| Interferons (IFNs),with the functions of anti-viral, antiproliferative.and immunomodulatory activities, are a family of inducible proteins secreted by cells. They are known as ones of the most effective drugs against virus clinically. But during clinical therapies, IFNs showed some disadvantages, such as, unstable properties, immunogenicity and a relatively short half-life in circulation. Modification of IFNs by covalent attachment of soluble and nonimmunogenic polymers (for example polyethylene glycol, PEG) results in species with dramatically increased plasma half lives and low immunogenicity. Consequently, PEGylation might improve IFNs'therapeutic benefits. Theinvestigation laid the basic and preclinical research of PEG-IFNα2b.PEG-IFNα2b was analyzed for the biochemical characterizations. Its concentration was 0.66mg/ml determined by Lowry Method; Ultraviolet spectrophotometry of IFNα2b and PEG-IFNα2b showed no perceptable difference in spectra, about 280nm. PEG had no effect on the ultraviolet spectrophotometry of IFNα2b; Relative molecular weight 61195 of PEG-IFNα2b was acquired by MALDI-TOF-MOS. The data showed that relative molecular weight of modified IFNα2b was increased for two-fold. Isoelectric focusing (pI) range of PEG-IFNα2b can be estimated to be 6.6. As a control, the pI of IFNα2b was determined in a manner similar to that employed for the PEG-IFNα2b, and was found to possess a pI of 5.5-6.8, in agreement with the established value. The two products had the similar pI, but pI of PEG-IFNα2b showed a broader band than one of IFNα2b.The anti-viral activity of PEG-IFNα2b was analyzed by VSV-Wish system. Its ability to protect wish cells had decreased for 15-fold, and its anti-viral activity remained 7% of unmodified IFNα2b. At the same time, anti-viral activity of PEG-IFNα2b used a cytopathic effect assay. In this assay, 2BS cell were exposed to hepatitis A virus (HAV) and varicella zoster virus (VZV). IFNα2b was much more effective at the same concentrations than PEG-IFNα2b. At the same specific activity (IU/ml), the extent of protection was the same.IFNα2b and PEG-IFNα2b were tested for their effects on the growth of K562 and HP1 cells. At the same concentrations and specific activity, PEG-IFNα2b had a greater antiproliferative effect than IFNα2b on K562 and HP1 cells (p>0.05). After PEGylation, PEG-IFNα2b had a lower anti-viral activity than IFNα2b, but it had a greater antiproliferative effect than IFNα2b. And at the same concentrations, PEG-IFNα2b had antiproliferative effect 2 to 3-fold higher than that of IFNα2b in vitro.Trypsin degradation of PEG- IFNα2b and IFNα2b had been done. The results showed that anti-viral activity of IFNα2b decreased from 8.8×107IU/ml to 939IU/ml, however PEG-IFNα2b's from 4.8×106IU/ml to 6×105IU/ml on the effect of trypsin at 37℃for 1h. And PEG-IFNα2b had a much slower decrement trend on antiviral activity than that of IFNα2b in the trypsin degradation reaction at first 30min.PEG- IFNα2b and IFNα2b were put at 56℃to assay their heat resistance. Their antiviral activity had no obvious changes until 12 hours. At the 36th hour, antiviral activity of IFNα2b had decreased to below 105IU/ml, and that of PEG-IFNα2b remained above 106IU/ml. The results indicated that PEG-IFNα2b had greater resistance to heat than that of IFNα2b.Protective additive of PEG-IFNα2b for freez-drying formulation was screening from several groups of prescriptions,PH,time of dissolution and anti-viral.One of the prescriptions was coming to confirmed that could take the place of human albumin as protective additive in the freez-drying formulation The mean ingredients of it are Mannitol,low concentrated glutamic acid and Arginin srcrose or fucose.It had been confirmed in this study that this freez-drying formulation was stabie at 4℃and 25℃in one-year time,the same effect as that of human albumin...
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