Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong> Vol. 6 (2) 229-240 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) Table 5. ANOVA table for 2 3 full factorial design. Source <strong>of</strong> variation a p-value for HPLC p-value for results Ninhydrin test results Model
Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong> Vol. 6 (2) 229-240 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) Figure 3 (b-e) show intact HeLa cells which have normal shapes <strong>and</strong> sizes. Figure 3f shows the biological activity <strong>of</strong> HeLa cells in the presence <strong>of</strong> virus <strong>and</strong> absence <strong>of</strong> IFN-β. In the absence <strong>of</strong> IFN-β the cells were detected at the bottom <strong>of</strong> the microplate (indicating the cytopathic effect <strong>of</strong> virus). In Table 7, by considering image b as representing 100% protein biological activity <strong>and</strong> addition <strong>of</strong> the virus to the growth medium, IFN-β biological activity decreases by about 70% (image c). By PEGylating the protein, its biological activity, as compared to image b, has decreased by about 40% (image d). Then, by adding virus to the growth medium it is obvious that the IFNβ biological activity in image e, compare to image c, has calculated about 80%. The studies that have been published regarding PEGylated IFNα-2b (PEG-Intron) <strong>and</strong> PEGylated IFN-α-2a (Pegasys) support our observation that nontargeted PEGylation has a deleterious effect on the activity <strong>of</strong> interferons. In the case <strong>of</strong> PEG- Intron, reaction involving succinimidyl carbonate is used to attach the linear 12 kDa PEG (35), while for Pegasys an N-hydroxysuccinimide ester derivative <strong>of</strong> the branched 40 kDa PEG was used to modify the protein [13]. The use <strong>of</strong> such chemical reactors has resulted in the modification <strong>of</strong> numerous individual sites as well as a significant reduction in the in vitro specific antiviral activity. With regard to the PEG-Intron, lysine, tyrosine, histidine, serine, <strong>and</strong> cysteine residues are modified, resulting in 14 different monoPEGylated positional isomers. The antiviral activity <strong>of</strong> the mixture is 28% <strong>of</strong> that <strong>of</strong> the unmodified protein <strong>and</strong> ranges from 6 to 37% for the individual species (36). In the case <strong>of</strong> Pegasys, only lysine residues are modified, resulting in 6 different monoPEGylated positional isomers, with the mixture having an antiviral activity <strong>of</strong> only 7% <strong>of</strong> the unmodified protein (13). For rat IFN-β, the PEGylated protein, which retains essentially full in vitro antiviral activity, had improved pharmacokinetic parameters as Interferon Beta-1a PEGylation 237 compared to the unmodified protein (23). Baker et al. have shown that PEGylated IFN-β-1a retains approximately 50% <strong>of</strong> its activity when compared to the unmodified protein (1). Relative to native IFN-β-1b, the monoPEGylated compounds possess excellent activity. The retention <strong>of</strong> about 20-70% <strong>of</strong> antiviral activity in these derivatives compares favorably to the marketed PEGylated IFN-R drugs, PEG- INTRON <strong>and</strong> Pegasys, where in vitro antiviral activities are about 28% or 7% <strong>of</strong> the unmodified protein, respectively (13,14). It is apparent that the conjugates with the highest molecular weight, such as the 40 kDa PEG polymers have lower antiviral activity in vitro (25). Conclusions mPEG-SPAs <strong>of</strong> 5, 20 <strong>and</strong> 40 kDa were covalently attached to IFN-β-1a <strong>and</strong> the optimum conditions for this reaction were determined by different methods <strong>of</strong> experimental design. For 5 <strong>and</strong> 20 kDa mPEGs, the optimum molecular weight, pH <strong>and</strong> the ratio <strong>of</strong> protein to mPEG were found to be 20 kDa, 8 <strong>and</strong> 1:40, respectively. Under optimum conditions, the percentages <strong>of</strong> protein modifications were obtained 45.5% <strong>and</strong> 46.8% by HPLC <strong>and</strong> Ninhydrin methods, respectively. For 40 kDa mPEG reactions, the optimum molecular weight, pH <strong>and</strong> protein to mPEG molar ratio were found to be 40 kDa, 8 <strong>and</strong> 1:40, respectively. Under optimum conditions, the percentage protein modifications were obtained 46.5% <strong>and</strong> 47.7% HPLC <strong>and</strong> Ninhydrin methods, respectively. Further increases in polymer concentration, may increase the extent <strong>of</strong> protein coating. Based on the results <strong>of</strong> the present <strong>and</strong> previous studies (1,24,30), it can be concluded that the optimum conditions for PEGylation <strong>of</strong> IFNs vary with changing molecular weight <strong>of</strong> mPEG or using different activating agents. The biological activity test showed that the PEGylated protein retained about 80% <strong>of</strong> its activity, were compared to that <strong>of</strong> the unmodified protein, While, in other studies the maximum biological activity were obtained about 50%.
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