Improving and engineering cell-free systems for glycoprotein production

Hershewe, Jasmine  Marie

doi:https://doi.org/10.21985/n2-1htx-x413

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Improving and engineering cell-free systems for glycoprotein production

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The need to rapidly develop and produce life-saving vaccines and therapeutics is critical for overcoming pandemics in a global economy. Recent advances in automation and cell-free systems have opened new avenues for expediting optimization and production of biologic vaccines and therapeutics. A key consideration for the development of protein biologics is glycosylation, the enzymatic modification of amino acid sidechains with sugar moieties. Protein glycosylation occurs on over half of FDA-approved therapeutics, with glycosylation patterns profoundly impacting bioactivity of protein biologics. We recently developed a platform for cell-free glycoprotein synthesis (CFGpS) that enables on-demand, decentralized biomanufacturing of glycoconjugate vaccines. This platform overcomes inefficiencies in traditional, centralized manufacturing processes because it does not incur cold-chain storage costs, can be performed using a single microbial host, and can synthesize vaccine in ~ 1 hour at the point of care. Our CFGpS platform, which is driven by crude extracts of Escherichia coli, is highly modular because the glycosylation machinery is programmed with heterologous enzymes. The platform modularity is a major advantage for producing a palette of pathogen-specific conjugate vaccines. This platform, however, has limitations including 1) low glycoprotein yields, and 2) a lack of a high throughput testbed for discovering optimized glycosylation systems (i.e., identifying optimized enzymes that carry out defined glycosylation reactions). This work addresses both issues. Here, we significantly improve glycoprotein yields in CFGpS by optimizing the methods for preparing crude E. coli extracts enriched with glycosylation machinery. We characterize, for the first, time the biological catalyst of CFGpS reactions—nanoscale lipid vesicles with embedded glycosylation machinery. We use these insights to optimize the CFGpS platform, significantly increasing glycosylation efficiency, enhancing glycoprotein titers by up to 90%, and enabling glycoprotein titers of > 100 µg/mL. Notably, the methods used to improve glycosylation efficiency also cut processing time and cost, further improving the CFGpS platform efficiency. To expedite characterization of new glycosylation pathways, we also develop a platform for rapid, high-yielding cell-free synthesis and testing of critical glycosylation enzymes. We express high titers of glycosylation enzymes in vitro, and show that these enzymes can be characterized downstream using a powerful, high-throughput mass spectrometry technique. This platform promises to expedite the discovery and engineering of enzymes to improve glycosylation efficiency in the CFGpS platform. Taken together, these works alleviate research bottlenecks toward more efficient, agile, and cost-effective production of vaccines that combat pathogens.

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