摘要：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 (CH 1 : C177–CL : C160) and one cysteine at the C-terminal end (mutSS Fab SH ) was designed to maintain Fab thermostability and for site-specific PEGylation. MutSS Fab SH was expressed in Pichia pastoris and purified mutSS Fab SH 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 Fab SH . To confirm the influence of PEGylation on the pharmacokinetic behavior of the Fab, PEGylated mutSS Fab SH was administered to rats via tail vein injection. Analysis of the mean serum concentration of the PEGylated mutSS Fab SH 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..
