Trimeric SARS-CoV-2 Spike Proteins Produced from CHO Cells in Bioreactors Are High-Quality Antigens
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Abstract
The spike protein of the pandemic human corona virus is essential for its entry into human cells. In fact, most neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) are directed against the Virus-surface exposed spike protein, making it the antigen of choice for use in vaccines and diagnostic tests. In the current pandemic context, global demand for spike proteins has rapidly increased and could exceed hundreds of grams to kilograms annually. Coronavirus spikes are large heavily glycosylated homo-trimeric complexes, with inherent instability. The poor manufacturability now threatens the availability of these proteins for vaccines and diagnostic tests. Here, we outline scalable, Good Manufacturing Practice (GMP) compliant, and chemically defined processes for the production of two cell-secreted stabilized forms of the trimeric spike proteins (Wuhan and D614G variant). The processes are chemically defined and based on clonal suspension-CHO cell populations and on protein purification via a two-step scalable downstream process. The trimeric conformation was confirmed using electron microscopy and HPLC analysis. Binding to susceptible cells was shown using a virus-inhibition assay. The diagnostic sensitivity and specificity for detection of serum SARS-CoV-2-specific-immunoglobulin molecules was found to exceed that of spike fragments (Spike subunit-1, S1 and Receptor Binding Domain, RBD). The process described here will enable production of sufficient high-quality trimeric spike protein to meet the global demand for SARS-CoV-2 diagnostic tests and potentially vaccines.
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SciScore for 10.1101/2020.11.15.382044: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Supernatants of high-density transient transfections under suspension culture were harvested after 10 days, mimicking a fed-batch process with ExcellGene’s animal component-free and protein-free CHO4Tx® PM (production medium) in 50 mL TubeSpin® bioreactor 50 tubes (TPP, Trasadingen, Switzerland). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Supernatants Recombinant CHO cell lines: After transfections, suspension cells were stringently selected with puromycin. CHOsuggested: CLS Cat# …SciScore for 10.1101/2020.11.15.382044: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Supernatants of high-density transient transfections under suspension culture were harvested after 10 days, mimicking a fed-batch process with ExcellGene’s animal component-free and protein-free CHO4Tx® PM (production medium) in 50 mL TubeSpin® bioreactor 50 tubes (TPP, Trasadingen, Switzerland). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Supernatants Recombinant CHO cell lines: After transfections, suspension cells were stringently selected with puromycin. CHOsuggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)This mixture was inoculated onto Vero E6 cells were inoculated with SARS-CoV-2 and at 48 hours post infection, SARS-CoV-2 infection was visualized using virus nucleocapsid antigen specific staining (red) and by a cell nucleus specific staining (blue), as described in [10]. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Contrast transfer function estimation and particle picking were performed in cisTEM [16]. cisTEMsuggested: (cisTEM, RRID:SCR_016502)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.
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