Development of a Liquid Chromatography High Resolution Mass Spectrometry (LC-

31 Background: Ebola virus like particles; (EBOV VLPs, eVLPs), are produced by expressing the 32 viral transmembrane glycoprotein (GP) and the structural matrix protein VP40 in mammalian 33 cells. When expressed, these proteins self-assemble and bud from ‘host’ cells displaying 34 morphology similar to infectious virions. Several studies have shown that rodents and non35 human primates vaccinated with eVLPs are protected from lethal EBOV challenge. The mucin36 like domain of envelope glycoprotein (GP1) serves as the major target for a productive humoral 37 immune response. Therefore GP1 concentration is a critical quality attribute of EBOV vaccines 38 and accurate measurement of the amount of GP1 present in eVLP lots is crucial to understanding 39 variability in vaccine efficacy. 40 Methods: After production, eVLP’s are characterized by determining total protein concentration 41 and by western blotting, which only provides semi-quantitative information for GP1. Therefore, a 42 liquid chromatography high resolution mass spectrometry (LC-HRMS) approach for accurately 43 measuring the concentration of GP1 in eVLP’s was developed. The method employs isotope 44 dilution mass spectrometry using 4 target peptides from 2 regions of the GP1 protein. Purified 45 recombinant GP1 was generated to serve as an assay standard. GP1 quantitation in 5 eVLP lots 46 was performed on an LTQ-Orbitrap Elite MS and the final quantitation was derived by 47 comparing the relative response of the 200 fmol AQUA peptide standards to the analyte response 48 at 4 ppm. 49 Results: Conditions were optimized to ensure complete tryptic digestion of eVLP, however, 50 persistent missed cleavages were observed in the target peptides. Additionally, N-terminal 51 truncated forms of the GP1 protein were observed in all eVLP lots, making peptide selection 52 crucial. The LC-HRMS strategy resulted in the quantitation of GP1 with a lower limit of 53 TR-16-141 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.