| dc.description.abstract | Monoclonal antibodies (mAbs) represent the largest class of biopharmaceuticals, accounting for over 50% of new drug approvals and 65% of global biopharmaceutical sales. Due to their complexity, mAbs are typically produced in mammalian cells. In particular, Chinese hamster ovary (CHO) cells are used in over 70% of mAb production processes. However, despite their widespread use, CHO cells are not naturally metabolically efficient. In this work, carbon-13 metabolic flux analysis (13C MFA) and metabolic engineering approaches were applied to industrial mAb-producing CHO cell lines to elucidate desirable metabolic phenotypes. First, a CHO cell line featuring an attenuated glutamine synthetase selection system was found to have reduced glutamine overflow, increased mAb production, and altered glutamine and glutamate metabolism, while central carbon metabolism was unaffected. Next, a CHO cell line overexpressing PGC-1⍺, which was previously shown to exhibit increased specific productivity and consumption of extracellular metabolites, was further characterized through protein and mRNA expression assays. Then, a metabolic model of glycosylation precursor production was developed and validated through a study comparing glucose-fed cultures to galactose-fed cultures. Finally, the use of tricarboxylic acid (TCA) cycle intermediate feeding during a fed-batch culture was found to increase TCA cycle flux and reduce the production of waste products such as lactate and ammonia. Together, these studies investigate different desirable aspects of CHO cell culture, such as improved productivity or decreased byproduct formation, and explore the relationship between these culture traits and metabolism. Overall, these studies provide evidence that optimizing metabolic efficiency can lead to increased mAb productivity and decreased byproduct formation. Furthermore, this work highlights a wide range of potential applications of 13C MFA to guide host cell engineering and develop novel feeding strategies for controlling CHO metabolism. | |
