Effect of ammonia production on intracellular pH: Consequent effect on adenovirus vector production

In this study, the effect of ammonia production on intracellular pH (pHi) and consequently inhibition of AdV production at high cell densities is assessed. Different specific ammonia production rates were obtained for 293 cells adapted to grow in glutamate supplemented medium (non-ammoniagenic medium) as compared with 293 cells growing in glutamine supplemented medium (ammoniagenic medium); pHi was observed to be lower during cell growth and AdV production at both high and low CCI in the ammoniagenic medium, where the specific ammonia production rate is higher. In addition, after infection at CCI of 3 × 106 cell/ml, the cell viability decreased significantly in the ammoniagenic medium, attributed to the activation of an acidic pathway of apoptosis. Furthermore, AdV DNA was observed to be degraded at the observed pHi in the ammoniagenic medium, decreasing significantly the amount of AdV DNA available for encapsulation. To elucidate the pHi effect upon AdV production, 293 cells were infected at a CCI of 1 × 106 cell/ml in the non-ammoniagenic medium with a manipulated pHi as observed at the time of infection at CCI of 3 × 106 cell/ml in the ammoniagenic (pHi 7.0) and non-ammoniagenic (pHi 7.3) media; AdV volumetric productivities were observed to be lower when the cells were exposed to the lower pHi. Thus, the importance of controlling all the factors contributing to pHi on AdV production, such as ammonia production, has been established.