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Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor binding domain in engineered Komagataella phaffii

Neil C. Dalvie1, Andrew M. Biedermann1, Sergio A. Rodriguez-Aponte1, Christopher A Naranjo1, Harish D Rao2, Meghraj P Rajurkar2, Rakesh R Lothe2, Umesh Shaligram2, Ryan S Johnston1, Laura E. Crowell1, Seraphin Castelino1, Mary Kate Tracey1, Charles A. Whittaker1, J. Christopher Love1 •
Massachusetts Institute of Technology1, Serum Institute of India (India)2
15 Apr 2021-bioRxiv (Cold Spring Harbor Laboratory)-
TL;DR: In this article, an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor binding domain (RBD) was reported, which is a key antigen for several reported vaccine candidates.
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 on-going 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 scale up to a 1,200 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.

Summary (2 min read)

Jump to: [Manuscript] – [Yeast strains] – [Cultivations] – [Analytical assays for protein characterization] and [Transcriptome analysis]

Manuscript

  • As new variants of SARS-CoV-2 emerge, continued development of diagnostics, vaccines, and reagents remains essential to address the COVID-19 pandemic.
  • Vaccine candidates based on protein subunits are also important ones for enabling interventions for the pandemic in low-and middle-income countries due to existing large-scale manufacturing facilities and less stringent temperature and storage requirements for distribution (Dai et al., 2020) .
  • Given these results with reduced quantities of methanol in these batch cultivations, the authors hypothesized that they could achieve efficient secretion of the RBD with no methanol.
  • Strains engineered for use without methanol and increased productivity could facilitate manufacturing of RBD and other antigens for vaccine candidates at large volumes and low costs to enable accessible and affordable vaccines for global use.

Yeast strains

  • All strains were derived from wild-type Komagataella phaffii (NRRL Y-11430).
  • The gene containing the RBD was codon optimized, synthesized (Integrated DNA Technologies), and cloned into a custom vector.
  • Transcription factors mit1 and mxr1 were integrated into the genome near genomic loci GQ67_02967 and GQ67_04576, respectively, using a markerless CRISPR-Cas9 system described previously (Dalvie et al., 2019) .
  • Both mit1 and mxr1 were under control of the PCAT1 promoter from K. phaffii.

Cultivations

  • Strains for initial characterization and titer measurement were grown in 3 mL culture in 24-well deep well plates (25°C, 600 rpm), and strains for protein purification were grown in 200 mL culture in 1 L shake flasks (25°C, 250 rpm).
  • Cells were cultivated in Rich Defined Media, described previously (Matthews et al., 2018) .
  • Cells were inoculated at 0.1 OD600, outgrown for 24 h with 4% glycerol feed, pelleted, and resuspended in fresh media with methanol or sorbitol feed to induce recombinant gene expression.
  • Supernatant samples were collected after 24 h of production, filtered, and analyzed.
  • InSCyT bioreactors and purification modules were operated as described previously (Crowell et al., 2018; Dalvie et al., 2021) .

Analytical assays for protein characterization

  • Purified protein concentrations were determined by absorbance at A280 nm.
  • SDS-PAGE was carried out as described previously (Crowell et al., 2018) .
  • Supernatant titers were measured by reverse phase liquid chromatography as described previously (Dalvie et al., 2021) , and normalized by cell density, measured by OD600.
  • Intact mass spectrometry was performed as described previously (Dalvie et al., 2021) .

Transcriptome analysis

  • RNA was extracted and purified according to the Qiagen RNeasy kit (cat #74104) and RNA quality was analyzed to ensure RNA Quality Number >6.5.
  • Sequenced mRNA transcripts were demultiplexed using sample barcodes and PCR duplicates were removed by selecting one sequence read per Unique Molecular Identifier (UMI) using a custom python script.
  • Expression was visualized using log2(Counts per Million + 1) values.
  • 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) .

