Proteins are very important molecules as therapeutics. They have several advantages over small molecule drugs but their use is limited by their circulation short half life, immunogenicity and physicochemical instability. Several approaches have been developed to improve the pharmacokinetics of proteins. One that is clinically proven is the covalent conjugation of poly (ethylene glycol) (PEG) to the protein. This is known as
PEGylation. Problems associated with PEGylation include product heterogenicity that is due to position isomerism and low conjugation efficiency. Several strategies are being developed to overcome these problems. One new approach that is being developed for site-specific PEGylation is called disulfide bridging PEGylation. This approach to
PEGylation relies on the presence of accessible, native disulfide bonds in therapeutic proteins. The main principle is to reduce a native disulfide bond in the protein molecule followed by PEGylation using a bis-alkylation PEG reagent that can insert a three carbon bridge that connects to the two sulphurs that formed the disulfide. In the case of proteins with multiple disulfides, a potential limitation in disulfide bridging PEGylation is that proteins with multiple, accessible disulfides may give many PEG products. We therefore examined the possibility of controlled reduction and disulfide bridging
PEGylation of 2 proteins with more than one disulfide. The first protein was ribonuclease A (RNase A) which has 4 disulfides buried within its structure. The second protein transferrin, which has 19 disulfides some that are close to the surface and are accessible. With RNase A no reduction was observed using (100 mM) DTT unless a denaturant was added, and then complete reduction was achieved using either urea or guanidine as denaturant. Using an immobilized reductant colum also give no reduction without denaturant. It was difficult to control the conditions for partial reduction and partial denaturation of RNase A as a means to reduce 1-2 disulfides in RNase A. For transferring partial reduction was achieved using (0.5 mM) DTT which is considered to be mild reducing conditions. No denaturant was required for the partial reduction of transferring. Mono-PEGylation was achieved with a small amount of di-PEGylation being observed. Holo-Tf (which is bound to iron) and apo-Tf (which is iron free) were compared to see if bounded iron had any effect on protein conformation that could influence disulfide reduction or PEGylation. Both forms of Tf give the same results by
SDS PAGE indicating that iron had no effect. Using higher equivalents of the PEG reagent was unnecessary; we obtained good mono-PEGylation by adding 2 equivalents of PEG to the protein. These results demonstrated the possibility of controlled reduction of multiple disulfide proteins under mild conditions.