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

View ORCID ProfileNeil C. Dalvie, Andrew M. Biedermann, Sergio A. Rodriguez-Aponte, Christopher A. Naranjo, Harish D. Rao, Meghraj P. Rajurkar, Rakesh R. Lothe, Umesh S. Shaligram, Ryan S. Johnston, Laura E. Crowell, Seraphin Castelino, Mary Kate Tracey, Charles A. Whittaker, View ORCID ProfileJ. Christopher Love
doi: https://doi.org/10.1101/2021.04.15.440035
Neil C. Dalvie
1Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
2The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 01239, United States
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  • ORCID record for Neil C. Dalvie
Andrew M. Biedermann
1Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
2The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 01239, United States
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Sergio A. Rodriguez-Aponte
2The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 01239, United States
3Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
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Christopher A. Naranjo
2The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 01239, United States
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Harish D. Rao
4Serum Institute of India Pvt. Ltd., Pune, India
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Meghraj P. Rajurkar
4Serum Institute of India Pvt. Ltd., Pune, India
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Rakesh R. Lothe
4Serum Institute of India Pvt. Ltd., Pune, India
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Umesh S. Shaligram
4Serum Institute of India Pvt. Ltd., Pune, India
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Ryan S. Johnston
2The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 01239, United States
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Laura E. Crowell
1Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
2The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 01239, United States
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Seraphin Castelino
1Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
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Mary Kate Tracey
2The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 01239, United States
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Charles A. Whittaker
2The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 01239, United States
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J. Christopher Love
1Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
2The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 01239, United States
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  • ORCID record for J. Christopher Love
  • For correspondence: clove@mit.edu
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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 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.

Competing Interest Statement

L.E.C. and J.C.L. have filed patents related to the InSCyT system and methods. L.E.C. is a current employee at Sunflower Therapeutics PBC. J.C.L. has interests in Sunflower Therapeutics PBC, Pfizer, Honeycomb Biotechnologies, OneCyte Biotechnologies, QuantumCyte, Amgen, and Repligen. J.C.L's interests are reviewed and managed under MIT's policies for potential conflicts of interest. H.D.R., M.P.R., R.R.L., and U.S.S. are employees of Serum Institute of India Pvt. Ltd.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted April 15, 2021.
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Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor binding domain in engineered Komagataella phaffii
Neil C. Dalvie, Andrew M. Biedermann, Sergio A. Rodriguez-Aponte, Christopher A. Naranjo, Harish D. Rao, Meghraj P. Rajurkar, Rakesh R. Lothe, Umesh S. Shaligram, Ryan S. Johnston, Laura E. Crowell, Seraphin Castelino, Mary Kate Tracey, Charles A. Whittaker, J. Christopher Love
bioRxiv 2021.04.15.440035; doi: https://doi.org/10.1101/2021.04.15.440035
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Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor binding domain in engineered Komagataella phaffii
Neil C. Dalvie, Andrew M. Biedermann, Sergio A. Rodriguez-Aponte, Christopher A. Naranjo, Harish D. Rao, Meghraj P. Rajurkar, Rakesh R. Lothe, Umesh S. Shaligram, Ryan S. Johnston, Laura E. Crowell, Seraphin Castelino, Mary Kate Tracey, Charles A. Whittaker, J. Christopher Love
bioRxiv 2021.04.15.440035; doi: https://doi.org/10.1101/2021.04.15.440035

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