Establishing Novel Cell-free Platforms for the Synthesis of Protein-based Biologics

Rey William Martin

doi:https://doi.org/10.21985/N2JJ1C

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Establishing Novel Cell-free Platforms for the Synthesis of Protein-based Biologics

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There is a need for alternative biomanufacturing solutions to produce novel medicines and help solve long-standing issues regarding difficult-to-treat diseases. Cell-free protein synthesis (CFPS) provides scientists with a new paradigm for producing proteins with an unprecedented freedom and control over the biomolecular machinery involved. In this document, I describe my PhD work on establishing novel cell-free systems for expanding the genetic code, as well as accelerating design-build-test cycles of important protein-based biologics. In my first aim, I focus on establishing a cell-free system derived from genomically recoded Escherichia coli strains for the synthesis of proteins containing non-canonical amino acids (ncAAs) at high yield and purity. This work first focused on genomic engineering efforts to improve CFPS yields, as well as biochemical and system biology analysis of reactions to understand the mechanistic impact of the mutations made. Yields surpassed the gram per liter range for a variety of wild-type proteins and, importantly, our engineered CFPS system demonstrated the highest level of ncAA incorporation to date at high yield and purity. Furthermore, in collaboration with Leidos, we demonstrate the engineered system’s utility towards on-demand biomanufacturing of therapeutic proteins. Next, I apply my knowledge of cell-free systems towards the development of CHO-based CFPS system for accelerating the design-build-test cycles of monoclonal antibodies (mAbs). First, we demonstrate the synthesis of over 100 milligrams of mAb per liter in an active conformation. Second, we compare expression yields of our in vitro system to conventional in vivo systems and demonstrate a good predictive correlation. The work described throughout this dissertation has implications for the accelerated synthesis of novel protein-based biologics to treat diseases both on-demand and at manufacturing scale. Current state-of-the-art in vivo expression systems struggle to meet these demands due to slower production timelines (on the order of days to weeks) starting from DNA.

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