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

作者:Nakamura Hitomi; Oda Ueda Naoko; Ueda Tadashi; Ohkuri Takatoshi*
来源:Biochemical and Biophysical Research Communications, 2018, 495(1): 7-11.
DOI:10.1016/j.bbrc.2017.10.140

摘要

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 CH1 and CL domains was deleted by substitution of Cys with Ala (Fab(Delta ss)). DSC measurements showed that the Tm values of Fab(Delta ss) were approximately 5 degrees 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 inter chain disulfide bond outside the C-terminal region to increase the stability of Fab(Delta ss). The resulting Fab (mutSS Fab(Delta ss)) had the mutations H:V177C and L:Q160C in Fab(Delta ss), confirming the formation of the disulfide bond between CH1 and CL. The thermostability of mutSS Fab(Delta ss) was approximately 5 degrees C higher than that of Fab(Delta ss). Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of Fab(Delta ss) 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.

  • 出版日期2018-1-1