الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 178 |  |  |
| | | from | 178 | | |
Abstract
Recombinant human interferon- alpha 2 (rhIFN-α2) is FDA approved for treatment of some viral and malignant diseases. Pegylated rhIFN-α2 (Pegasys and PEG-Intron) have highly improved pharmacokinetics, pharmacodynamics and therapeutic efficiency compared to native protein. In this work, we studied the pegylation of purified properly refolded rhIFN -α2b using linear (20kDa) PEG-NHS to prepare pegylated rhIFN-α2b with high stability and activity. The effect of different parameters like rhIFN-α2b final concentration, pH, rhIFN-α2b/PEG molar ratios and reaction time on the efficiency of pegylation (high percentage of monopegylated rhIFN-α2b) have been studied in small scale (100µl) pegylation reaction trials. Study of the percentages of different components of these reactions (mono, di, polypegylated rhIFN-α2b and unpegylated rhIFN-α2b) indicated that, 2h is optimum time to complete the reaction. The pegylation efficiency increased at pH 8 (57.9%) by reducing the protein concentration to 1mg/ml and reducing the rhIFN-α2b/PEG ratio to 1:2. Using the larger scale (2ml) pegylation reaction (65% efficiency), ion exchange chromatography method has been optimized to prepare and purify the monopegylated rhIFN-α2b with high purity (96%). The prepared monopegylated rhIFN-α2b had apparent Mwt of approximately 65 kDa and high in vitro antiviral activity (2.1x107± 0.8 x107 IU/mg). Although it retained approximately 8.4 % of the antiviral activity of the unpegylated rhIFN-α2b, its activity still high compared to other pegylated rhIFN-α2 developed using similar approach or higher molecular weight branched PEG like Pegasys. Further evaluation of the in vivo stability and activity is required for full evaluation of the therapeutic efficacy for the prepared monopegylated rhIFN-α2b.
KEYWORDS: Pegylation, rhIFN-α2b, monopegylated rhIFN-α2b, Pegylation efficiency and antiviral activity.