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 technology platforms are used for expression screening, but these can be time- and resource-intensive and may also suffer from performance variability. Here, we have developed a cell-free protein synthesis (CFPS) platform utilizing a commercially available CHO extract for the rapid <i>in vitro</i> synthesis of active mAbs. Specifically, we optimized reaction conditions to maximize protein yields, established an oxidizing environment to enable disulfide bond formation, and demonstrated the importance of chaperone supplementation and 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 cell-free protein synthesis of biologically active, intact mAb using CHO cell extracts. 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 to obtain mAb product, using our CHO-based CFPS platform, mAb product can be 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 and has positive implications for the synthesis of difficult-to-express and toxic proteins.

Development of a CHO-based cell-free platform for the synthesis of active monoclonal antibodies as a potential high-throughput screening tool

253rd National Meeting (2017)

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