Elimination of the free sulfhydryl group in the human serum albumin (HSA) moiety of human interferon-α2b and HSA fusion protein increases its stability against mechanical and thermal stresses

Abstract

Interferon-α2b (IFN-α2b) and human serum albumin (HSA) fusion protein (IFN-α2b–HSA) is a promising long acting formulation of IFN-α2b for the treatment of hepatitis C. However, accelerated mechanical and thermal stress tests revealed that IFN-α2b–HSA was prone to disulfide-linked aggregation. The formation of aggregates was associated with an increase in immunogenicity in mice. The addition of non-ionic surfactant Tween 80 increased the stability of IFN-α2b–HSA against agitation, but its thermal stability was not improved. Moreover, Tween 80 prompted the aggregation of IFN-α2b–HSA during quiescent storage. To increase the stability of IFN-α2b–HSA, the unpaired cysteine residue in this fusion protein was substituted with serine by site-directed mutagenesis. The resultant fusion protein was designated as IFN-α2b–HSA(C34S). IFN-α2b–HSA(C34S) had significant higher stability over IFN-α2b–HSA, which was evidenced by the facts that after agitation for 72 h or incubation at 60 °C for 2 h, more than 90% of IFN-α2b–HSA(C34S) remained monomeric. Consistent with its improved stability, the immunogenicity of IFN-α2b–HSA(C34S) increased less significantly after agitation. Pharmacokinetics studies in rats revealed that both fusion proteins had similar pharmacokinetic behavior, both with a half-life of about 50 h.