Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing cost. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. 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. 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.

Conventional manufacturing of protein biopharmaceuticals in centralized, large-scale single-product facilities is not well-suited to the agile production of drugs for small patient populations or individuals. Solutions for small-scale manufacturing are potentially more nimble, though previous systems are limited in both process reproducibility and product quality, owing to complicated means of protein expression and purification 1 – 4 . We describe an automated bench-top multi-product manufacturing system, called Integrated Scalable Cyto-Technology (InSCyT), for the end-to-end production of hundreds to thousands of doses of clinical-quality protein biologics in about three days. We also demonstrate that InSCyT can accelerate process development from sequence to purified drug in 12 weeks. We produced hGH, IFNα-2b, and G-CSF using highly similar processes on InSCyT and found that the purity and potency of these products is comparable to that of marketedreference products.

Komagataella phaffii, also known as Pichia pastoris, is a common host for the production of biologics and enzymes, due to fast growth, high productivity, and advancements in host engineering. Several K. phaffii variants are commonly used as interchangeable base strains, which confounds efforts to improve this host. In this study, genomic and transcriptomic analyses of Y‐11430 (CBS7435), GS115, X‐33, and eight other variants enabled a comparative assessment of the relative fitness of these hosts for recombinant protein expression. Cell wall integrity explained the majority of the variation among strains, impacting transformation efficiency, growth, methanol metabolism, and secretion of heterologous proteins. Y‐11430 exhibited the highest activity of genes involved in methanol utilization, up to two‐fold higher transcription of heterologous genes, and robust growth. With a more permeable cell wall, X‐33 displayed a six‐fold higher transformation efficiency and up to 1.2‐fold higher titers than Y‐11430. X‐33 also shared nearly all mutations, and a defective variant of HIS4, with GS115, precluding robust growth. Transferring two beneficial mutations identified in X‐33 into Y‐11430 resulted in an optimized base strain that provided up to four‐fold higher transformation efficiency and three‐fold higher protein titers, while retaining robust growth. The approach employed here to assess unique banked variants in a species and then transfer key beneficial variants into a base strain should also facilitate rational assessment of a broad set of other recombinant hosts.

There is growing interest in the use of nonmodel microorganisms as hosts for biopharmaceutical manufacturing. These hosts require genomic engineering to meet clinically relevant product qualities and titers, but the adaptation of tools for editing genomes, such as CRISPR-Cas9, has been slow for poorly characterized hosts. Specifically, a lack of biochemical characterization of RNA polymerase III transcription has hindered reliable expression of guide RNAs in new hosts. Here, we present a sequencingbased strategy for the design of host-specific cassettes for modular, reliable, expression of guide RNAs. Using this strategy, we achieved up to 95% gene editing efficiency in the methylotrophic yeast Komagataella phaff ii. We applied this approach for the rapid, multiplexed engineering of a complex phenotype, achieving humanized product glycosylation in two sequential steps of engineering. Reliable extension of simple gene editing tools to nonmodel manufacturing hosts will enable rapid engineering of manufacturing strains tuned for specific product profiles and potentially decrease the costs and timelines for process development.

No abstract