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Figures (4)

Content maybe subject to copyright    Report

1
Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor binding domain in
engineered Komagataella phaffii
Neil C. Dalvie
1,2
^, Andrew M. Biedermann
1,2
^, Sergio A. Rodriguez-Aponte
2,3
, Christopher A.
Naranjo
2
, Harish D. Rao
4
, Meghraj P. Rajurkar
4
, Rakesh R. Lothe
4
, Umesh S. Shaligram
4
, Ryan
S. Johnston
2
, Laura E. Crowell
1,2
, Seraphin Castelino
1
, Mary Kate Tracey
2
, Charles A.
Whittaker
2
, J. Christopher Love
1,2
*
1
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139, United States
2
The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology,
Cambridge, Massachusetts 01239, United States
3
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139, United States
4
Serum Institute of India Pvt. Ltd., Pune, India
^Contributed equally
*Correspondence to: clove@mit.edu
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted April 15, 2021. ; https://doi.org/10.1101/2021.04.15.440035doi: bioRxiv preprint
2
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 on-going 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 scale up to a 1,200 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.
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted April 15, 2021. ; https://doi.org/10.1101/2021.04.15.440035doi: bioRxiv preprint
3
Manuscript
As new variants of SARS-CoV-2 emerge, continued development of diagnostics,
vaccines, and reagents remains essential to address the COVID-19 pandemic. The SARS-CoV-2
spike protein is an essential reagent for serological assays, and a component of several protein-
based vaccines (Guebre-Xabier et al., 2020; Tian et al., 2020). Vaccine candidates based on
protein subunits are also important ones for enabling interventions for the pandemic in low- and
middle-income countries (LMICs) due to existing large-scale manufacturing facilities and less
stringent temperature and storage requirements for distribution (Dai et al., 2020). We and others
have reported vaccine designs based on the receptor binding domain (RBD) of the spike protein
(Dalvie et al., 2021). In these designs, the RBD can be produced independently, and
subsequently displayed on protein or lipid nanoparticles for enhanced immunogenicity (Cohen et
al., 2021; Walls et al., 2020). The 201 amino acid RBD is an especially promising antigen for
accessible vaccines because it can be manufactured at low cost and high volumes in microbial
hosts (Chen et al., 2020; Pollet et al., 2020). Here, we report an engineered yeast strain with
enhanced secretion of the SARS-CoV-2 RBD from the circulating variants of Wuhan Hu-1,
B.1.1.7, and B.1.351 strains of the virus. This engineered host has been successfully deployed at
1,200 L scale to produce a vaccine component currently in clinical trials.
The methylotrophic yeast Komagataella phaffii (Pichia pastoris) is routinely used for the
production of therapeutic proteins at large volumes because of its high-capacity eukaryotic
secretory pathway (Love et al., 2018). Another key advantage of this production host is the
strong, tightly regulated, methanol-inducible promoter, P
AOX1
, used for expression of the
recombinant gene
(Ahmad et al., 2014). This promoter enables outgrowth to high cell densities
with inexpensive feedstock like glycerol before induction of the recombinant gene with methanol
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted April 15, 2021. ; https://doi.org/10.1101/2021.04.15.440035doi: bioRxiv preprint
4
feed. Methanol can pose challenges, however, in large-scale facilities, including high heat
generation during fermentation and flammability concerns while in storage (Potvin et al., 2012).
The impact of these challenges is that facilities require specific designs or modifications to
handle methanol. This requirement could limit the number of manufacturing facilities available
for the production vaccine components like the RBD antigens in K. phaffii in a pandemic. We
sought to reduce or eliminate the requirement for methanol for efficient secretion of the RBD.
We previously reported the production of the SARS-CoV-2 RBD (Wuhan-Hu-1
sequence) in an engineered variant of K. phaffii (Brady et al., 2020; Dalvie et al., 2021). To
assess the feasibility of methanol-free production, we cultivated the strain expressing RBD
regulated under the native AOX1 promoter, and induced expression of the recombinant gene
with varying amounts of methanol (Fig. 1A). Interestingly, the secreted titers of RBD increased
as the concentrations of methanol were reduced. We also induced protein production with a
combination of methanol and sorbitol—a supplementary carbon source that does not repress
P
AOX1
expression—and observed a further increase in titer.
Given these results with reduced quantities of methanol in these batch cultivations, we
hypothesized that we could achieve efficient secretion of the RBD with no methanol. Expression
of genes regulated by P
AOX1
in wild type K. phaffii in the absence of methanol is inconsistent,
even with non-repressive carbon sources like sorbitol (Vogl et al., 2018). Several studies,
however, have demonstrated that constitutive overexpression of activating transcription factors
can lead to consistent activation of P
AOX1
without methanol (Shi et al., 2019; Vogl et al., 2018).
To test production of RBD without methanol, we integrated additional copies of the endogenous
transcription factors mit1 and mxr1 into the K. phaffii genome under a glycerol-repressible
promoter (Dalvie et al., 2019). We cultivated these strains for protein production by feeding with
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted April 15, 2021. ; https://doi.org/10.1101/2021.04.15.440035doi: bioRxiv preprint
5
only sorbitol (Fig. 1B). We observed a >3-fold increase in specific productivity in all strains,
particularly with a strain containing only one extra copy of the transcription factor mit1 (>5-
fold). To assess the potential source of improved productivity, we examined the transcriptomes
of the methanol-fed initial strain and the modified, sorbitol-fed mit1+ strain by RNA-sequencing,
and analyzed the variations by gene set enrichment analysis (Fig. 1C). We observed significantly
higher expression of genes associated with protein folding stress in the methanol-fed condition
compared to the sorbitol-fed mit1+ condition (family-wise error p=0.003). These results
suggested that sorbitol-fed mit1+ may improve productivity by mitigating protein folding stress
associated with RBD production.
After comparing the specific productivity of the methanol-free strain (mit1+) to the
methanol-induced (base) strain, we assessed the production of RBD using both strains on
InSCyT, a continuous, automated, perfusion-based manufacturing platform (Crowell et al.,
2018). The base strain exhibited low titers (~30 mg/L) in perfusates and significant cell lysis
after ~120 h of fermentation in perfusion (Fig. 2A-B). In contrast, the mit1+ strain maintained
protein secretion at >50 mg/L/day for the duration of a >200 h campaign. RBD purified from the
perfusates produced by the base strain also contained more host-related impurities than RBD
from the mit1+ campaign (Fig. 2C). These results from the sustained production of RBD,
including the cell lysis observed in the base strain, are consistent with the observations for
increased cellular stress relative to the mit1+ strain, and suggest the transcriptional changes
observed also translated into variation in protein expression as well.
From these data for the improved production of RBD in bioreactors with the modified
strain without methanol, we then generated an mit1+ strain that expressed RBD with a C-
terminal fusion of SpyTag, a short peptide that can mediate a transpeptidation reaction with a
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted April 15, 2021. ; https://doi.org/10.1101/2021.04.15.440035doi: bioRxiv preprint
Citations
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Scientific rationale for developing potent RBD-based vaccines targeting COVID-19.

