a b s t r a c tMetabolic flux analysis (MFA) is widely used to estimate intracellular fluxes. Conventional MFA, however, is limited to continuous cultures and the mid-exponential growth phase of batch cultures. Dynamic MFA (DMFA) has emerged to characterize time-resolved metabolic fluxes for the entire culture period. Here, the linear DMFA approach was extended using B-spline fitting (B-DMFA) to estimate mass balanced fluxes. Smoother fits were achieved using reduced number of knots and parameters. Additionally, computation time was greatly reduced using a new heuristic algorithm for knot placement. B-DMFA revealed that Chinese hamster ovary cells shifted from 37°C to 32°C maintained a constant IgG volumespecific productivity, whereas the productivity for the controls peaked during mid-exponential growth phase and declined afterward. The observed 42% increase in product titer at 32°C was explained by a prolonged cell growth with high cell viability, a larger cell volume and a more stable volume-specific productivity.

Lactate is one of the key waste metabolites of mammalian cell culture. High lactate levels are caused by high aerobic glycolysis, also known as the Warburg effect, and are usually associated with adverse culture performance. Therefore, reducing lactate accumulation has been an ongoing challenge in the cell culture development to improve growth, productivity, and process robustness. The pyruvate dehydrogenase complex (PDC) plays a crucial role for the fate of pyruvate, as it converts pyruvate to acetyl coenzyme A (acetyl-CoA). The PDC activity can be indirectly increased by inhibiting the PDC inhibitor, pyruvate dehydrogenase kinase, using dichloroacetate (DCA), resulting in less pyruvate being available for lactate formation. Here, Chinese hamster ovary cells were cultivated either with 5 mM DCA or without DCA in various batch and fed-batch bioreactor processes. In all cultures, DCA increased peak viable cell density (VCD), culture length and final antibody titer. The strongest effect was observed in a fed batch with media and glucose feeding in which peak VCD was increased by more than 50%, culture length was extended by more than 3 days, and the final antibody titer increased by more than twofold. In cultures with DCA, lactate production and glucose consumption during exponential growth were on average reduced by approximately 40% and 35%, respectively. Metabolic flux analysis showed reduced glycolytic fluxes, whereas fluxes in the tricarboxylic acid (TCA) cycle were not affected, suggesting that cultures with DCA use glucose more efficiently. In a proteomics analysis, only few proteins were identified as being differentially expressed, indicating that DCA acts on a posttranslational level. Antibody quality in terms of aggregation, charge variant, and glycosylation pattern was unaffected. Subsequent bioreactor experiments with sodium lactate and sodium chloride feeding indicated that lower osmolality, rather than lower lactate concentration itself, improved culture performance in DCA cultures. In conclusion, the addition of DCA to the cell culture improved culture performance and increased antibody titers without any disadvantages for cell-specific productivity or antibody quality.

Aerobic glycolysis is an inefficient metabolic phenotype displayed by many rapidly proliferating cells during growth. It is characterized by high glycolytic activity and only partial oxidation of glucose resulting in the production of high amounts of lactate. This phenotype was originally reported by Otto Warburg in 1927 as a hallmark of cancer andwhile it is now known to occur in other fast growing cells as well -it remains an interesting target for cancer therapy. Aerobic glycolysis also has major implications for biopharmaceutical production, since lactate accumulation can be growth inhibiting, limiting the cell density that can be achieved in culture.Due to its association with various diseases and being an unfavorable metabolic phenotype in industrial applications, reducing the Warburg effect and analyzing accompanying effects on the cell as a whole are of great interest. Whereas in cancer therapy the objective is to kill cells relying on aerobic glycolysis, the aim in industrial applications is to reduce aerobic glycolysis without inducing cell death or inhibiting cell growth. Pyruvate dehydrogenase complex (PDC) is a mitochondrial gatekeeping enzyme determining how much pyruvate is converted to acetyl-CoA and subsequently enters the TCA cycle. PDC activity is regulated by reversible phosphorylation catalyzed by pyruvate dehydrogenase kinase (PDK) (phosphorylation → inactivation) and pyruvate dehydrogenase phosphatase (dephosphorylation → activation). PDC activity can be increased by inhibiting PDK using dichloroacetate (DCA) a known PDK inhibitor, hereby reducing aerobic glycolysis.The objective in this thesis was twofold; i) analyzing metabolic as well as growth inhibitory effects of DCA in human embryonic kidney 293 (HEK293) cells, and ii) investigating the effects of a non growth inhibiting DCA concentration using Chinese hamster ovary (CHO) cells in industrial relevant bioprocesses. In both studies aerobic glycolysis decreased with increasing DCA concentration characterized by reduced glucose consumption and lactate production. At lower DCA concentrations cell growth was unaffected. Furthermore, no increase in oxidative metabolism was detected at low DCA concentration indicating that the cells adopt a more energy efficient metabolism without directing more pyruvate into the TCA cycle. However, it appears that the cytoplasmic pyruvate fraction is reduced as not only less lactate but also less alanine is produced. The metabolic changes observed were mostly attributable to post-translational regulation since transcriptomics and proteomics analyses revealed only minor changes to metabolic enzymes. However, in the absence of iv increased TCA cycle activity, allosteric regulation of glycolytic enzymes did not readily explain reduced glycolysis.Cell growth in HEK293 cells was reduced only at higher DCA concentration when increased cellular stress and TCA cycle activity were detected. Since DCA was found to depolarize mitochondria the increased TCA cycle activity may not result in higher ATP productio...