Development of a liquid chromatography high resolution mass spectrometry method for the quantitation of viral envelope glycoprotein in Ebola virus-like particle vaccine preparations.

Background: Ebola virus like particles (EBOV VLPs, eVLPs), are produced by expressing the viral transmembrane glycoprotein (GP) and structural matrix protein VP40 in mammalian cells. When expressed, these proteins self-assemble and bud from 'host' cells displaying morphology similar to infectious virions. Several studies have shown that rodents and non-human primates vaccinated with eVLPs are protected from lethal EBOV challenge. The mucin-like domain of envelope glycoprotein GP₁ serves as the major target for a productive humoral immune response. Therefore GP₁ concentration is a critical quality attribute of EBOV vaccines and accurate measurement of the amount of GP₁ present in eVLP lots is crucial to understanding variability in vaccine efficacy. Methods: After production, eVLPs are characterized by determining total protein concentration and by western blotting, which only provides semi-quantitative information for GP₁. Therefore, a liquid chromatography high resolution mass spectrometry (LC-HRMS) approach for accurately measuring GP₁ concentration in eVLPs was developed. The method employs an isotope dilution strategy using four target peptides from two regions of the GP₁ protein. Purified recombinant GP₁ was generated to serve as an assay standard. GP₁ quantitation in 5 eVLP lots was performed on an LTQ-Orbitrap Elite and the final quantitation was derived by comparing the relative response of 200 fmol AQUA peptide standards to the analyte response at 4 ppm. Results: Conditions were optimized to ensure complete tryptic digestion of eVLP, however, persistent missed cleavages were observed in target peptides. Additionally, N-terminal truncated forms of the GP₁ protein were observed in all eVLP lots, making peptide selection crucial. The LC-HRMS strategy resulted in quantitation of GP₁ with a lower limit of quantitation of 1 fmol and an average percent coefficient of variation (CV) of 7.6%. Unlike western blot values, the LC-HRMS quantitation of GP₁ in 5 eVLP vaccine lots exhibited a strong linear relationship (positive correlation) with survival (after EBOV challenge) in mice. Conclusions: This method provides a means to rapidly determine eVLP batch quality based upon quantitation of antigenic GP₁. By monitoring variability in GP₁ content, the eVLP production process can be optimized, and the total amount of GP₁ needed to confer protection accurately determined. [ABSTRACT FROM AUTHOR]