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

Clinical Proteomics

Lisa H. Cazares³  Paul Demond¹  Christopher R. Mahone⁴  Karen A. O. Martins⁴  Trevor Glaros²  Ernst E. Brueggemann⁴  Jonathan E. Nuss⁴  Michael D. Ward⁴  Sina Bavari⁴  Tara Kenny⁴ 

[1]BioSciences Division, Biodefense Branch, Edgewood Chemical Biological Center, Gunpowder, USAExcet, Inc., Springfield, USABioSciences Division, Biodefense Branch, Edgewood Chemical Biological Center, Gunpowder, USABioSciences Division, Biodefense Branch, Edgewood Chemical Biological Center, Gunpowder, USAExcet, Inc., Springfield, USAExcet, Inc., Springfield, USABioSciences Division, Biodefense Branch, Edgewood Chemical Biological Center, Gunpowder, USAExcet, Inc., Springfield, USA

[2]BioSciences Division, Biodefense Branch, Edgewood Chemical Biological Center, Gunpowder, USABioSciences Division, Biodefense Branch, Edgewood Chemical Biological Center, Gunpowder, USABioSciences Division, Biodefense Branch, Edgewood Chemical Biological Center, Gunpowder, USA

[3]Molecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, USADOD Biotechnology High Performance Computing Software Applications Institute, Telemedicine and Advanced Technology Research Center, US Army Medical Research and Materiel Command, Fort Detrick, USAMolecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, USAMolecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, USADOD Biotechnology High Performance Computing Software Applications Institute, Telemedicine and Advanced Technology Research Center, US Army Medical Research and Materiel Command, Fort Detrick, USADOD Biotechnology High Performance Computing Software Applications Institute, Telemedicine and Advanced Technology Research Center, US Army Medical Research and Materiel Command, Fort Detrick, USAMolecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, USADOD Biotechnology High Performance Computing Software Applications Institute, Telemedicine and Advanced Technology Research Center, US Army Medical Research and Materiel Command, Fort Detrick, USA

[4]Molecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, USAMolecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, USAMolecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, USA

关键词: Ebola virus;    Virus like particles;    High resolution mass spectrometry;    Stable isotope dilution quantitation;   

DOI  :  10.1186/s12014-016-9119-8

来源: Humana Press Inc

【 摘 要 】

Abstract

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.

【 授权许可】

Unknown