Enoxaparin sodium is a low molecular weight heparin sodium derived from the esterification of heparin sodium extracted from pig intestine membrane. It is composed of many complex unknown oligosaccharides. So what are the preparation methods of enoxaparin sodium?
1. Into salt. Weigh 30g crude heparin sodium, add purified water to dissolve, get heparin sodium solution; 75g benzethonium chloride was weighed and dissolved in purified water to obtain benzethonium chloride solution. Slowly add benzathonium chloride solution to heparin sodium solution, stir while adding, stir evenly, stand for 2 hours. Then centrifuge, centrifuge the solid obtained with purified water washing, and finally the wet heparin benzyl chloro-ammonium salt vacuum drying, to obtain heparin benzyl chloro-ammonium salt.
2. The esterification. To join in the heparin benzyl ammonium chloride five times the amount of methylene chloride, stir to dissolve and add in 1 times the amount of benzyl chloride, keep the temperature at 35 ℃, mixing reaction for 24 hours, after the reaction, add six times the amount of heparin benzyl chloride ammonium salt of 10% sodium acetate methanol solution, stirring for 10 minutes, let stand for 4 hours, precipitation, filtration precipitation repeatedly with methanol washing filtering twice, Wet crude heparin benzyl ester is obtained. Add 8% sodium chloride aqueous solution 3 times the weight of heparin benzyl chloride ammonium salt to the wet crude heparin benzyl chloride, stir until completely dissolved, then add methanol 4 times the volume of sodium chloride solution, stir again for 10 minutes, stand for 2 hours, filter, repeat this operation 3 times to obtain the wet heparin benzyl chloride. The wet product of heparin benzyl ester was vacuum dried to obtain heparin benzyl ester.
3. The depolymerization. Add 20 times the weight of heparin benzyl ester purified water, stir to dissolve, heat up to 60℃. The sodium hydroxide, which accounts for 10% of the weight of heparin benzyl ester, was dissolved in pure water, and then slowly added to the heparin benzyl ester solution, the temperature was kept at 60℃, and the reaction was stirred for 2 hours. Then, the pH value of the solution was adjusted to 6.0-6.5 with appropriate amount of hydrochloric acid, and the depolymerization solution was obtained after cooling to room temperature. Add an appropriate amount of sodium chloride to the depolymerization solution, make its concentration 10%, stir until completely dissolved, then add methanol 60 times the weight of heparin benzyl ester in the stirred state, stir evenly, stand for 5 hours, separate the supernatant, get wet enoxaparin sodium crude product.
4. Oxidation alcohol precipitation. Add purified water 3 times the weight of enoxaparin sodium wet crude, stir until completely dissolved, add purified water to make the solution concentration of 20%. Then add 1% of the volume of sodium chloride solution, stir, heat to 35℃, adjust the solution pH to 9.5-10.0 with sodium hydroxide; Then add 1% of the volume of hydrogen peroxide solution (30% concentration), maintain temperature and pH, oxidation for 8 hours, to obtain the oxidation solution. The oxidation solution was filtered with a 0.45μm filter element, and the pH was adjusted to 6.0~6.5 with hydrochloric acid. Methanol, 5 times the volume of the filtrate, was added after agitation, and stirred evenly. The supernatant was separated for 5 hours to obtain the oxidized alcohol precipitate of enoxaparin sodium.
5. Sterilization and filtration. Add purified water to the oxidized alcohol precipitate at a concentration of 25%, stir until it is completely dissolved, and then filter the solution through the filter element. The sterilized filtrate was dried, crushed and mixed with lyophilizer to obtain refined enoxaparin sodium.
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