Scalable, methanol‐free manufacturing of the SARS‐CoV‐2 receptor‐binding domain in engineered Komagataella phaffii
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Abstract
Prevention of COVID‐19 on a global scale will require the continued development of high‐volume, low‐cost platforms for the manufacturing of vaccines to supply ongoing demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large‐scale production facilities that use microbial hosts like Komagataella phaffii ( Pichia pastoris ). Here, we report an improved and scalable manufacturing approach for the SARS‐CoV‐2 spike protein receptor‐binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol induction of the recombinant gene. Methanol‐free production improved the secreted titer of the RBD protein by >5X by alleviating protein folding stress. Removal of methanol from the production process enabled to scale up to a 1200 L pre‐existing production facility. This engineered strain is now used to produce an RBD‐based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.
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SciScore for 10.1101/2021.04.15.440035: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources Transcripts were quantified with Salmon version 1.3.0 (Patro et al., 2017) and selective alignment using a target consisting of the K. phaffii transcripts, the RBD, and selectable marker transgene sequences and the K. Salmonsuggested: (Salmon, RRID:SCR_017036)Expression values were summarized with tximport version 1.12.3 (Soneson et al., 2016) and edgeR version 3.26.8 (McCarthy et al., 2012; Robinson et al., 2009). tximportsuggested: (tximport, RRID:SCR_016752)edgeRsuggested: …SciScore for 10.1101/2021.04.15.440035: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources Transcripts were quantified with Salmon version 1.3.0 (Patro et al., 2017) and selective alignment using a target consisting of the K. phaffii transcripts, the RBD, and selectable marker transgene sequences and the K. Salmonsuggested: (Salmon, RRID:SCR_017036)Expression values were summarized with tximport version 1.12.3 (Soneson et al., 2016) and edgeR version 3.26.8 (McCarthy et al., 2012; Robinson et al., 2009). tximportsuggested: (tximport, RRID:SCR_016752)edgeRsuggested: (edgeR, RRID:SCR_012802)Gene set enrichment analysis (GSEA) was performed with GSEA 4.1.0 using Wald statistics calculated by DESeq2 (Love et al., 2014) and gene sets from yeast GO Slim (Subramanian et al., 2005). Gene set enrichment analysissuggested: (Gene Set Enrichment Analysis, RRID:SCR_003199)GSEAsuggested: (SeqGSEA, RRID:SCR_005724)DESeq2suggested: (DESeq, RRID:SCR_000154)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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
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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