A process for efficiently removing slow-moving heparin from heparin byproducts

Abstract

The present application discloses a process for efficiently removing slow-moving heparin from heparin byproducts, comprising the following steps: S1, adding heparin byproducts to sodium chloride solution, heating and dissolving; S2, adding trypsin to the dissolved solution, stirring for more than 30min, and then heating to 40-50°C, and continuing to stir for more than 30min; S3, loading CH-90 resin column, and then separating the raw liquid obtained in step S1 through CH-90 resin column, collecting the lower discharge liquid; S4, the CH-90 resin column after separating the raw liquid in step S3, rinsing with purified water, collecting the lower discharge liquid; S5, merging the lower discharge liquid collected in step S3 and step S4, adding silica gel adsorbent adsorption treatment, 0.45μm filter membrane filtration, adding sodium chloride, precipitating with medicinal ethanol, collecting the precipitate, drying, and obtaining slow-moving heparin. The present application effectively improves the utilization rate of raw materials and improves the yield of heparin.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a process for efficiently removing slow-moving heparin from heparin byproducts. Background Art

[0002] Heparin, also known as heparin sodium, is an important biological drug with very important biological functions. Currently, the anti-thrombotic drugs used clinically are mainly low-molecular-weight heparin preparations.

[0003] Heparin is mainly distributed in the lung, liver, and small intestinal mucosal cells of mammals. At present, the raw materials for preparing heparin at home and abroad are mainly pig small intestinal mucosa, cattle lung, and pig lung. In the extraction process, three processes must be undergone: extraction of heparin protein complex, decomposition of heparin protein complex, and refining of crude heparin. my country's livestock and poultry by-products are extremely rich, with the annual slaughter of pigs and cattle reaching more than several billion heads, and the output of livestock and poultry organs is extremely high. However, the utilization rate of animal viscera for pharmaceutical production in China is still very low. Most of them are sold and processed in the form of food, and the added value is very low. The existing process for extracting heparin from livestock and poultry by-products in my country generally adopts the steps of beating, salting out, adsorption, impurity removal, protein precipitation, filtration, and drying. First, impurities such as cell protein and fat are removed, and then impurities are removed by salting out. Protein, finally, crude heparin is obtained after heparin is purified by oxidation. The above process is mainly based on processing crude heparin, and there are problems such as low product titer, low product yield, low production efficiency, and high production cost.

[0004] In recent years, with the upgrading and improvement of heparin processing technology in my country, the use of enzymatic hydrolysis technology and ion exchange, gel column chromatography, affinity column chromatography and other purification technologies can purify crude heparin and effectively improve the quality of heparin products. However, the existing process still has problems such as low heparin yield and low raw material utilization. Summary of the invention

[0005] In order to solve at least one of the above technical problems, a heparin preparation process is developed to effectively improve the utilization rate of raw materials and the yield of heparin. The present application provides a process for efficiently removing slow-moving heparin from heparin by-products.

[0006] The present application provides a process for efficiently removing slow-moving heparin from heparin byproducts, comprising the following steps:

[0007] S1. adding the heparin byproduct into a sodium chloride solution, heating and dissolving, and preparing a solution;

[0008] S2, adding trypsin to the solution prepared in step S1, stirring for more than 30 minutes, and then heating to 40-50° C., and continuing to stir for more than 30 minutes to obtain a raw material solution;

[0009] S3, loading CH-90 resin into a column, and then separating the raw material liquid obtained in step S1 through a CH-90 resin column, detecting the lower discharge liquid with azure dye, and collecting the lower discharge liquid if it is red;

[0010] S4, washing the CH-90 resin column after separating the raw material liquid in step S3 with purified water, detecting the lower discharge with azure dye, and collecting the lower discharge if it is red and continue to wash with purified water until the lower discharge is no longer red when detected with azure dye;

[0011] S5. The lower discharge liquid collected in step S3 and step S4 is combined, adsorbed with silica gel adsorbent, and then filtered with a 0.45 μm filter membrane. After sodium chloride is added to dissolve completely, it is precipitated with medicinal ethanol and allowed to stand for more than 24 hours. The precipitate is collected and dried to obtain slow-moving heparin.

[0012] Optionally, in step S1, a sodium chloride solution with a concentration of 5-10% is used to prepare a dissolving solution with a heparin by-product concentration of 10%.

[0013] Optionally, in step S2, the amount of trypsin added is 1-3% of the mass of the dissolving solution.

