(27ci) Multi-Omic Characterization of CHO Cells Reveals Fluxomic Diversity and Amino Acid Utilization Bottlenecks in High-producer Clones

Bioprocess optimization and cell line development benefits from host cell line characterization to understand their metabolic capabilities, responses to the reactor conditions, and product-specific demands. While process data reveals nutrient limitations arising from depletion, it does not provide any insights into bottlenecks in cellular metabolism that currently affect antibody productivity. Here, we present a multi-omic characterization of three independently selected antibody-producing Chinese Hamster Ovary (CHO) cell lines to reveal the key metabolic reprogramming correlated with increased antibody production as well as the metabolic bottlenecks limiting antibody production in high-producer clones for each cell line. Analysis of spent media and product formation in a fed-batch process reveals that high-producer clones exhibit diverse phenotypes with increased antibody titer and no clear correlation between final cell density and titer. All producer clones exhibited increased antibody productivity in the later process phases, but individual cell lines extended growth phase or improved antibody production by decreasing nutrient channeling towards biomass production. When compared to their associated pools, clone phenotypes were associated with changes in overall glycine and serine consumption and overall lactate, alanine, aspartate, and glutamate secretion over the four identified phases of the bioprocess. Cell line-specific metabolic models revealed differences in lipid and cofactor metabolism, glycosylation, and transporters; meanwhile, central, amino acid, nucleotide, and energy metabolism were largely conserved across cell lines and process phases. Computed metabolic fluxes demonstrated substantial changes in the pentose phosphate pathway and lactate secretion, especially following clone selection from pools. Generally, glycolytic metabolism was fueled by glucose uptake and TCA was fueled by the degradation of asparagine, glutamine, and other essential amino acids. Clones exhibited changes in glycine and serine consumption and lactate, alanine, aspartate, and glutamate secretion, and on average, degraded 65% of the consumed amino acids. The uptake of arginine, proline, asparagine, glutamine, phenylalanine, valine, and isoleucine limited cell growth and antibody production in different cell lines. Thus, a wide range of metabolic activities can exist, even with similar cell line development strategies, and a multi-omic analysis of high-producer clones can guide cell engineering and bioprocess optimization.