New developments in online OUR monitoring and its application to animal cell cultures.

The increasing demand for biopharmaceuticals produced in mammalian cells has driven the industry to enhance the productivity of bioprocesses through intensification of culture process. Fed-batch and perfusion culturing strategies are considered the most attractive choices, but the application of these processes requires the availability of reliable online measuring systems for the estimation of cell density and metabolic activity. This manuscript reviews the methods (and the devices used) for monitoring of the oxygen consumption, also known as oxygen uptake rate (OUR), since it is a straightforward parameter to estimate viable cell density and the physiological state of cells. Furthermore, as oxygen plays an important role in the cell metabolism, OUR has also been very useful to estimate nutrient consumption, especially the carbon (glucose and glutamine) and nitrogen (glutamine) sources. Three different methods for the measurement of OUR have been developed up to date, being the dynamic method the golden standard, even though DO and pH perturbations generated in the culture during each measurement. For this, many efforts have been focused in developing non-invasive methods, such as global mass balance or stationary liquid mass balance. The low oxygen consumption rates by the cells and the high accuracy required for oxygen concentration measurement in the gas streams (inlet and outlet) have limited the applicability of the global mass balance methodology in mammalian cell cultures. In contrast, stationary liquid mass balance has successfully been implemented showing very similar OUR profiles compared with those obtained with the dynamic method. The huge amount of studies published in the last years evidence that OUR have become a reliable alternative for the monitoring and control of high cell density culturing strategies with very high productivities.

A new strategy for fed-batch process control of HEK293 cell cultures based on alkali buffer addition monitoring: comparison with O.U.R. dynamic method.

The increasing demand for biopharmaceuticals produced in mammalian cells has driven the industry to enhance productivity of bioprocesses through different strategies. This is why fed-batch and perfusion cultures are considered more attractive choices than batch processes. In this context, the availability of reliable online measuring systems for cell density and metabolic activity estimation will help the application of these processes. The present work focuses on the comparison of two different monitoring tools for indirect estimation of biomass concentration in a HEK293 cell cultures producing IFN-γ: on one side, the oxygen uptake rate (O.U.R.) determination, by means of application of the dynamic method measurement which is already a widely used tool and, on the other side, a new robust online monitoring tool based on the alkali buffer addition used to maintain the pH set point. Both strategies allow a proper monitoring of cell growth and metabolic activity, with precise identification of the balanced cell growth and the most important action in the process, as is the media feeding. The application of these monitoring systems in fed-batch processes allows extending the growth of HEK293 cells, which in turn results in higher final cell concentrations compared with Batch strategy (7 · 106 cells mL-1), achieving 14 · 106 cells mL-1 for the fed-batch based on O.U.R. and 19 · 106 cells mL-1 for the fed-batch based on the alkali addition. Product titter is also increased in respect of the batch strategy (3.70 mg L-1), resulting in 8.27 mg L-1 when fed-batch was based on O.U.R. and 11.49 mg L-1 when it was based on the alkali buffer strategy. Results prove that fed-batch strategy based on the alkali buffer addition is a robust online monitoring method that has shown its great potential to optimize the feeding strategy in HEK293 fed-batch cultures.

Effect of continuous feeding of CO2 and pH in cell concentration and product titers in hIFNγ producing HEK293 cells: Induced metabolic shift for concomitant consumption of glucose and lactate.

Although pH control at physiological levels is generally considered as the optimal culture condition, in some cases other strategies should be taken into account for their beneficial effects on process performance. pH and CO2 levels are chemical variables that have a major impact in cell growth and product titers in cell culture since their effect on key metabolic routes. HEK293 cells expressing recombinant hIFNγ showed different metabolic behavior when cultured in shake flask compared to pH-controlled bioreactors, in which a decrease in cell density and product titer were observed. This yield loss observed in bioreactor cultures could be reverted by adding 1% CO2 to air inlet flow in a non-controlled pH bioprocess. With this strategy, a significant outcome of 4-fold increase in terms of maximum cell density and 2-fold increase in volumetric concentration of recombinant protein (hIFNγ) when compared to the pH-controlled culture in bioreactor (standard culture conditions) has been obtained. Results evidenced the importance of pH and CO2 concentration in this case, in order to reproduce the behavior observed in optimization experiments performed in shake flasks. Thus, it was demonstrated that not always constant controlled variable setpoint (like pH or CO2 addition) becomes the best bioprocess performance strategy.

Analysis of CHO cells metabolic redistribution in a glutamate-based defined medium in continuous culture.

The effect of glutamine replacement by glutamate and the balance between glutamate and glucose metabolism on the redistribution of t-PA-producing recombinant CHO cells metabolism is studied in a series of glucose shift down and shift up experiments in continuous culture. These experiments reveal the existence of multiple steady states, and experimental data are used to perform metabolic flux analysis to gain a better insight into cellular metabolism and its redistribution. Regulation of glucose feed rate promotes a higher efficiency of glucose and nitrogen source utilization, with lower production of metabolic byproducts, but this reduces t-PA specific production rate. This reduction under glucose limitation can be attributed to the fact that the cells are forced to efficiently utilize the carbon and energy source for growth, impairing the production of dispensable metabolites. It is, therefore, the combination of growth rate and carbon and energy source availability that determines the level of t-PA production in continuous culture.