6.3.1 Inhibit enzyme over expression in the cells.
Trx is the terminal enzyme that is responsible for disulfide bond
reduction. If the cells cannot overexpress Trx, the enzyme system
activity is significantly inhibited. Koterba et al. demonstrated the
reduced disulfide reduction by using Trx knock out CHO cells. Their
research showed that inhibiting Trx overexpression did not affect the
overall cell growth (Koterba et al., 2012). By incubating cell lysate
with intact IgG for 24 hours, there is still higher than 50% intact IgG
remaining in engineered cell lysate (without Trx overexpression) pool,
while in the control cell lysate (with Trx overexpressed) the intact IgG
is close to 0%. This result showed that it is possible to inhibit the
Trx overexpression in cells and minimize disulfide bond reduction caused
by cell lysis. However, this way of controlling disulfide reduction
depends on concentrations of other enzymes: if concentrations of other
enzymes are high, the redox reaction still can be catalyzed efficiently
and result in high level of disulfide bond reduction.