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.