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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.

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). 1 Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. 2 Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing costs. 3 These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. 4-6 Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. 7,8 Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge., Competing Interests: Competing interests L.E.C., K.R.L., and J.C.L. have filed patents related to the InSCyT system and methods. N.C.D., S.R.A., and J.C.L. have filed a patent related to the RBD-L452K-F490W sequence. K.R.L., L.E.C., and M.K.T. are current employees 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. J.M.S. is an employee of the Bill & Melinda Gates Medical Research Institute. H.K. is an employee of the Bill & Melinda Gates Foundation.