Issue 22, 2023

An isotope dilution mass spectrometry assay to track Norovirus-like particles in vaccine process intermediates by quantifying capsid protein VP1

Abstract

The coronavirus disease (COVID-19) pandemic shows the rapid pace at which vaccine development can occur which highlights the need for more fast and efficient analytical methodologies to track and characterize candidate vaccines during manufacturing and purification processes. The candidate vaccine in this work comprises plant-derived Norovirus-like particles (NVLPs) which are structures that mimic the virus but lack any infectious genetic material. Presented here is a liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology for the quantification of viral protein VP1, the main component of the NVLPs in this study. It combines isotope dilution mass spectrometry (IDMS) with multiple reaction monitoring (MRM) to quantify targeted peptides in process intermediates. Multiple MRM transitions (precursor/product ion pairs) for VP1 peptides were tested with varying MS source conditions and collision energies. Final parameter selection for quantification includes three peptides with two MRM transitions each offering maximum detection sensitivity under optimized MS conditions. For quantification, a known concentration of the isotopically labeled version of the peptides to be quantified was added into working standard solutions to serve as an internal standard (IS); calibration curves were generated for concentration of native peptide vs. the peak area ratio of native-to-isotope labeled peptide. VP1 peptides in samples were quantified with labeled versions of the peptides added at the same level as that of the standards. Peptides were quantified with limit of detection (LOD) as low as 1.0 fmol μL−1 and limit of quantitation (LOQ) as low as 2.5 fmol μL−1. NVLP preparations spiked with known quantities of either native peptides or drug substance (DS) comprising assembled NVLPs produced recoveries indicative of minimal matrix effects. Overall, we report a fast, specific, selective, and sensitive LC-MS/MS strategy to track NVLPs through the purification steps of the DS of a Norovirus candidate vaccine. To the best of our knowledge, this is the first application of an IDMS method to track virus-like particles (VLPs) produced in plants as well as measurements performed with VP1, a Norovirus capsid protein.

Graphical abstract: An isotope dilution mass spectrometry assay to track Norovirus-like particles in vaccine process intermediates by quantifying capsid protein VP1

Supplementary files

Article information

Article type
Paper
Submitted
17 Mar 2023
Accepted
14 May 2023
First published
15 May 2023
This article is Open Access
Creative Commons BY-NC license

Anal. Methods, 2023,15, 2729-2735

An isotope dilution mass spectrometry assay to track Norovirus-like particles in vaccine process intermediates by quantifying capsid protein VP1

J. R. Jhingree, J. Boisvert and G. Mercier, Anal. Methods, 2023, 15, 2729 DOI: 10.1039/D3AY00411B

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