|dc.description.abstract||Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry towards production of biotherapeutics of great value to the mankind. These cells possess a unique feature of providing a human- compatible post-translational modification pattern which makes them the most suitable mammalian hosts for the industry. Understanding the metabolism of various simple and complex molecules present in their culture environment is essential in order to envision ways to improve the productivity of these cells. It is possible to decipher this metabolism through various ‘omics techniques such as metabolomics, proteomics and transcriptomics. In order to understand how CHO cells, utilize simple molecules such as amino acids, 13C-tracer metabolomics was enforced which is a technique helping researchers look using a magnifying glass in and around the cells for molecules of interest and their derivatives for a long time now. In the current study, we replaced each amino acid with its 13C enriched form to elucidate the path it takes in CHO cell cultures and subsequently utilized this knowledge towards various applications such as deducing an optimized amino acid nutrient composition for these cells to not just conserve these biomolecules, but also to limit the release of growth and productivity inhibiting molecules in high cell density cultures. Other techniques to understand cellular metabolism such as global metabolomics using LC-MS were employed to identify culture performance inhibitors. Successfully limiting their production in the cell culture using media design strategies such as design of experiments enabled expand the production window of these cell lines. Complex medium additives such as plant hydrolysates are often used to enhance culture performance although their mechanism of action in enhancing cell performance is not well understood. In this study, cottonseed hydrolysates were added to CHO batch cell cultures, enhancing cell growth, IgG titers, and productivities. Extracellular metabolomics coupled with TMT proteomics revealed several metabolic phases in the entire culture duration and clearly highlighted differences in key central metabolism players potentially driving the performance changes in the presence of cottonseed hydrolysate. As a raw material for CHO cells, cottonseed hydrolysate added alternate substrates such as galactose and peptides in addition to the predominant energy sources like glucose and free amino acids that are in the basal medium. As a result, these cultures demonstrated a shift from production to consumption and vice-versa in the metabolism of various glycolysis and TCA (Tricarboxylic Acid) cycle precursors/by-products such as serine, glycine, lactate, pyruvate, glutamate and aspartate suggesting reincorporation to combat nutrient deprivation. Quantitative proteomics revealed 5521 proteins and numerous differentially abundant proteins related to growth, metabolism, oxidative stress, protein productivity, and apoptosis/cell death at day 5 and day 6 in hydrolysate cultures. Differential abundance of amino acid transporter proteins and catabolism enzymes such as BCAT1 (Branched Chain Amino Acid Transaminase 1) and FAH (Fumarylacetoacetate Hydrolase) can alter availability and utilization of several amino acids. Also, pathways involved in growth including the polyamine biosynthesis through higher ODC1 (Ornithine decarboxylase 1) abundance and hippo signaling were upregulated and downregulated, respectively. Central metabolism rewiring was indicated by GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) downregulation, which corresponded with re-uptake of secreted lactate in the cottonseed-supplemented cultures. Overall, cottonseed hydrolysate supplementation modifies culture performance by altering cellular activities critical to growth and protein productivity including metabolism, transport, mitosis, transcription, translation, protein processing, and apoptosis. And finally, it is imperative to design more efficient CHO cell processes for the biopharmaceutical industry, in possession of the abovementioned wide range of information on cellular metabolism of CHO cells through ‘omics techniques.
Graduate Advisor: Dr. Michael Betenbaugh
Dissertation Readers: Dr. Marc Donohue, Dr. Alan Stone||