Chemical Modification of Recombinant Human Interferon Beta-1a Using Linear and Branched mPEGs

Order of Publishing in Issue: 
Volume :6
Issue :2
April, 2012
Page No: 
Ahmad Abolhasani [1], Sameereh Hashemi-Najafabadi [1]*, Mahvash Khodabandeh Shahraki*[2] and Zohreh-Azita Sadigh [3]
[1] Biotechnology Group, Department of Chemical Engineering, Tarbiat Modares University, P. O. Box: 14115/114, Tehran, I. R. Iran
[2] National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, I. R. Iran
[3] Razi Vaccine and Serum Research Institute, Tehran, I. R. Iran

Interferon-beta-1a (IFN-ß-1a) is used clinically in the treatment of multiple sclerosis. Similar to other biological molecules, IFN-ß-1a has a relatively short serum half-life and is rapidly detected by the host’s immune system. PEGylation is a common approach to increase the blood circulation time of therapeutic proteins. In the present study, IFN-ß-1a was PEGylated using linear methoxy polyethylene glycols (mPEGs) with molecular weights of 5 and 20 kDa and also 40 kDa branched mPEG-SPA. Prior to PEGylation, the mPEGs were activated by succinimidyl propionic acid (SPA). PEGylation was evaluated by size-exclusion HPLC (SEHPLC) and Ninhydrin method. In the designed experiments, the factors of mPEG molecular weight, pH, and the molecular ratio of protein to mPEG fractions were studied. The results were analyzed using Design-Expert statistical software and the significant factors were determined. Then, in order to find the optimum conditions, Taguchi method (L9 array) was used by considering the significant factors. Consequently, the optimum conditions for PEGylation, using 20 kDa linear mPEG-SPA was found to be at pH 8 and the protein to mPEG molar ratio of 1/40. The extents of protein modification were obtained 45.5% and 46.8% by HPLC and Ninhydrin methods, respectively. Optimum PEGylation with 40 kDa branched mPEG-SPA was obtained at pH 8 and protein/mPEG of 1/40. In this case, the extents of protein modification were obtained 46.5% and 47.7% by HPLC and Ninhydrin methods, respectively. The biological activity test showed that the PEGylated protein retained about 80% of its activity, were compared to that of the unmodified protein.
Key words:

Beta interferon, Biological activity, mPEG-SPA, PEGylation, Taguchi method.
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