Engineering nutrient and by-product metabolism of CHO cells

Chinese Hamster Ovary (CHO) cells are the preferred hosts for the production of therapeutic glycoproteins used for treating severe health conditions. It is of interest to improve the production of such proteins in CHO cells to cut the production costs without compromising on product quality, which is critical for patient safety.

However, a major challenge is that CHO cells have an inefficient metabolism, characterized by the build-up of toxic metabolites, such as lactate and ammonia. These impair cell growth and decrease productivity during cell cultivations. Recent advances in the field, such as sequencing of Chinese hamster (Cricetulus griseus) and CHO cell lines genomes, the publishing of accurate metabolic models and appearance of new precise genome editing tools, such as the CRISPR/Cas9 system, create a favorable landscape for the rational engineering of CHO cells towards optimal nutrient and by-product metabolism.

Overall, this thesis aims to identify and study metabolites that are toxic and inhibit cell growth in a similar way as the well investigated by-products of the mammalian metabolism, followed by cell line engineering approaches to generate cells with improved phenotypes. First, a review article describing metabolites that are biomarkers of the metabolic status of the cells and are linked to cell growth inhibition or cell death is presented.

The second part of this work covers applications of cell line engineering tools to target the cell metabolism. Thus, as we have identified targets participating in amino acid catabolic pathways, engineering of the nutrient metabolism is described as a strategy to obtain enhanced cell factories. The single or combinatorial disruption of eleven genes using the CRISPR/Cas9 system was carried out to decrease by-product formation and increase amino acid availability for protein biosynthesis and important cellular processes.

Moreover, this section includes the study of the effects of engineering the co-factor metabolism via G6pd overexpression. The findings related to cell physiology and resistance to induced cellular stress are also described. Finally, to understand how ammonium is affecting the CHO cells used in-house, the study of dose- dependent effects of ammonia chloride in cell growth is presented.

Altogether, this thesis compiles a set of studies employing state-of-the-art methods for cell line development and metabolic engineering. Concluding remarks and future perspectives are presented to close this work.