Figure 5. Hypotheses of culture pH and time effects on mAb glycosylation, charge variants and aggregation.
Left panel: At low pHculture, increased v-ATPases and PAM (pH sensor) levels help remove excess H+ which has entered the cytoplasm from the culture media. This leads to perturbation of the luminal pHER/Golgi gradient, with a larger effect on the more basic compartments of the early secretory pathway as suggested by the increased expression of N-glycosylation enzymes in the ER and cis/medial Golgi. Concomitant increase in ER/Golgi trafficking not only increases the rate of excess H+ removal but also reduces residence (and processing) time of cargo proteins, resulting in increased relative abundance of early mAb glycoforms. The increase in basic variants is a result of protein modification (distinct from pH sensing function) by elevated PAM level and possibly its activity too as PAM has an acidic pH optimum.
Right panel: With increasing culture time regardless of pHculture, prolonged production of mAb results in increasing unfolded proteins and ER stress, as well as increasing exposure to reactive oxygen species (ROS) derived from oxidative protein folding and fatty acid β-oxidation. The consequent disruption of ER homeostasis leads to failure of ER quality control and further accumulation of unfolded proteins which can self-associate to form aggregation nuclei that promote HMW species. The concomitant increase in oxidative stress leads to the accumulation of oxidative damage to glycosylation enzymes which in turn decreases late mAb glycoforms.