TY - JOUR
T1 - A novel engineered interchain disulfide bond in the constant region enhances the thermostability of adalimumab Fab
AU - Nakamura, Hitomi
AU - Oda-Ueda, Naoko
AU - Ueda, Tadashi
AU - Ohkuri, Takatoshi
N1 - Funding Information:
This work was supported by JSPS KAKENHI Grant Number 16K08922 . We would like to thank Editage ( www.editage.jp ) for Englishi language editing.
PY - 2018/1/1
Y1 - 2018/1/1
N2 - 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 (FabΔSS). DSC measurements showed that the Tm values of FabΔSS 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 FabΔSS. The resulting Fab (mutSS FabΔSS) had the mutations H:V177C and L:Q160C in FabΔSS, confirming the formation of the disulfide bond between CH1 and CL. The thermostability of mutSS FabΔSS was approximately 5 °C higher than that of FabΔSS. Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of FabΔSS 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.
AB - 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 (FabΔSS). DSC measurements showed that the Tm values of FabΔSS 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 FabΔSS. The resulting Fab (mutSS FabΔSS) had the mutations H:V177C and L:Q160C in FabΔSS, confirming the formation of the disulfide bond between CH1 and CL. The thermostability of mutSS FabΔSS was approximately 5 °C higher than that of FabΔSS. Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of FabΔSS 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.
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U2 - 10.1016/j.bbrc.2017.10.140
DO - 10.1016/j.bbrc.2017.10.140
M3 - Article
C2 - 29097200
AN - SCOPUS:85034990751
SN - 0006-291X
VL - 495
SP - 7
EP - 11
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 1
ER -