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Harry Kleanthous1, Judith M. Silverman, Karen W. Makar1, In-Kyu Yoon, Nicholas Jackson, D. W. Vaughn1 •
Bill & Melinda Gates Foundation1
28 Oct 2021
TL;DR: In this paper, the authors reviewed the data supporting the non-inferiority of RBD as a vaccine immunogen compared to full-length S-protein vaccines with respect to humoral and cellular immune responses against both the prototype pandemic SARS-CoV-2 isolate and emerging variants of concern.
Abstract: Vaccination of the global population against COVID-19 is a great scientific, logistical, and moral challenge. Despite the rapid development and authorization of several full-length Spike (S) protein vaccines, the global demand outweighs the current supply and there is a need for safe, potent, high-volume, affordable vaccines that can fill this gap, especially in low- and middle-income countries. Whether SARS-CoV-2 S-protein receptor-binding domain (RBD)-based vaccines could fill this gap has been debated, especially with regards to its suitability to protect against emerging viral variants of concern. Given a predominance for elicitation of neutralizing antibodies (nAbs) that target RBD following natural infection or vaccination, a key biomarker of protection, there is merit for selection of RBD as a sole vaccine immunogen. With its high-yielding production and manufacturing potential, RBD-based vaccines offer an abundance of temperature-stable doses at an affordable cost. In addition, as the RBD preferentially focuses the immune response to potent and recently recognized cross-protective determinants, this domain may be central to the development of future pan-sarbecovirus vaccines. In this study, we review the data supporting the non-inferiority of RBD as a vaccine immunogen compared to full-length S-protein vaccines with respect to humoral and cellular immune responses against both the prototype pandemic SARS-CoV-2 isolate and emerging variants of concern.

