Development of delivery system for transferrin using chemical modification approach

الملخص EN

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 and low conjugation efficiency.

A new approach is applied in this study which is site-specific PEGylation and it is called disulfide bridging PEGylation.

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 sulphur that had formed the disulfide.

In this study controlled reduction and disulfide bridging PEGylation of transferrin (which has 19 disulfides some that are close to the surface and are accessible) was conducted.

For transferrin (Tf) 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 transferrin.

Mono-PEGylation was achieved with a small amount of di-PEGylation being observed.

Holo-Tf (which is Tf bound to iron) and apo-Tf (which is Tf 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 Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (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.

The results approved by Size exclusion chromatography –High performance liquid chromatography (SEC-HPLC) analysis.

These results demonstrate the possibility of controlled reduction of multiple disulfide proteins under mild conditions which may improve the stability, prolong the half life and improve pharmacokinetics of Tf to present it as medicine.