TY - JOUR
T1 - C-terminal cysteine PEGylation of adalimumab fab with an engineered interchain SS bond
AU - Nakamura, Hitomi
AU - Anraku, Makoto
AU - Oda-Ueda, Naoko
AU - Ueda, Tadashi
AU - Ohkuri, Takatoshi
N1 - Funding Information:
Acknowledgments This study was supported by JSPS
Funding Information:
This study was supported by JSPS KAKENHI (16K08922).
Publisher Copyright:
© 2020 The Pharmaceutical Society of Japan
PY - 2020/3/1
Y1 - 2020/3/1
N2 - Conjugation with polyethylene glycol (PEG) is performed to increase serum half-life of the Fab for clinical applications. However, current designs for recombinant Fab only allow PEGylation at the interchain SS bond (disulfide bond) at the C-terminal end of the heavy chain and light chain of the Fab, which the decrease of thermostability occurred by partial reduction of the interchain SS bond. An adalimumab Fab mutant with a novel interchain SS bond (CH1:C177–CL:C160) and one cysteine at the C-terminal end (mutSS FabSH) was designed to maintain Fab thermostability and for site-specific PEGylation. MutSS FabSH was expressed in Pichia pastoris and purified mutSS FabSH was conjugated with 20-kDa PEG targeted at the free cysteine. Based on enzyme-linked immunosorbent assay (ELISA), PEGylation did not affect the binding capacity of the mutSS FabSH. To confirm the influence of PEGylation on the pharmacokinetic behavior of the Fab, PEGylated mutSS FabSH was administered to rats via tail vein injection. Analysis of the mean serum concentration of the PEGylated mutSS FabSH versus time through ELISA indicated an increase in half-life compared to that of non-PEGylated wild-type Fab. Consequently, we have successfully demonstrated that a Fab mutant with a novel interchain SS bond and one free cysteine at the C-terminal end can be PEGylated without changes in functionality. This design can potentially be used as a platform for modification of other recombinant Fabs.
AB - Conjugation with polyethylene glycol (PEG) is performed to increase serum half-life of the Fab for clinical applications. However, current designs for recombinant Fab only allow PEGylation at the interchain SS bond (disulfide bond) at the C-terminal end of the heavy chain and light chain of the Fab, which the decrease of thermostability occurred by partial reduction of the interchain SS bond. An adalimumab Fab mutant with a novel interchain SS bond (CH1:C177–CL:C160) and one cysteine at the C-terminal end (mutSS FabSH) was designed to maintain Fab thermostability and for site-specific PEGylation. MutSS FabSH was expressed in Pichia pastoris and purified mutSS FabSH was conjugated with 20-kDa PEG targeted at the free cysteine. Based on enzyme-linked immunosorbent assay (ELISA), PEGylation did not affect the binding capacity of the mutSS FabSH. To confirm the influence of PEGylation on the pharmacokinetic behavior of the Fab, PEGylated mutSS FabSH was administered to rats via tail vein injection. Analysis of the mean serum concentration of the PEGylated mutSS FabSH versus time through ELISA indicated an increase in half-life compared to that of non-PEGylated wild-type Fab. Consequently, we have successfully demonstrated that a Fab mutant with a novel interchain SS bond and one free cysteine at the C-terminal end can be PEGylated without changes in functionality. This design can potentially be used as a platform for modification of other recombinant Fabs.
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U2 - 10.1248/bpb.b19-00612
DO - 10.1248/bpb.b19-00612
M3 - Article
C2 - 31866612
AN - SCOPUS:85081072777
SN - 0918-6158
VL - 43
SP - 418
EP - 423
JO - Biological and Pharmaceutical Bulletin
JF - Biological and Pharmaceutical Bulletin
IS - 3
ER -