[0014] Optionally, in step S2, the solution is heated to 40-50°C, stirred for more than 30 minutes, and then ultrasonically treated for more than 15 minutes.

[0015] Further optionally, in step S2, the power of the ultrasonic treatment is 200-300W.

[0016] Optionally, in step S3, after the CH-90 resin is loaded into the column, the pH value is adjusted to 6.0-7.0.

[0017] Optionally, in step S3, when CH-90 column is loaded, the solid-liquid ratio is 15 mL of CH-90 resin per 1 g of heparin by-product.

[0018] Further optionally, in step S3, the specific steps of separating the raw material liquid through the CH-90 resin column are as follows: close the valve at the bottom of the column, add 5 to 10 ml of raw material liquid each time, and after there is no liquid at the upper end of the resin, open the bottom valve to collect the lower discharge liquid, and the separation of the raw material liquid is completed.

[0019] Optionally, in step S5, the amount of silica gel adsorbent added is 3-5% of the mass of the heparin by-product.

[0020] Optionally, in step S5, the amount of sodium chloride added is 1% of the mass of the combined liquid, and the amount of medicinal ethanol used is 2 times the volume of the combined liquid.

[0021] In summary, the present invention includes at least one of the following beneficial technical effects:

[0022] 1. The present application can effectively separate slow-moving heparin from heparin by-products by adding sodium chloride solution to dissolve the heparin by-products, and then use a CH-90 resin column to separate the heparin by-products after the protease hydrolyzes them again, thereby effectively improving the utilization rate of raw materials for heparin production.

[0023] 2. The present application uses heparin byproducts to further extract and prepare heparin. The purity of the obtained heparin is relatively high, and the yield of heparin can be effectively improved, and the cost is relatively low. DETAILED DESCRIPTION

[0024] The present application is further described in detail below with reference to the embodiments.

[0025] The present application provides a process for efficiently removing slow-moving heparin from heparin byproducts, comprising the following steps:

[0026] S1. adding the heparin byproduct into a sodium chloride solution, heating and dissolving, and preparing a solution;

[0027] S2, adding trypsin to the solution prepared in step S1, stirring for more than 30 minutes, and then heating to 40-50° C., and continuing to stir for more than 30 minutes to obtain a raw material solution;

[0028] S3, loading CH-90 resin into a column, and then separating the raw material liquid obtained in step S1 through a CH-90 resin column, detecting the lower discharge liquid with azure dye, and collecting the lower discharge liquid if it is red;

[0029] S4, washing the CH-90 resin column after separating the raw material liquid in step S3 with purified water, detecting the lower discharge with azure dye, and collecting the lower discharge if it is red and continue to wash with purified water until the lower discharge is no longer red when detected with azure dye;

[0030] S5. The lower discharge liquid collected in step S3 and step S4 is combined, adsorbed with silica gel adsorbent, and then filtered with a 0.45 μm filter membrane. After sodium chloride is added to dissolve completely, it is precipitated with medicinal ethanol and allowed to stand for more than 24 hours. The precipitate is collected and dried to obtain slow-moving heparin.

[0031] Preferably, in step S5, after the lower drainage liquids are combined, the pH value is adjusted to 7.5-8.0.

[0032] The design of the present application re-extracts the existing heparin by-products, effectively utilizes waste, obtains a relatively high heparin yield, and greatly improves the utilization rate of heparin raw materials.

[0033] The raw materials used in the examples of the present application are basically purchased from the market, trypsin is purchased from Beijing Aoboxing Biotechnology Co., Ltd., and azure dye is purchased from Shanghai Reagent Factory.

[0034] The heparin byproducts processed in the examples of the present application are byproducts obtained from the same batch of heparin production, which are derived from the production line for preparing heparin from pig small intestine. The heparin byproducts processed in the examples of the present application are all 10 g.

