Circumventing the heterogeneity and instability of human serum albumin-interferon-alpha2b fusion protein by altering its orientation

J Biotechnol. 2007 Sep 15;131(3):245-52. doi: 10.1016/j.jbiotec.2007.04.016. Epub 2007 Apr 29.


Albuferon is a novel long-acting interferon resulted from the direct genetic fusion of human albumin and interferon-alpha2b (HSA-IFN-alpha2b). Albuferon, co-developed by Human Genome Sciences Inc. and Novartis, is currently in late stage development for the treatment of hepatitis C. It was unexpected that HSA-IFN-alpha2b secreted from Pichia pastoris migrated as doublets on non-reducing SDS-PAGE and was prone to form covalent aggregates in aqueous solution. The heterogeneity and instability of HSA-IFN-alpha2b lowered its recovery rate to about 10% and necessitated lyophilized formulation. Site-directed mutagenesis revealed that the heterogeneity and instability of HSA-IFN-alpha2b was caused by the incomplete disulfide bridge formation between Cys1 and Cys98 of IFN-alpha2b. To alleviate the structural perturbation of IFN-alpha2b by HSA, IFN-alpha2b-HSA fusion protein, in which IFN-alpha2b was located at the N-terminus, was created. IFN-alpha2b-HSA was shown to be homogeneous and stable at 37 degrees C for at least 10 days. The improved homogeneity and stability of IFN-alpha2b-HSA increased the recovery rate by 2.5-fold and made the development of stable solution formulation possible. In vitro antiviral assays showed that both fusion proteins retained the activity of IFN-alpha2b, and the EC(50) of HSA-IFN-alpha2b, and IFN-alpha2b-HSA was calculated to be 120+/-12.5, and 160+/-1 1.3ng/ml, respectively. The increased recovery rate and the possibility of solution formulation of IFN-alpha2b-HSA may compensate for its slightly decreased in vitro activity, and makes it to be a promising therapeutic agent that deserves further evaluation.

MeSH terms

  • Antiviral Agents / chemistry
  • Antiviral Agents / pharmacology
  • Drug Design
  • Drug Stability
  • Humans
  • Interferon alpha-2
  • Interferon-alpha / chemistry*
  • Interferon-alpha / metabolism
  • Interferon-alpha / pharmacology*
  • Protein Conformation
  • Protein Engineering / methods*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Fusion Proteins / pharmacology
  • Recombinant Proteins
  • Serum Albumin / chemistry*
  • Serum Albumin / genetics
  • Serum Albumin / metabolism
  • Serum Albumin / pharmacology*
  • Vesicular stomatitis Indiana virus / drug effects*


  • Antiviral Agents
  • Interferon alpha-2
  • Interferon-alpha
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Serum Albumin