drugBiopharmaceutical manufacturers are required to demonstrate the consistency of theprotein HOS conformation to the regulatory agenciesMoreoverIdentifying the binding site of a protein to itscorresponding antigenknown as epitope mappingis critical for the development of new therapeuticsvaccines and diagnosticsFood and Drug AdministrationFDAguidelines require specific binding siteinformation between a drug and its target for the regulatory filingCurrent techniques for protein HOScharacterization and epitope mapping are slowexpensive and difficult to performWe have developed atechnology called Plasma Induced Modification to Biomolecules that addresses the need of the industry forroutine structuralmass spectrometrybased protein HOS analysisPLIMB generates sub microsecond burstsof hydroxylOHradicals from water to label proteins in solutionThe OH radicals covalently labels the solventaccessible regions of the protein and subsequent mass spectrometric analysis reveals single amino acid levelstructural informationA way to quickly and efficiently analyze higher order structures of proteins on a benchtopscalePLIMB will enable faster development of protein therapeuticsthroughout the drug discovery processInSBIR Phase Iwe will prove the feasibility of a hydroxyl radical detection system that will tightly control the OHradical dose generated by PLIMB to yield highly reproducible HOS dataAfter completion of the SBIR Phase Iprojectwe will continue the development and validation of the PLIMB system for commercial useUltimatelyPLIMB will enable fasthigh resolution structural analysis of proteinsa capability which is highly sought after inthe pharmaceutical industryProject NarrativeThe goal of this SBIR is to develop a highly reproduciblebenchtop instrument to perform Hydroxyl RadicalProtein FootprintingHRFfor analysis of three dimensional structures in protein therapeuticsThis instrumentwill enable structural characterization of proteins at a much higher throughput than traditional techniques tobetter drive the discovery and development of protein therapeutics