[0035] Example 1

[0036] The process for efficiently removing slow-moving heparin from heparin byproducts of this embodiment comprises the following steps:

[0037] S1. Add 10 g of heparin by-product to a 5% sodium chloride solution, heat and dissolve completely to prepare a solution with a heparin by-product concentration of 10%;

[0038] S2, adding 1% trypsin by weight of the dissolving solution to the dissolving solution prepared in step S1, stirring for 30 minutes, and then heating to 40° C., and continuing stirring for 30 minutes to obtain a raw material solution;

[0039] S3, 150mL CH-90 resin was loaded into the column, the pH value was adjusted to 7.0, and then the raw material solution obtained in step S1 was separated by a CH-90 resin column, and the lower discharge was detected with azure dye, and the lower discharge was collected if it was red;

[0040] S4, washing the CH-90 resin column after separating the raw material liquid in step S3 with purified water, detecting the lower discharge with azure dye, and collecting the lower discharge if it is red and continue to wash with purified water until the lower discharge is no longer red when detected with azure dye;

[0041] S5. Combine the lower liquid collected in step S3 and step S4, adjust the pH value to 7.5, add silica gel adsorbent accounting for 3% of the mass of heparin by-product for adsorption treatment, and then filter with a 0.45μm filter membrane, add sodium chloride accounting for 1% of the mass of the combined feed liquid, stir until the sodium chloride is completely dissolved, add medicinal ethanol twice the volume of the combined feed liquid for precipitation and let it stand for more than 24 hours, collect the precipitate, and dry it to obtain slow-moving heparin.

[0042] Example 2

[0043] The difference between this embodiment and embodiment 1 is that in step S1, a sodium chloride solution with a concentration of 5% is used.

[0044] Example 3

[0045] The difference between this embodiment and embodiment 1 is that in step S1, 3% trypsin is added based on the mass of the dissolving solution.

[0046] Example 4

[0047] The difference between this embodiment and embodiment 3 is that in step S2, the mixture is heated to 50°C and stirred for 30 minutes.

[0048] Example 5

[0049] The difference between this embodiment and embodiment 4 is that in step S3, the pH value is adjusted to 6.0.

[0050] Example 6

[0051] The difference between this embodiment and embodiment 5 is that in step S4, the specific steps of rinsing with purified water are: close the valve at the bottom of the column, add 10-20 ml of purified water as the raw material liquid each time, and after no purified water is present at the upper end of the resin, open the valve at the bottom to collect the lower discharge liquid, and detect the lower discharge liquid with azure dye. If it is red, collect the lower discharge liquid and continue to rinse with purified water until the lower discharge liquid is no longer red when detected by azure dye.

[0052] Example 7

[0053] The difference between this embodiment and embodiment 6 is that in step S5, a silica gel adsorbent accounting for 5% of the mass of the heparin by-product is added for adsorption treatment.

[0054] Example 8

[0055] The difference between this embodiment and embodiment 7 is that in step S5, the pH value is adjusted to 8.0.

[0056] Example 9

[0057] The difference between this embodiment and embodiment 8 is that in step S2, after heating and continuing stirring, the dissolved liquid is ultrasonically treated for 15 minutes with an ultrasonic power of 200W.

[0058] Example 10

[0059] The difference between this embodiment and embodiment 8 is that in step S2, after heating and continuing stirring, the dissolved liquid is ultrasonically treated for 35 minutes with an ultrasonic power of 300W.

[0060] Comparative Example 1

[0061] The process for efficiently removing slow-moving heparin from heparin byproducts in this comparative example comprises the following steps:

[0062] S1. Add 10 g of heparin by-product to a 10% sodium chloride solution, heat to dissolve completely, and prepare a solution with a heparin by-product concentration of 10%;

[0063] S2, adding 3% trypsin by weight of the dissolving solution to the dissolving solution prepared in step S1, stirring for 30 minutes, and then heating to 50° C., and continuing stirring for 30 minutes to obtain a raw material solution;

[0064] S3, the raw material liquid of step S2 is adjusted to pH 7.5, and a silica gel adsorbent accounting for 3% of the mass of the heparin by-product is added for adsorption treatment, and then filtered through a 0.45 μm filter membrane, and then sodium chloride accounting for 1% of the mass of the combined feed liquid is added, and stirred until the sodium chloride is completely dissolved, and then medicinal ethanol twice the volume of the combined feed liquid is added for precipitation and allowed to stand for more than 24 hours, and the precipitate is collected and dried to obtain slow-moving heparin.

[0065] The slow-moving heparin obtained by the processes of Examples 1 to 10 of the present application and Comparative Example 1 was tested for quality and purity, and the test results are shown in Table 1. The purity test of heparin was carried out according to the method recorded in the Chinese Pharmacopoeia.

