Description:

Speaker: Rey Martin, Jewett Lab

Title: Development of a CHO-based Cell-free Biomanufacturing Platform for the Synthesis of Active Monoclonal Antibodies as a Potential High-Throughput Screening Tool

Abstract
Chinese Hamster Ovary (CHO) cells are routinely optimized to stably express monoclonal antibodies (mAbs) at high titers. At the early stages of lead isolation and optimization, hundreds of sequences for the target protein of interest are screened. Typically, cell-based transient expression technology platforms are used for expression screening, but these can be time- and resource-intensive. I will describe our work towards developing a cell-free protein synthesis (CFPS) platform utilizing a commercially available CHO extract for the rapid in vitro synthesis of active, aglycosylated mAbs. Specifically, we optimized reaction conditions to maximize protein yields, established an oxidizing environment to enable disulfide bond formation, and demonstrated the importance of temporal addition of heavy chain and light chain plasmids for intact mAb production. Using our optimized platform, we demonstrate for the first time to our knowledge the CFPS of biologically active, intact mAb at >100 mg/L using a eukaryotic-based extract. We then also explored the utility of our system as a tool for ranking yields of candidate antibodies. Unlike stable or transient transfection-based screening, which requires a minimum of 7 days for setup and execution, results using our CHO-based CFPS platform are attained within 2 days and it is well-suited for automation. Further development would provide a tool for rapid, high throughput prediction of expression ranking of mAb producers to accelerate design-build-test cycles required for antibody expression and engineering. Looking forward, the CHO-based CFPS platform could facilitate the synthesis of toxic proteins as well.

Speaker: Quentin Dudley, Jewett Lab

Title: Bio-breadboarding: using cell-free mixing of crude extracts to prototype isoprenoid biosynthesis

Abstract
Metabolic engineering of microorganisms to produce useful compounds from renewable substrates is a promising means for sustainable, on-demand production of chemicals. However, efforts to design and engineer microbial cell factories are constrained by slow “build” times in which each genetic variation requires re-engineering a new strain for each iteration. To alleviate this challenge, we have built a plug-and-play prototyping system for isoprenoid biosynthesis. Isoprenoids are a promising class of target molecules with over 40,000 known structures and potential uses as pharmaceuticals, flavors, fragrances, pesticides, disinfectants, and chemical feedstocks. By mixing together multiple crude extracts, each enriched in a single pathway enzyme, we can recapitulate isoprenoid metabolism in a test tube which allows easy manipulation of the reaction conditions and quick testing of enzyme ratios and pathway configurations. To further minimize the time required to test enzyme homologs, we have used cell-free protein synthesis (CFPS) to synthesize pathway enzymes directly in the lysate by simply adding the appropriate DNA template along with energy molecules, cofactors, and amino acid substrate. By running nine separate CFPS reactions and mixing them together with a glucose substrate, we can generate 300 mg/L limonene (an isoprenoid fragrance molecule). This approach shortens the time from ordering genes to characterizing active enzymes to ~3 days and allows precise measurement and control of enzyme concentration. Our platform opens the possibility of extensive testing of enzymes levels and physiochemical conditions in order to prototype and accelerate in vivo metabolic engineering efforts.