Pegylation Of Defibrase

Defibrase is extensively used in domestic clinic for Thromb disease such as acute myocardial infarction and cerebral thrombosis on account of its satisfactory treatment effect in thrombokinesis, fat, tensional blood vessel, microcirculation, while accompanied inevitable such common defects as high immunogenicity, low stability, short half-life time as a pharmaceutical grade protein.The technology of peptide and protein PEGylation has gained great progress during the last 15 years. A lot of research in this field were emerged. Many therapeutic grade proteins coupled PEG have been applied to therapy of various kinds of diseases in clinic and revealed the immunogenic depression, stability elevation and half-life time extension. Such improved Defibrase is expected to obtain by PEGylation with the purpose of and convenient administration through amelioration of Defibrase's pharmacokinetics in this paper.During the test of Defibrase PEGylation, many kinds of PEG including mPEG-ALD, mPEG-SPA, mPEG-SBA, mPEG2-NHS et cetera were tried. Concerning enzyme activity, modification rate and purity of products, we apply mPEG-SPA as the optimal PEG category for the PEGylation of Defibrase. Orthogonal experiments were used to optimize the condition of Defibrase PEGylation and to evaluate the effect of the four factors. Optimization condition is buffer pH 6.5, 1:20molar ratio of Defibrase and mPEG , one hour reaction time, 37°C reaction temperature. The highest modification rate of Defibrase was achieved while high enzyme activity was still remained if the PEGylation proceeded under this condition. Molar ratio is the most significant factor with decibel 0.191, while the reaction time is opposite with decibel 0.045 among the four factors. The method of purification and detection such as HPLC, superdex75, SDS-PAGE, for Defibrase and its PEGylation products,was established. Two single PEGylation products, Df-mPEG -SPA5000 and Df-mPEG -SPA30000, remained high enzyme activity were purified by molecular sieve column chromatography. The modification rate of Defibrase with mPEG-SPA5000 is 46.7%, recovery rate of Defibrase is 45.2%; while with mPEG-SPA 30000 is 38.2%, recovery rate of Defibrase is 34.1 %.The quality of Defibrase and its purified PEGylation products was studied. The result has disclosed that the expecting objective of Defibrase PEGylation is achieved in this work.Remaining rate of enzyme activity of Df-mPEG -SPA5000 was as high as 42.3 %, while Df-mPEG -SPA30000 37.9%. The immunogenicity of Df-mPEG -SPA500(h Df-mPEG -SPA30000 was steped down. The stability and action time on fibrinogen of Df-mPEG -SPA5000 was improved. Preliminary study was carried out on affirmation of modification site.Some outcomes of this work were applied for invention patent of China, Appl. No.is 200410017779.4.