Production of trimeric SARS‐CoV‐2 spike protein by CHO cells for serological COVID‐19 testing

  1. Yusuf B. Johari
  2. Stephen R. P. Jaffé
  3. Joseph M. Scarrott
  4. Abayomi O. Johnson
  5. Théo Mozzanino
  6. Thilo H. Pohle
  7. Sheetal Maisuria
  8. Amina Bhayat‐Cammack
  9. Giulia Lambiase
  10. Adam J. Brown
  11. Kang Lan Tee
  12. Philip J. Jackson
  13. Tuck Seng Wong
  14. Mark J. Dickman
  15. Ravishankar B. Sargur
  16. David C. James

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Abstract

We describe scalable and cost‐efficient production of full length, His‐tagged severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike glycoprotein trimer by Chinese hamster ovary (CHO) cells that can be used to detect SARS‐CoV‐2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both human embryonic kidney (HEK) and CHO cells mediated by polyethyleneimine was increased significantly (up to 10.9‐fold) by a reduction in culture temperature to 32°C to permit extended duration cultures. Based on these data GS‐CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9‐fold to 53 mg/L. Purification of recombinant spike by Ni‐chelate affinity chromatography initially yielded a variety of co‐eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in enzyme‐linked immunosorbent assay format to detect immunoglobulin G antibodies against SARS‐CoV‐2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (COVID‐19) symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS‐CoV‐2 trimer which can be used as antigen for mass serological testing.

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  1. Version published to 10.1002/bit.27615
  2. ScreenIT

    SciScore for 10.1101/2020.08.07.20169441: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Membranes were blocked in 5% milk/TBS-T before being incubated with HRP-conjugated anti-HisTag antibody (Bio-Rad) and visualized by enhanced chemiluminescence (ECL; ThermoFisher).
    anti-HisTag
    suggested: (RevMAb Biosciences Cat# 54-1161-00, RRID:AB_2716428)
    A complementary quantification of spike in culture supernatant was performed using CR3022 antibody ELISA (Supplementary Figure S4)
    CR3022
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Transient Production in HEK and CHO Cells: pCAGGS-spike was provided by the Krammer Laboratory (Icahn School of Medicine at Mount Sinai), amplified, and purified using QIAGEN Plasmid Plus kit (Qiagen).
    HEK
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Software and Algorithms
    SentencesResources
    Proteins were identified by converting the MS data into Mascot Generic Format (MGF) files and analyzed against human and Chinese hamster reference proteome databases with the spike glycoprotein construct sequence inserted (www.uniprot.org) using Mascot Daemon v.
    Mascot Daemon
    suggested: None
    2.5.1 with Mascot server v.2.5
    Mascot
    suggested: (Mascot, RRID:SCR_014322)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2020.08.07.20169441 on medRxiv
  4. Version published to 10.22541/au.159665063.38244714