Mammalian cells represent the most widely used host system for the industrial production of recombinant therapeutic proteins. In order to increase productivity, chemically defined media are often used and optimized to provide the cells with the necessary nutrients. However, a deregulated cell metabolism is often observed in these culture conditions. In particular, carbon sources are fast and inefficiently consumed with a consequent accumulation of byproducts, mainly lactate and ammonia. Lactate, in particular, causes a decrease of the medium pH which can be detrimental for cells growth and productivity. Its metabolic profile is normally characterized by a first production phase, which is associated to the cells exponential growth. Afterwards, when the cells enter into the stationary phase, a shift to net lactate consumption can occur under optimal culture conditions. However, such a metabolic shift is not easily controlled, since the mechanisms modulating lactate production in cell culture are still under investigation. This work aims to understand which factors could influence the lactate metabolic shift in CHO cell culture, focusing, in particular, on the mitochondrial role. To this purpose, cell lines with opposite lactate profiles were compared. The initial lactate production phase was common to all the fast growing cultures and was concomitant to a rapid glutamine consumption. After glutamine depletion, two different scenario occurred. In one case, the cells started to consume lactate until complete depletion. Alternatively, lactate continued to be accumulated, causing a decrease of media pH. The mitochondrial oxidative capacity was hypothesized as a possible cause of the different lactate profile. Indeed, mitochondrial membrane potential and oxygen consumption measurements highlighted a correlation between a reduced oxidative metabolism and a state of high lactate production. This correlation was confirmed by evaluating other cell lines or media compositions which resulted in the same divergence of lactate metabolism. Afterwards, the expression of selected genes was analysed in correlation with the observed lactate profile. Among 22 genes, two were identified as significantly downregulated (absolute fold change ≥2) in conditions of high lactate accumulation; namely, the mitochondrial aspartate-glutamate carrier (aralar1) and the translocase of the inner mitochondrial membrane 8 (timm8a). Aralar1, in particular, is an important component of the malate-aspartate shuttle (MAS). This system promotes the recycling of the cytosolic NADH pool, which is necessary for maintaining the glycolytic flux. Alternatively, NADH can be oxidised through pyruvate conversion into lactate, catalysed by the lactate dehydrogenase enzyme. Therefore, an inefficient MAS activity can engender an increased lactate accumulation. Finally, stable cell lines, which overexpressed aralar1 or timm8a, were generated. Clones derived from a lactate-producing cell line showed an improvement of lactate metabolism. In particular, they switched more easily to lactate consumption compared to the untransfected control, while maintaining a similar glucose consumption rate. On the other hand, no impact of the transgene expression was observed in the clones derived from the lactate-consuming cell line, since the switch to lactate consumption was maintained and no other effect on cell growth or glucose metabolism was detected. The data obtained indicate that lactate consumption was most likely promoted by a better NADH oxidation through the malate-aspartate shuttle, which resulted in a more efficient link between glycolysis and the mitochondrial oxidative metabolism. In conclusion, the results presented in this thesis indicate that the mitochondrial oxidative capacity plays a central role in the control of lactate production. Media composition and intrinsic cell characteristics have both an impact on mitochondrial activity. Moreover, the expression of specific genes can also be influenced by the culture media. In particular, aralar1 and timm8a have been identified as promising targets for the generation of a host cell line with an improved lactate metabolism.
EPFL2012
