Resumen de Development of tools for monitoring and controlling cell cultures through the metabolic analysis
Mammalian cells are well established systems for the production of a wide range of high added value proteins. About 60-70% of all biopharmaceuticals are produced in mammalian cells due to the capacity of these cells to perform complex post-translational modifications to yield biologically active proteins.
One of the most important limitations of mammalian cells is their inefficient metabolism, characterized by the consumption of large quantities of glucose and concomitant production of similar amounts of by-products, like ammonia and lactate, which can detrimentally affect the cell growth. Interestingly, we have observed that under certain culture conditions mammalian cells are able to co-consume both glucose and lactate during the exponential growth phase.
Chapters 3 and 4 are focused on presenting the different glucose and lactate metabolisms in cultures of HEK293 and CHO. Three different glucose and lactate metabolisms have been obtained: Phase 1: glucose consumption and lactate production (exponentially growth), Phase 2: glucose and lactate simultaneous consumption (exponentially growth), and Phase 3: lactate consumption as a sole carbon source (no cell growth). These different metabolic phases were observed mainly depending on two cell culture conditions: the pH and the lactate concentration. In order to perform a deeper study of the different phases presented, an analysis of the intracellular flux distribution for the different phases have been performed for both cell lines by means of Flux Balance Analysis (FBA).
FBA showed that, in Phase 1, lactate is produced because pyruvate is converted to lactate to fulfill the NADH regeneration requirements in the cytoplasm and only a small amount of pyruvate is introduced into TCA through Acetyl-CoA. In glucose-lactate concomitant consumption (Phase 2), glucose uptake was significantly reduced and a balance between glycolysis and TCA cycle fluxes was reached, yielding a more efficient substrate consumption.
Once understood the metabolism of mammalian cells in culture, the next step is to apply this knowledge in the engineering bioprocess area. To this end, a new robust on-line monitoring tool based on the alkali buffer addition used to maintain the pH set-point is presented in Chapter 5. This new tool is compared with a widely used monitoring tool based on the Oxygen Uptake Rate (O.U.R.) determination, by means of application of the dynamic method.
The two alternatives presented have shown clear advantages in respect to final product titer and, especially, volumetric productivities. But better results have been obtained with the alkali addition strategy, increasing the total viable cell concentration and product titer by 178% and 257% respectively, and obtaining a 109% increment of the process volumetric productivity in respect to the batch culture. This is due to the culture constant distortions of the pO2 and pH performed in every O.U.R. dynamic measurement.
To close the work performed, a different non-invasive method for O.U.R. determination based on the stationary liquid mass balance was presented and tested in batch culture in Chapter 6. The results demonstrated to be a reliable alternative to monitor the metabolic activity in bioreactors, being a useful tool for high cell density culture strategies implementation based on O.U.R. monitoring.
