13C metabolic flux analysis of industrial Chinese hamster ovary (CHO) cell cultures

Abstract

Chinese hamster ovary (CHO) cells are the most commonly utilized host cell line for biomanufacturing monoclonal antibody (mAb) drugs and other therapeutic proteins. This dissertation describes the application of 13C metabolic flux analysis (MFA) to characterize CHO cell metabolism under various experimental conditions that are relevant to industrial bioprocesses. First, isotopically nonstationary (INST)-MFA was utilized to assess the metabolism of an industrial CHO cell line engineered to constitutively express PGC1α—a transcriptional co-activator previously reported to enhance mitochondrial metabolism in animal cells. Overexpression of PGC1α successfully increased specific productivity (qP) by a range of 2.6- to 5.2-fold over the parental cell line and increased overall carbon flux through the citric acid cycle (CAC). This finding is consistent with prior studies that have correlated increased CAC flux with elevated mAb productivity in CHO cell cultures. Next, we applied 13C MFA to industrial CHO cell cultures exposed to a novel proprietary growth medium designed to decrease excess ammonia production by roughly 40%. We determined that the low-ammonia-producing medium did not significantly alter central carbon metabolism of CHO cell cultures. Additionally, the presence of excess ammonia in the culture was determined to have no significant effect on metabolic phenotype. Therefore, it was concluded that nitrogen metabolism could be effectively manipulated through media redesign without significantly altering important bioenergetic fluxes. Next, a fed-batch 13C MFA study of CHO cells during stationary growth phase uncovered that anti-apoptotic gene expression increased glucose and lactate consumption rates and peak viable cell densities while reducing cell death rates. Because mAb productivities are typically highest when culture growth rate is minimal, attempts to prolong the stationary growth phase and enhance metabolism during this phase are important for maximizing overall product yields. Finally, the metabolism of a CHO cell clone producing a degraded mAb product was compared to a CHO cell clone producing the intact product. We hypothesized that an overactive oxidative pentose phosphate pathway (OPPP) was responsible for increased product degradation by fueling the thioredoxin system with NADPH, thus leading to disulfide bond reduction. However, the two CHO cell clones exhibited similar OPPP activities as quantified with 13C MFA. Therefore, we concluded that differences in OPPP activity were not responsible for the increase in product degradation. Overall, this dissertation details comprehensive metabolic studies of multiple industrial CHO cell lines providing actionable insights into further advances in biopharmaceuticals manufacturing.