79 citations

Journal Article•DOI•
SARS-CoV-2 receptor binding domain displayed on HBsAg virus–like particles elicits protective immunity in macaques

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Neil C. Dalvie, Lisa H. Tostanoski, Sergio A. Rodriguez-Aponte, Kawaljit Kaur, S Bajoria, Ozan S. Kumru, Amanda J. Martinot, Abishek Chandrashekar, Katherine McMahan, Noe B. Mercado, Jingyou Yu, Aiquan Chang, Victoria M. Giffin, Felix Nampanya, Shivani A. Patel, Lesley A.H. Bowman, C. A. Naranjo, Dongsoo Yun, Zach Flinchbaugh, Laurent Pessaint, Renita Brown, Jason Velasco, Elyse Teow, Anthony Cook, Hanne Andersen, Mark A. Lewis, Danielle L. Camp, Judith M. Silverman, Gaurav S Nagar, Harish D Rao, Rakesh R. Lothe, Rahul Chandrasekharan, Meghraj P. Rajurkar, Umesh Shaligram, Harry Kleanthous, Sangeeta B. Joshi, David B. Volkin, Sumi Biswas, J. Christopher Love, Dan H. Barouch 
01 Mar 2022-Science Advances
TL;DR: Here, a clinical-stage vaccine candidate comprising a SARS-CoV-2 receptor binding domain–hepatitis B surface antigen virus–like particle elicited protective immunity in cynomolgus macaques, and the potential benefit of this design for a low-cost modular vaccine platform is supported.
Abstract: Authorized vaccines against SARS-CoV-2 remain less available in low- and middle-income countries due to insufficient supply, high costs, and storage requirements. Global immunity could still benefit from new vaccines using widely available, safe adjuvants, such as alum and protein subunits, suited to low-cost production in existing manufacturing facilities. Here, a clinical-stage vaccine candidate comprising a SARS-CoV-2 receptor binding domain–hepatitis B surface antigen virus–like particle elicited protective immunity in cynomolgus macaques. Titers of neutralizing antibodies (>104) induced by this candidate were above the range of protection for other licensed vaccines in nonhuman primates. Including CpG 1018 did not significantly improve the immunological responses. Vaccinated animals challenged with SARS-CoV-2 showed reduced median viral loads in bronchoalveolar lavage (~3.4 log10) and nasal mucosa (~2.9 log10) versus sham controls. These data support the potential benefit of this design for a low-cost modular vaccine platform for SARS-CoV-2 and other variants of concern or betacoronaviruses.

20 citations

Posted Content•DOI•
A modular protein subunit vaccine candidate produced in yeast confers protection against SARS-CoV-2 in non-human primates

[...]

