Developing tools for rational engineering of the secretory pathway in CHO cells

Recombinant therapeutic proteins provide considerable benefits to human health in treating severe conditions such as cancer, inflammatory and autoimmune diseases. Furthermore, therapeutic proteins hold the potential to treat a wide range of devastating diseases that are currently untreatable. Industrial manufacturing of these high-value biopharmaceuticals is predominated by mammalian production cells, and especially the Chinese Hamster Ovary (CHO) cells have been prevalently used and favored in production processes. Significant advancements in CHO cell engineering have paved the way for remarkable improvements in production yields and quality. However, some therapeutic proteins exhibit production limitations in CHO cells, and with the emergence of new artificial protein formats, this may become an even greater issue in the future. The productivity constraints arise in what is known as the secretory pathway, which carries out protein folding, modification, and trafficking processes. While some efforts to engineer CHO cells for increased secretion capacity have been successful, it has become clear that these processes are frequently product-specific, making it difficult to find generalized solutions.

This thesis suggests new methods for identifying product-specific engineering targets to help aid the production of challenging proteins. The first approach is based on proximity labeling, which we demonstrate can be used to identify and quantify the protein-protein interactions that a therapeutic protein encounters during secretion. We employed this technique in both HEK293 and CHO cell lines to identify differentiated interactions and transfer potentially required interactors to CHO cells to enable secretion. In the second approach, we screened all dormant genes in the CHO genome to pinpoint effects that could alleviate intracellular stress originating from the production of challenging proteins. Lastly, we present an alternative and license-friendly CRISPR enzyme, which is indispensable for the contemporary engineering of CHO cells.

In summary, this thesis presents new strategies to improve the production process of challenging therapeutic proteins in CHO cells, thereby contributing to the goal of providing novel and more accessible medicine for everyone.