Enhance Methanol Tolerability of M. Extorquens AM1 By Adaptive Evolution
- Conference: Translational Medicine and Bioengineering Conference
- Year: 2016
- Proceeding: Translational Medicine and Bioengineering Conference
- Group: Poster Submissions
- Time: Saturday, November 12, 2016 - 5:00pm-7:00pm
Chinese Hamster Ovary (CHO) cells are routinely optimized to stably express monoclonal antibodies (mAbs) at high titers. However, CHO cell technology can be unwieldy for rapid, high-throughput production and screening of candidate therapeutic proteins. Here, we have developed a cell-free protein biomanufacturing platform utilizing a commercially available CHO extract for the cell-free synthesis of mAbs. Our specific aims were to: (1) optimize cell-free reaction parameters to maximize protein yields, (2) screen additives to enhance disulfide bond formation for intact mAb production, and (3) establish the extract as a predictable tool for ranking yields of candidate antibodies. Optimization of the reaction conditions increased protein yields 8-fold to 700 mg/L using green fluorescent protein (GFP) as a reporter. Additives such as oxidized glutathione established an oxidizing cell-free environment for disulfide bond formation, and chaperones with disulfide isomerase activity further increased intact mAb yields 2-fold. Lastly, light and heavy chain expression was optimized to minimize aggregation products. Using our optimized platform, we demonstrate for the first time the cell-free synthesis of active, intact mAb using CHO extract. Additionally, the cell-free system demonstrated the capacity to rank known high and low producing mAb sequences in the same order as seen from CHO cell-based platforms. Unlike stable or transient transfection-based screening, which requires 8-14 days for setup and execution, results using our CHO-based CFPS system are attained within 2 days. Further development of the customizable cell-free platform would provide a tool for rapid, high-throughput ranking of mAb producers and has implications for the synthesis of difficult-to-express proteins.