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. 2022 May:303:114498.
doi: 10.1016/j.jviromet.2022.114498. Epub 2022 Feb 22.

SARS-CoV-2 spike antigen quantification by targeted mass spectrometry of a virus-based vaccine

Osnat Rosen  1 , Avital Jayson  2 , Eyal Dor  2 , Eyal Epstein  2 , Arik Makovitzki  2 , Lilach Cherry  2 , Edith Lupu  2 , Arik Monash  2 , Sarah Borni  2 , Tzadok Baruchi  2 , Orly Laskar  3 , Shlomo Shmaya  3 , Ronit Rosenfeld  4 , Yinon Levy  4 , Ofir Schuster  5 , Liron Feldberg  6
Affiliations

SARS-CoV-2 spike antigen quantification by targeted mass spectrometry of a virus-based vaccine

Osnat Rosen et al. J Virol Methods. 2022 May.

Abstract

The spike glycoprotein mediates virus binding to the host cells and is a key target for vaccines development. One SARS-CoV-2 vaccine is based on vesicular stomatitis virus (VSV), in which the native surface glycoprotein has been replaced by the SARS-CoV-2 spike protein (VSV-ΔG-spike). The titer of the virus is quantified by the plaque forming unit (PFU) assay, but there is no method for spike protein quantitation as an antigen in a VSV-based vaccine. Here, we describe a mass spectrometric (MS) spike protein quantification method, applied to VSV-ΔG-spike based vaccine. Proof of concept of this method, combining two different sample preparations, is shown for complex matrix samples, produced during the vaccine manufacturing processes. Total spike levels were correlated with results from activity assays, and ranged between 0.3-0.5 μg of spike protein per 107 PFU virus-based vaccine. This method is simple, linear over a wide range, allows quantification of antigen within a sample and can be easily implemented for any vaccine or therapeutic sample.

Keywords: Mass spectrometry; Quantification; SARS-CoV-2; Spike; Vaccine.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Quantification of VSV-ΔG-spike markers using LC–MS/MS(MRM) analysis. (A) LC–MS/MS(MRM) chromatogram for the identification of three tryptic peptides derived from tryptic digestion of 107 PFU/mL VSV-ΔG-spike. An example for one MRM transition for each peptide is shown (MRM transition from precursor to its fragment ion 570 > 440, 613 > 206, 475 > 711 for FLPFQQFGR, SFIEDLLFNK, HTPINLVR respectively). (B) The assay's linearity in the range of 106–108 PFU/mL with precision lower than 20 % is shown.
Fig. 2
Fig. 2
Calibration curves for tryptic digest of purified spike protein in ammonium bicarbonate buffer (squares) vs. formulation buffer (circles). Results are shown for SFIEDLLFNK peptide, 613 > 206.
Fig. 3
Fig. 3
Immuno-magnetic separation of VSV-ΔG-spike (107 PFU/mL) using polyclonal-anti-SARS-CoV-2 Abs. Peak intensities of the three selected spike markers from IMS samples using anti-spike (blue hatched bars) and anti SARS-CoV-2 (hatched purple bars) comparing to direct analysis without capture (light blue bars).
See this image and copyright information in PMC

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