A novel engineered interchain disulfide bond in the constant region enhances the thermostability of adalimumab Fab.

We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH and CL domains was deleted by substitution of Cys with Ala (Fab). DSC measurements showed that the Tm values of Fab were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of Fab. The resulting Fab (mutSS Fab) had the mutations H:V177C and L:Q160C in Fab, confirming the formation of the disulfide bond between CH and CL. The thermostability of mutSS Fab was approximately 5 °C higher than that of Fab. Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of Fab and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation.