CHO cell multigene engineering by directed evolution

Chinese hamster ovary (CHO) cells are the dominant production platform for the production of recombinant biotherapeutics, and with continuing advances in host cell, transgene vector and process engineering product titers can reach up to 10g/L. Despite this, many proteins of potential therapeutic use remain difficult to express, and are unable to be developed for commercial use. An increased interest in the production of novel engineered antibody formats has created further challenges in this arena. This thesis presents a set of engineering strategies based on the use of bioactive small molecules to enhance the productivity of CHO cells producing difficult-to-express (DTE) monoclonal and bispecific antibodies.
A literature search was first performed to identify small molecules with the potential to relieve or prevent endoplasmic reticulum stress in non-CHO cell lines, and tested against a CHO cell line producing a bispecific antibody with the aim of repurposing these molecules as novel chemical chaperones to enhance productivity in CHO cell production processes. Next, a transcriptomic dataset of CHO cell lines producing a variety of easy-to-express (ETE) and DTE products was compiled and analysed using differential expression and weight gene co-expression network analysis to identify molecular targets for
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further small molecule-based engineering efforts. Following this, a panel of small molecule selection agents were identified and used to chemically filter transfectant pools expressing a DTE monoclonal antibody in order to enrich the pool for high-expressing clones. Finally, the chemical filtering process was applied to CHO hosts prior to transfection to determine the feasibility of generating banks of pre-filtered host cells for later transfection.
The engineering strategies presented here propose a generically applicable approach that may be used to derive product-specific engineering solutions while avoiding the need for lengthy troubleshooting processes.