[0066] Table 1 Test results of Examples 1 to 10 and Comparative Example 1

[0067]

[0068] From the data in Table 1, it can be seen that the heparin obtained in Examples 1 to 10 of the present application has a significantly improved purity and quality compared to the heparin obtained in Comparative Example 1. It can be seen that the use of the resin separation step of the present application can greatly improve the purity of the extracted heparin.

[0069] It can also be seen from the data in Table 1 that the heparin obtained in Examples 1 to 10 of the present application has a significantly improved yield compared to the heparin obtained in Comparative Example 1. It can be seen that after purification by the resin separation step, the efficiency of salt dissolution and alcohol precipitation can be effectively improved, and the interference of impurities in the above steps can be effectively avoided, thereby greatly improving the yield of heparin.

[0070] From the data in Table 1, it can be seen from the data comparison of the heparin obtained in Examples 1 to 10 that after optimizing the process parameters of the present application, the yield and purity of heparin can be further improved. In addition, after adding the ultrasonic treatment step, the yield of heparin can be further greatly improved, and the purity is also improved; it can be seen that after ultrasonic treatment, the proteolysis effect can be greatly improved, thereby effectively improving the yield and quality of heparin.

[0071] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Therefore, any equivalent changes made according to the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A process for efficiently removing slow-moving heparin from heparin byproducts, characterized in that it comprises the following steps: S1. adding the heparin byproduct into a sodium chloride solution, heating and dissolving, and preparing a solution; S2, adding trypsin to the solution prepared in step S1, stirring for more than 30 minutes, and then heating to 40-50° C., and continuing to stir for more than 30 minutes to obtain a raw material solution; S3, loading CH-90 resin into a column, and then separating the raw material liquid obtained in step S1 through a CH-90 resin column, detecting the lower discharge liquid with azure dye, and collecting the lower discharge liquid if it is red; S4, washing the CH-90 resin column after separating the raw material liquid in step S3 with purified water, detecting the lower discharge with azure dye, and collecting the lower discharge if it is red and continue to wash with purified water until the lower discharge is no longer red when detected with azure dye; S5. The lower discharge liquid collected in step S3 and step S4 is combined, adsorbed with silica gel adsorbent, and then filtered with a 0.45 μm filter membrane. After sodium chloride is added to dissolve completely, it is precipitated with medicinal ethanol and allowed to stand for more than 24 hours. The precipitate is collected and dried to obtain slow-moving heparin.

2. A process for efficiently removing slow-moving heparin from heparin by-products according to claim 1, characterized in that: In the step S1, a sodium chloride solution with a concentration of 5-10% is used to prepare a dissolving solution with a heparin byproduct concentration of 10%.

3. A process for efficiently removing slow-moving heparin from heparin by-products according to claim 1, characterized in that: In step S2, the amount of trypsin added is 1-3% of the mass of the dissolving solution.

4. A process for efficiently removing slow-moving heparin from heparin by-products according to claim 1, characterized in that: In the step S2, the solution is heated to 40-50°C, stirred for more than 30 minutes, and then subjected to ultrasonic treatment for more than 15 minutes.

5. A process for efficiently removing slow-moving heparin from heparin by-products according to claim 4, characterized in that: In step S2, the power of the ultrasonic treatment is 200-300W.

6. A process for efficiently removing slow-moving heparin from heparin by-products according to claim 1, characterized in that: In the step S3, after the CH-90 resin is loaded into the column, the pH value is adjusted to 6.0-7.

0.

7. A process for efficiently removing slow-moving heparin from heparin by-products according to claim 1, characterized in that: In step S3, when CH-90 is loaded into the column, the solid-liquid ratio is 15 mL of CH-90 resin per 1 g of heparin byproduct.

8. A process for efficiently removing slow-moving heparin from heparin by-products according to claim 7, characterized in that: In step S3, the specific steps of separating the raw material liquid through the CH-90 resin column are as follows: close the valve at the bottom of the column, add 5-10 ml of raw material liquid each time, and when there is no liquid at the top of the resin, open the bottom valve to collect the lower discharge liquid, and the separation of the raw material liquid is completed.

9. A process for efficiently removing slow-moving heparin from heparin by-products according to claim 1, characterized in that: In the step S5, the amount of silica gel adsorbent added is 3-5% of the mass of the heparin by-product.

10. A process for efficiently removing slow-moving heparin from heparin by-products according to claim 1, characterized in that: In step S5, the amount of sodium chloride added is 1% of the mass of the combined liquid, and the amount of medicinal ethanol used is 2 times the volume of the combined liquid.

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