Site-Specific PEGylation at Residue 86 Improves Pharmacokinetic Properties of Interferon α-2b

Interferon alpha-2b, a therapeutic protein, suffers from short half-life. PEGylation extends its circulation time but reduces activity. Site-specific PEGylation on cysteine residues, offers a strategy for improved conjugates.

This study explores cysteine analogs of interferon for site-specific PEGylation and assesses their pharmacokinetic properties.

Surface-exposed amino acids, identified through in silico studies, were selected for cysteine substitution based on the spatial accessibility of the introduced cysteine for site-specific PEGylation. The resulting mutant genes were synthesized, cloned into the pET26-b vector, and expressed in E. coli. The recombinant proteins were purified by immobilized metal chelate affinity chromatography (IMAC) and their biological activity was assessed via cell-based assays. PEGylation was performed using a 5 kDa Methoxy PEG Maleimide, and the resulting conjugate was purified by ion-exchange chromatography. The pharmacokinetic properties of both the PEGylated and unmodified interferon were then evaluated in rat blood circulation.

The in-silico modeling of interferon α-2b identified surface-exposed residues Glu78 and Thr86 as candidates for cysteine substitution. Successful gene synthesis and cloning in E. coli was achieved, followed by protein expression in E. coli with subsequent purification exceeding 95% homogeneity. Activity analysis revealed that the IFN_E78C variant suffered a substantial loss of activity, while IFN_T86C retained 83% of the native interferon's activity. Following PEGylation, the IFN_T86C conjugate retained a high specific activity and exhibited improved pharmacokinetic properties in rats. The differential activities observed between IFN_E78C and IFN_T86C highlight the impact of amino acid positioning on protein function, as IFN_T86C demonstrated significantly better activity retention compared to IFN_E78C. Although PEGylation resulted in some reduction in biological activity, PEG5-IFN_T86C still exhibited superior pharmacokinetic stability compared to its non-PEGylated counterpart.

PEGylation at position 86 improves early interferon pharmacokinetic properties, but longer-term stability requires investigation of higher molecular weight or branched PEG. These findings underline the importance of strategic amino acid modifications and PEGylation in optimizing therapeutic proteins for improved efficacy and stability.