Abstract

ChAdOx1 nCov-19 and Ad26.COV2.S are approved vaccines inducing protective immunity against SARS-CoV-2 infection in humans by expressing the Spike protein of SARS-CoV-2. We analyzed protein content and protein composition of ChAdOx1 nCov-19 and Ad26.COV2.S by biochemical methods and by mass-spectrometry. Four out of four tested lots of ChAdOx1 nCoV-19 contained significantly higher than expected levels of host cell proteins (HCPs) and of free viral proteins. The most abundant contaminating HCPs belonged to the heat-shock protein (HSP) and cytoskeletal protein families. The HCP content exceeded the 400 ng specification limit per vaccine dose, as set by the European Medicines Agency (EMA) for this vaccine, by at least 25-fold and the manufacturer's batch-release data in some of the lots by several hundred-fold. In contrast, three tested lots of the Ad26.COV2.S vaccine contained only very low amounts of HCPs. As shown for Ad26.COV2.S production of clinical grade adenovirus vaccines of high purity is feasible at an industrial scale. Correspondingly, purification procedures of the ChAdOx1 nCov-19 vaccine should be modified to remove protein impurities as good as possible. Our data also indicate that standard quality assays, as they are used in the manufacturing of proteins, have to be adapted for vectored vaccines.

Data availability

All data supporting the findings of this study are available within this paper. An overview of protein identifications and quantifications based on LC/MS analysis is shown in the source data (Fig. 2 - Source Data 1, Fig. 3 - Source Data 1, and Fig. 4 - Source Data 1).LC/MS-raw data and search results have been deposited at the Mass Spectrometry Interactive Virtual Environment(MassIVE; https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp) data lake and are publicly available under ID MSV000089634.

The following data sets were generated

Article and author information

Author details

  1. Lea Krutzke

    Department of Gene Therapy, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4092-4131
  2. Reinhild Rösler

    Core Unit Mass Spectrometry and Proteomics, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Ellen Allmendinger

    Department of Gene Therapy, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Tatjana Engler

    Department of Gene Therapy, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Sebastian Wiese

    Core Unit Mass Spectrometry and Proteomics, University of Ulm, Ulm, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Stefan Kochanek

    Department of Gene Therapy, University of Ulm, Ulm, Germany
    For correspondence
    stefan.kochanek@uni-ulm.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7494-1602

Funding

German Federal Ministry of Education and Research and Federal States of Germany Grant Innovative Hochschule"" (FKZ3IHS024D)

  • Lea Krutzke
  • Reinhild Rösler
  • Ellen Allmendinger
  • Tatjana Engler
  • Sebastian Wiese
  • Stefan Kochanek

German Research Foundation (SFB1074)

  • Lea Krutzke
  • Reinhild Rösler
  • Ellen Allmendinger
  • Tatjana Engler
  • Sebastian Wiese
  • Stefan Kochanek

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: Animal experiments were approved by the Animal Care Commission of the Government Baden-Württemberg. Reference number: TVA #1508.

Copyright

© 2022, Krutzke et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 14,504
    views
  • 1,327
    downloads
  • 28
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Lea Krutzke
  2. Reinhild Rösler
  3. Ellen Allmendinger
  4. Tatjana Engler
  5. Sebastian Wiese
  6. Stefan Kochanek
(2022)
Process- and product-related impurities in the ChAdOx1 nCov-19 vaccine
eLife 11:e78513.
https://doi.org/10.7554/eLife.78513

Share this article

https://doi.org/10.7554/eLife.78513

Further reading

    1. Immunology and Inflammation
    2. Microbiology and Infectious Disease
    Ainhoa Arbués, Sarah Schmidiger ... Damien Portevin
    Research Article

    The members of the Mycobacterium tuberculosis complex (MTBC) causing human tuberculosis comprise 10 phylogenetic lineages that differ in their geographical distribution. The human consequences of this phylogenetic diversity remain poorly understood. Here, we assessed the phenotypic properties at the host-pathogen interface of 14 clinical strains representing five major MTBC lineages. Using a human in vitro granuloma model combined with bacterial load assessment, microscopy, flow cytometry, and multiplexed-bead arrays, we observed considerable intra-lineage diversity. Yet, modern lineages were overall associated with increased growth rate and more pronounced granulomatous responses. MTBC lineages exhibited distinct propensities to accumulate triglyceride lipid droplets—a phenotype associated with dormancy—that was particularly pronounced in lineage 2 and reduced in lineage 3 strains. The most favorable granuloma responses were associated with strong CD4 and CD8 T cell activation as well as inflammatory responses mediated by CXCL9, granzyme B, and TNF. Both of which showed consistent negative correlation with bacterial proliferation across genetically distant MTBC strains of different lineages. Taken together, our data indicate that different virulence strategies and protective immune traits associate with MTBC genetic diversity at lineage and strain level.

    1. Microbiology and Infectious Disease
    Yue Sun, Jingwei Li ... Xin Deng
    Research Article

    The model Gram-negative plant pathogen Pseudomonas syringae utilises hundreds of transcription factors (TFs) to regulate its functional processes, including virulence and metabolic pathways that control its ability to infect host plants. Although the molecular mechanisms of regulators have been studied for decades, a comprehensive understanding of genome-wide TFs in Psph 1448A remains limited. Here, we investigated the binding characteristics of 170 of 301 annotated TFs through chromatin immunoprecipitation sequencing (ChIP-seq). Fifty-four TFs, 62 TFs, and 147 TFs were identified in top-level, middle-level, and bottom-level, reflecting multiple higher-order network structures and direction of information flow. More than 40,000 TF pairs were classified into 13 three-node submodules which revealed the regulatory diversity of TFs in Psph 1448A regulatory network. We found that bottom-level TFs performed high co-associated scores to their target genes. Functional categories of TFs at three levels encompassed various regulatory pathways. Three and 25 master TFs were identified to involve in virulence and metabolic regulation, respectively. Evolutionary analysis and topological modularity network revealed functional variability and various conservation of TFs in P. syringae (Psph 1448A, Pst DC3000, Pss B728a, and Psa C48). Overall, our findings demonstrated a global transcriptional regulatory network of genome-wide TFs in Psph 1448A. This knowledge can advance the development of effective treatment and prevention strategies for related infectious diseases.