title: Circumventing the heterogeneity and instability of human serum albumin-interferon-alpha2b fusion protein by altering its orientation.
abstract: 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.
authors: Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, People's Republic of China.
authors: Hong Liang
authors: HL
authors: Zhao
authors: Chong
authors: C
authors: Xue
authors: Yang
authors: Y
authors: Wang
authors: Xian Yu
authors: XY
authors: Li
authors: Xiang Hua
authors: XH
authors: Xiong
authors: Xue Qin
authors: XQ
authors: Yao
authors: Zhi Min
authors: ZM
authors: Liu
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology; D015195:Drug Design; D004355:Drug Stability; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins; D012709:Serum Albumin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D014721:Vesicular stomatitis Indiana virus / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012709:Serum Albumin