Circumventing the heterogeneity and instability of human serum albumin-interferon-alpha2b fusion protein by altering its orientation.
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.
Zhao
Hong Liang
HL
Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, People's Republic of China.
Xue
Chong
C
Wang
Yang
Y
Li
Xian Yu
XY
Xiong
Xiang Hua
XH
Yao
Xue Qin
XQ
Liu
Zhi Min
ZM
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*
D000998:Antiviral Agents; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012709:Serum Albumin