Elimination of the free sulfhydryl group in the human serum albumin (HSA) moiety of human interferon-alpha2b and HSA fusion protein increases its stability against mechanical and thermal stresses.
Interferon-alpha2b (IFN-alpha2b) and human serum albumin (HSA) fusion protein (IFN-alpha2b-HSA) is a promising long acting formulation of IFN-alpha2b for the treatment of hepatitis C. However, accelerated mechanical and thermal stress tests revealed that IFN-alpha2b-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-alpha2b-HSA against agitation, but its thermal stability was not improved. Moreover, Tween 80 prompted the aggregation of IFN-alpha2b-HSA during quiescent storage. To increase the stability of IFN-alpha2b-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-alpha2b-HSA(C34S). IFN-alpha2b-HSA(C34S) had significant higher stability over IFN-alpha2b-HSA, which was evidenced by the facts that after agitation for 72 h or incubation at 60 degrees C for 2 h, more than 90% of IFN-alpha2b-HSA(C34S) remained monomeric. Consistent with its improved stability, the immunogenicity of IFN-alpha2b-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.
Zhao
Hong Liang
HL
Department of Microorganism Engineering, Beijing Institute of Biotechnology, Beijing, People's Republic of China.
Xue
Chong
C
Wang
Yang
Y
Sun
Bo
B
Yao
Xue Qin
XQ
Liu
Zhi Min
ZM
D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins; D012709:Serum Albumin / Q000737:chemistry* / Q000235:genetics; D013314:Stress, Mechanical; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D017931:DNA Primers; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012709:Serum Albumin; D013438:Sulfhydryl Compounds