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. 2021 Feb;118(2):1013-1021.
doi: 10.1002/bit.27615. Epub 2020 Nov 11.

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

Yusuf B Johari  1 , Stephen R P Jaffé  1 , Joseph M Scarrott  1 , Abayomi O Johnson  1 , Théo Mozzanino  1 , Thilo H Pohle  1 , Sheetal Maisuria  2 , Amina Bhayat-Cammack  2 , Giulia Lambiase  1 , Adam J Brown  1 , Kang Lan Tee  1 , Philip J Jackson  1 , Tuck Seng Wong  1 , Mark J Dickman  1 , Ravishankar B Sargur  2 , David C James  1
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Free article

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

Yusuf B Johari et al. Biotechnol Bioeng. 2021 Feb.
Free article

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.

Keywords: CHO cells; COVID-19; SARS-CoV-2; bioproduction; serological assay; spike trimer.

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REFERENCES

    1. Amanat, F., Stadlbauer, D., Strohmeier, S., Nguyen, T. H. O., Chromikova, V., McMahon, M., Jiang, K., Arunkumar, G. A., Jurczyszak, D., Polanco, J., Bermudez-Gonzalez, M., Kleiner, G., Aydillo, T., Miorin, L., Fierer, D. S., Lugo, L. A., Kojic, E. M., Stoever, J., Liu, S. T. H., … Krammer, F. (2020). A serological assay to detect SARS-CoV-2 seroconversion in humans. Nature Medicine, 26, 1033-1036. https://doi.org/10.1038/s41591-020-0913-5
    1. Brown, A. J., Gibson, S. J., Hatton, D., & James, D. C. (2017). In silico design of context-responsive mammalian promoters with user-defined functionality. Nucleic Acids Research, 45, 10906-10919. https://doi.org/10.1093/nar/gkx768
    1. Cartwright, J. F., Arnall, C. L., Patel, Y. D., Barber, N. O. W., Lovelady, C. S., Rosignoli, G., Harris, C. L., Dunn, S., Field, R. P., Dean, G., Daramola, O., Gibson, S. J., Peden, A. A., Brown, A. J., Hatton, D., & James, D. C. (2020). A platform for context-specific genetic engineering of recombinant protein production by CHO cells. Journal of Biotechnology, 312, 11-22. https://doi.org/10.1016/j.jbiotec.2020.02.012
    1. Esposito, D., Mehalko, J., Drew, M., Snead, K., Wall, V., Taylor, T., Frank, P., Denson, J.-P., Hong, M., Gulten, G., Sadtler, K., Messing, S., & Gillette, W. (2020). Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays. Protein Expression and Purification, 174, 105686. https://doi.org/10.1016/j.pep.2020.105686
    1. Estes, B., Hsu, Y. R., Tam, L. T., Sheng, J., Stevens, J., & Haldankar, R. (2015). Uncovering methods for the prevention of protein aggregation and improvement of product quality in a transient expression system. Biotechnology Progress, 31, 258-267. https://doi.org/10.1002/btpr.2021

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