Neil C. Dalvie1, Lisa H. Tostanoski2, Sergio A. Rodriguez-Aponte1, Kawaljit Kaur3, Sakshi Bajoria3, Ozan S. Kumru3, Amanda J. Martinot2, Amanda J. Martinot4, Abishek Chandrashekar2, Katherine McMahan2, Noe B. Mercado2, Jingyou Yu2, Aiquan Chang2, Aiquan Chang5, Victoria M. Giffin2, Felix Nampanya2, Shivani A. Patel2, Lesley Bowman, Christopher A Naranjo1, Dongsoo Yun1, Zach Flinchbaugh, Laurent Pessaint, Renita Brown, Jason Velasco, Elyse Teow, Anthony L. Cook, Hanne Leth Andersen, Mark G. Lewis, Danielle L Camp1, Judith M. Silverman, Harry Kleanthous6, Sangeeta B. Joshi3, David B. Volkin3, Sumi Biswas6, J. Christopher Love1, J. Christopher Love7, Dan H. Barouch •
Massachusetts Institute of Technology1, Beth Israel Deaconess Medical Center2, University of Kansas3, Tufts University4, Harvard University5, Bill & Melinda Gates Foundation6, Ragon Institute of MGH, MIT and Harvard7
14 Jul 2021-bioRxiv
TL;DR: In this article, the SARS-CoV-2 receptor binding domain (RBD) and hepatitis B surface antigen virus-like particles (VLPs) were used for preclinical testing in cynomolgus macaques.
Abstract: Vaccines against SARS-CoV-2 have been distributed at massive scale in developed countries, and have been effective at preventing COVID-19. Access to vaccines is limited, however, in low- and middle-income countries (LMICs) due to insufficient supply, high costs, and cold storage requirements. New vaccines that can be produced in existing manufacturing facilities in LMICs, can be manufactured at low cost, and use widely available, proven, safe adjuvants like alum, would improve global immunity against SARS-CoV-2. One such protein subunit vaccine is produced by the Serum Institute of India Pvt. Ltd. and is currently in clinical testing. Two protein components, the SARS-CoV-2 receptor binding domain (RBD) and hepatitis B surface antigen virus-like particles (VLPs), are each produced in yeast, which would enable a low-cost, high-volume manufacturing process. Here, we describe the design and preclinical testing of the RBD-VLP vaccine in cynomolgus macaques. We observed titers of neutralizing antibodies (>10 4 ) above the range of protection for other licensed vaccines in non-human primates. Interestingly, addition of a second adjuvant (CpG1018) appeared to improve the cellular response while reducing the humoral response. We challenged animals with SARS-CoV-2, and observed a ~3.4 and ~2.9 log 10 reduction in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, compared to sham controls. These results inform the design and formulation of current clinical COVID-19 vaccine candidates like the one described here, and future designs of RBD-based vaccines against variants of SARS-CoV-2 or other betacoronaviruses.

14 citations

Journal Article•DOI•
Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor-binding domain in engineered Komagataella phaffii.

[...]

Neil C. Dalvie1, Andrew M. Biedermann1, Sergio A. Rodriguez-Aponte1, Christopher A Naranjo1, Harish D Rao2, Meghraj P Rajurkar2, Rakesh R Lothe2, Umesh Shaligram2, Ryan S Johnston1, Laura E. Crowell1, Seraphin Castelino1, Mary Kate Tracey1, Charles A. Whittaker1, J. Christopher Love1 •
Massachusetts Institute of Technology1, Serum Institute of India (India)2
15 Nov 2021-Biotechnology and Bioengineering
TL;DR: In this article, an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor-binding domain (RBD) was reported, which is a key antigen for several reported vaccine candidates.
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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An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen

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TL;DR: In this article , a vaccine based on the SARS-CoV-2 receptor-binding domain (RBD), produced in the yeast Pichia pastoris, was developed.

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Stony Brook University1, Harvard University2, Carnegie Mellon University3
01 Apr 2017-Nature Methods
TL;DR: Salmon is the first transcriptome-wide quantifier to correct for fragment GC-content bias, which substantially improves the accuracy of abundance estimates and the sensitivity of subsequent differential expression analysis.
Abstract: We introduce Salmon, a lightweight method for quantifying transcript abundance from RNA-seq reads. Salmon combines a new dual-phase parallel inference algorithm and feature-rich bias models with an ultra-fast read mapping procedure. It is the first transcriptome-wide quantifier to correct for fragment GC-content bias, which, as we demonstrate here, substantially improves the accuracy of abundance estimates and the sensitivity of subsequent differential expression analysis.

