| Low molecular weight heparin is a kind of sodium or calcium salts prepared through chemical or enzymatic degradation from un-fraction heparin. LMWH also can be defined as a upgrade of UFH. The average molecular weight of LMWH is about 5000D. Because the antiⅩa titer of LMWH is much higher than unfractionated heparin when subcutaneous injection. So the researches on low molecular weight heparin are much accounted more and more. Now LMWH had become a hot research interests at home and abroad. Because the chemical degradation methods feature low cost and easily to be industrialized, so the various chemical degradation methods of LMWH are widely studied in the field of LMWH.At present, nitrous acid degradation method,β-elimination degradation method and enzymatic degradation method had usually been used in the industrial preparation of LMWH, while the reports about hydrogen peroxide degradation method were less. Taking into account the superiority of the product prepared with the hydrogen peroxide degradation method is that the products have a higher degree of sulfuric acid, and its cost is lower than other methods. So this study employed hydrogen peroxide degradation to produce LMWH. We tried to improve the product composition of antiⅩa by affinity chromatography. This study will play an important practical application of industrial production reference value.Because the quality of crude heparin directly determines the quality of LMWH, we need to do a series of purification on crude heparin. Salt solution was used in this study. The purification includes decolorization with hydrogen peroxide, sodium chloride salt solution, resin adsorption, precipitation, drying and other processing steps. Meanwhile, in the refining process, we achieved the optimum process conditions through real-time monitoring the anticoagulant potency, pH, optical rotation, molecular weight and molecular weight distribution. The results show that the optimal process conditions are as follows: the concentration of sodium chloride is 2%, pH value is between 9 and 10, alcohol precipitation volume fraction is 60%, the concentration of hydrogen peroxide is 2%. The anticoagulant potency of refined products tripled and the recovery is 85%. So the quality of the heparin boutique meets the production requirements of LMWH.To prepare a new ultra-pure LMWH with high antiⅩa titer and low antiⅡa activity, this study used copper as the catalyst, and orthogonal design to obtain the optimized conditions. The factors of orthogonal design are temperature (A), time (B), peroxide hydrogen concentration (C) and solution pH (D). And we dectected the molecular weight and molecular weight distribution of LMWH by GPC. We used chromogenic substrate to determinate the titer of antiⅩa and antiⅡa. The results show that when the hydrogen peroxide concentration is 2%, the temperature is 60℃, the reaction time is 1h, pH value is 7, the molecular weight of LMWH is 3773D, and the proportion of more than 8000D is 2.1%. The proportion of less than 2000D is 5.4%. The titer of AntiⅩa of LMWH is 585 IU/mg, and the titer of antiⅡa is 11 IU/mg. The ratio of anti-factorⅩa activity against anti-factorⅡa potency is 53.2.This study established a new chemical degradation method of heparin. In addition, we screened effective component of antiⅩa by affinity chromatography. Thus, we developed a new antithrombotic heparin drugs with high thrombolytic activity and low anticoagulant activity. Our study lays a solid foundation for the further study of ultrapure low molecular weight heparin, and meanwhile provides the possibility for its clinical applications.
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