Developing Tools for Improved and Predictable Recombinant Protein Production in CHO Cells

Chinese hamster ovary (CHO) cells are the dominant cell factory for recombinant production of diverse biotherapeutics. The complexity of biotherapeutics and the increasing demand for their rapid, cost-effective and high-quality production have made the biomanufacturing in CHO cells a highly sophisticated process. To meet the demands for better and inexpensive biotherapeutics, further improvements of recombinant protein production in CHO cells seem necessary. In this thesis we present clone screening and gene engineering tools that aim to facilitate improved and more predictable recombinant protein production in the next-generation CHO cell factories. First, a reporter-based screening method for high production capacity was established, that is able to rank statically-grown clones according to either volumetric productivity (overall yield) or a proxy of specific productivity (per cell yield). By systematically comparing the two clone screening criteria, we demonstrated that they perform similarly in terms of isolating CHO clones with high volumetric productivity in suspension. Second, a toolbox combining the CRISPR/Cas9 system with a recombinase-based system was developed to generate cell lines with so-called landing pads integrated in defined genomic sites. The toolbox allows targeted gene integration, followed by streamlined recombination of the expression cassette into the landing pad of a genetically identical cell line. This approach is exploited for systematic evaluation of site-specific gene expression modulated by different expression cassette components. We demonstrated that distinct and robust recombinant gene expression can be achieved in defined integration sites in CHO cells. Third, ectopic expression of target genes for improving the secretory capacity of CHO cells was reviewed and studied. We identified three novel target genes increasing specific productivity of CHO cells, transiently expressing different monoclonal antibodies. We attempted to unveil the mechanism of novel target genes by examining the rate-limiting step in production of these monoclonal antibodies. Overall, the tools developed in this thesis can in the future enable more rational engineering of CHO cell factories for production of a variety of complex biotherapeutics.