6,095 citations

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01 May 2012-Nucleic Acids Research
TL;DR: A flexible statistical framework is developed for the analysis of read counts from RNA-Seq gene expression studies, and parallel computational approaches are developed to make non-linear model fitting faster and more reliable, making the application of GLMs to genomic data more convenient and practical.
Abstract: A flexible statistical framework is developed for the analysis of read counts from RNA-Seq gene expression studies. It provides the ability to analyse complex experiments involving multiple treatment conditions and blocking variables while still taking full account of biological variation. Biological variation between RNA samples is estimated separately from the technical variation associated with sequencing technologies. Novel empirical Bayes methods allow each gene to have its own specific variability, even when there are relatively few biological replicates from which to estimate such variability. The pipeline is implemented in the edgeR package of the Bioconductor project. A case study analysis of carcinoma data demonstrates the ability of generalized linear model methods (GLMs) to detect differential expression in a paired design, and even to detect tumour-specific expression changes. The case study demonstrates the need to allow for gene-specific variability, rather than assuming a common dispersion across genes or a fixed relationship between abundance and variability. Genewise dispersions de-prioritize genes with inconsistent results and allow the main analysis to focus on changes that are consistent between biological replicates. Parallel computational approaches are developed to make non-linear model fitting faster and more reliable, making the application of GLMs to genomic data more convenient and practical. Simulations demonstrate the ability of adjusted profile likelihood estimators to return accurate estimators of biological variability in complex situations. When variation is gene-specific, empirical Bayes estimators provide an advantageous compromise between the extremes of assuming common dispersion or separate genewise dispersion. The methods developed here can also be applied to count data arising from DNA-Seq applications, including ChIP-Seq for epigenetic marks and DNA methylation analyses.

4,127 citations

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Frequently Asked Questions (14)
Q1. What are the contributions mentioned in the paper "Scalable, methanol-free manufacturing of the sars-cov-2 receptor binding domain in engineered komagataella phaffii" ?

The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 01239, United States this paper. 

As new variants of SARS-CoV-2 emerge, continued development of diagnostics,vaccines, and reagents remains essential to address the COVID-19 pandemic. 

Another key advantage of this production host is the strong, tightly regulated, methanol-inducible promoter, PAOX1, used for expression of the recombinant gene (Ahmad et al., 2014). 

The SARS-CoV-2 spike protein is an essential reagent for serological assays, and a component of several proteinbased vaccines (Guebre-Xabier et al., 2020; Tian et al., 2020). 

The methylotrophic yeast Komagataella phaffii (Pichia pastoris) is routinely used for theproduction of therapeutic proteins at large volumes because of its high-capacity eukaryotic secretory pathway (Love et al., 2018). 

The authors demonstrated sustained productivity from the strain in a perfusion process, and scale-up to a large-scale, solvent-free fed batch process to produce avaccine component currently in clinical trials. 

This engineered host has been successfully deployed at 1,200 L scale to produce a vaccine component currently in clinical trials. 

These results from the sustained production of RBD, including the cell lysis observed in the base strain, are consistent with the observations for increased cellular stress relative to the mit1+ strain, and suggest the transcriptional changes observed also translated into variation in protein expression as well. 

RNA was extracted andpurified according to the Qiagen RNeasy kit (cat #74104) and RNA quality was analyzed to ensure RNA Quality Number >6.5. 

From these data for the improved production of RBD in bioreactors with the modifiedstrain without methanol, the authors then generated an mit1+ strain that expressed RBD with a Cterminal fusion of SpyTag, a short peptide that can mediate a transpeptidation reaction with acognate SpyCatcher polypeptide, which can be presented on protein nanoparticles for example (Reddington and Howarth, 2015). 

This result demonstrates that the engineered mit1+ strain could facilitate new cell lines for manufacturing other RBD variants without methanol for seasonal vaccine boosters or next-generation vaccine candidates for emerging variants. 

To test production of RBD without methanol, the authors integrated additional copies of the endogenous transcription factors mit1 and mxr1 into the K. phaffii genome under a glycerol-repressible promoter (Dalvie et al., 2019). 

In these designs, the RBD can be produced independently, and subsequently displayed on protein or lipid nanoparticles for enhanced immunogenicity (Cohen et al., 2021; Walls et al., 2020). 

These results suggested that sorbitol-fed mit1+ may improve productivity by mitigating protein folding stress associated with RBD production.