Heparin was purified by enzymatic hydrolysis combined with oxidation and the residual protein in crude heparin sodium was removed by low temperature centrifugation in the process. Trypsin was added into crude heparin sodium to hydrolyze these foreign proteins, and then the impurities were removed by a process of oxidation, so as to improve the titer and titer recovery of refined heparin sodium.
1. Process flow
Weed an appropriate amount of heparin sodium and added about 3% NaCI solution to dissolve it. 50 Water bath was heated, pH was adjusted to 8.0 with NaOH solution, and stood for 2 hours. Insoluble impurities were filtered and removed.
3, enzymatic hydrolysis method in addition to protein
Heparin in animals is in the form of the heparin protein complex, which is removed from the complex, free protein component of heparin by enzymes or salt solutions. However, in the actual production development process in China, the disintegration of complex and the removal of protein are incomplete, and there are always a certain amount of different proteins in the crude products. Crude heparin sodium must be refined to further remove the remaining binding proteins in the crude. Not only did the titer increase due to the removal of the foreign protein, but the higher activity was obtained due to the release of the "masked" heparin activity. Therefore, the enzymatic hydrolysis of protein has little effect on the total potency of heparin when removing protein, which is based on the characteristic of proteolytic enzyme specific hydrolysis of protein, combined with the method of acidity regulation and oxidizing protein removal.
A small amount of trypsin was added into heparin solution to decompose a small amount of heparin protein into small peptides, which were separated from the heparin protein complex, and then the protein was removed by acid-base modulation and oxidation, which improved the titer and effective recovery of heparin sodium to a certain extent.
The reaction conditions of enzymatic hydrolysis were as follows: 0.05% protease content, pH 7~9, temperature 40℃, reaction time 5h.
4. Adjust acid and remove protein
In order to prevent the deactivation of heparin during the removal of acid protein, sodium sulfate is added as a protective agent. The reactive protein was removed by centrifugation at low temperature.
5. Oxidation conditions
H2O2 dosage 2/ % pH8.5, temperature 25~27℃, time 15h.
Alcohol precipitation and drying: adjust the pH of the oxidation solution to 6.5 and add the same amount of 95% ethanol to precipitate. The supernatant was extracted to get the precipitate, and then anhydrous ethanol or acetone was added slowly to dehydrate the precipitate.
55℃, vacuum drying for more than 3 hours to get a fine.
Heparin sodium was purified by enzymatic hydrolysis combined with primary oxidation of hydrogen peroxide. The product was whiter, and the potency and potency recovery were higher than those of the secondary oxidation method. The potency was more than 150U/mg, and the potency recovery was more than 95%. Consult the Heparins Manufacturer for details