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.