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.