Process development and scale-up optimization of the SARS-CoV-2 receptor binding domain–based vaccine candidate, RBD219-N1C1
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Abstract
A SARS-CoV-2 RBD219-N1C1 (RBD219-N1C1) recombinant protein antigen formulated on Alhydrogel® has recently been shown to elicit a robust neutralizing antibody response against SARS-CoV-2 pseudovirus in mice. The antigen has been produced under current good manufacturing practices (cGMPs) and is now in clinical testing. Here, we report on process development and scale-up optimization for upstream fermentation and downstream purification of the antigen. This includes production at the 1-L and 5-L scales in the yeast, Pichia pastoris , and the comparison of three different chromatographic purification methods. This culminated in the selection of a process to produce RBD219-N1C1 with a yield of >400 mg per liter of fermentation with >92% purity and >39% target product recovery after purification. In addition, we show the results from analytical studies, including SEC-HPLC, DLS, and an ACE2 receptor binding assay that were performed to characterize the purified proteins to select the best purification process. Finally, we propose an optimized upstream fermentation and downstream purification process that generates quality RBD219-N1C1 protein antigen and is fully scalable at a low cost.
Key points
• Yeast fermentation conditions for a recombinant COVID-19 vaccine were determined .
• Three purification protocols for a COVID-19 vaccine antigen were compared .
• Reproducibility of a scalable, low-cost process for a COVID-19 vaccine was shown .
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SciScore for 10.1101/2020.12.30.424829: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources RBD219-N1C1 was detected using a rabbit monoclonal antibody against the SARS-CoV-2 Spike S1 protein (Sino Biological, Beijing, China; Cat#: 40150-R007) and goat anti-rabbit IgG secondary antibodies conjugated with horseradish peroxidase (Invitrogen, Carlsbad, USA; Cat#: G21234). anti-rabbit IgGsuggested: (Thermo Fisher Scientific Cat# G-21234, RRID:AB_2536530)pastoris HCP antibodies that recognize the N-glycans, which could result in an overestimation of true HCP, we performed quantitative ELISAs with a second-generation anti-Pichia pastoris HCP ELISA Kit (Cygnus, Southport, USA; Cat# F640) following … SciScore for 10.1101/2020.12.30.424829: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources RBD219-N1C1 was detected using a rabbit monoclonal antibody against the SARS-CoV-2 Spike S1 protein (Sino Biological, Beijing, China; Cat#: 40150-R007) and goat anti-rabbit IgG secondary antibodies conjugated with horseradish peroxidase (Invitrogen, Carlsbad, USA; Cat#: G21234). anti-rabbit IgGsuggested: (Thermo Fisher Scientific Cat# G-21234, RRID:AB_2536530)pastoris HCP antibodies that recognize the N-glycans, which could result in an overestimation of true HCP, we performed quantitative ELISAs with a second-generation anti-Pichia pastoris HCP ELISA Kit (Cygnus, Southport, USA; Cat# F640) following the manufacturer’s instructions. anti-Pichiasuggested: NoneSerially-diluted RBD219-N1C1 was loaded onto the strips (HCP standards range from 0-250 ng/mL) in the presence of HRP conjugated anti-P. pastoris antibodies. anti-Psuggested: None100 μL serially-diluted ACE2-hFc (LakePharma, San Carlos, USA; Cat # 46672) was added to the wells and incubated at room temperature for 2 hours and the binding was detected by adding 100 μL 1:10,000 diluted HRP conjugated anti-human IgG antibodies (GenScript, Piscataway, USA; Cat# A00166) with a 1-hour incubation period at room temperature. anti-human IgGsuggested: NoneResults 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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