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.
Molecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702 USA ; DOD Biotechnology High Performance Computing Software Applications Institute, Telemedicine and Advanced Technology Research Center, US Army Medical Research and Materiel Command, Fort Detrick, MD 21702 USA.
Lisa H
LH
Cazares
Molecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702 USA.
Michael D
MD
Ward
Molecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702 USA.
Ernst E
EE
Brueggemann
Molecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702 USA.
Tara
T
Kenny
BioSciences Division, Biodefense Branch, Edgewood Chemical Biological Center, Gunpowder, MD 21010 USA ; Excet, Inc., 8001 Braddock Road, Suite 105, Springfield, VA 22151 USA.
Paul
P
Demond
Molecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702 USA.
Christopher R
CR
Mahone
Molecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702 USA.
Karen A O
KA
Martins
Molecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702 USA.
Jonathan E
JE
Nuss
BioSciences Division, Biodefense Branch, Edgewood Chemical Biological Center, Gunpowder, MD 21010 USA.
Trevor
T
Glaros
Molecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702 USA.
Sina
S
Bavari
Ebola virus; High resolution mass spectrometry; Stable isotope dilution quantitation; Virus like particles