A method for preparing low molecular weight heparin sodium by enzymatic hydrolysis

Abstract

The invention relates to the technical field of enzymatic hydrolysis, and particularly to an enzymatic hydrolysis preparation method for low-molecular-weight heparin sodium. The method comprises the following steps: S1.1, pre-treating the sodium heparin and combining it with a protective agent to obtain a heparin complex precipitate; S1.2, performing a first enzymatic hydrolysis on the heparin complex precipitate to obtain a supernatant A and a precipitate; S1.3, adjusting the pH of the precipitate to release highly sulfated fragments to obtain a supernatant B; S1.4, performing a second enzymatic hydrolysis on the supernatant B to obtain a secondary enzymatic hydrolyzate, combining the supernatant A and the secondary enzymatic hydrolyzate, and filtering to obtain a retentate; S1.5, post-treating the retentate to obtain the low-molecular-weight heparin sodium; and using a composite protective agent prepared by covalently coupling citric acid-modified superparamagnetic Fe3O4 nanoparticles with aminocalixarene, accurately regulating the enzymatic hydrolysis path and achieving efficient separation, thereby solving the problems of uneven molecular weight, low yield, and complex purification in traditional processes.

Description

Technical Field

[0001] The present invention relates to the technical field of enzymolysis, in particular to a method for preparing low-molecular-weight heparin sodium by enzymolysis. Background Art

[0002] Low molecular weight heparin sodium is a type of polysaccharide anticoagulant drug derived from unfractionated heparin by chemical or enzymatic degradation. Compared with unfractionated heparin, low molecular weight heparin sodium has more stable pharmacokinetic properties, a longer half-life, and a lower risk of bleeding. Its core advantage lies in the optimization of the activity ratio of anticoagulant factors Xa and IIa by controlling the molecular weight distribution (usually 4000-6000 Da). Therefore, it is widely used clinically to prevent and treat thrombotic diseases (such as deep vein thrombosis, pulmonary embolism, etc.).

[0003] However, heparin is a highly sulfated linear polysaccharide with an uneven distribution of sulfation along the chain, forming high-sulfation regions and low-sulfation regions. During enzymatic hydrolysis, substrate recognition by heparinase (such as heparinase I, II, and III) depends on the conformation and charge environment of specific glycosidic bonds. The dense negative charge in the highly sulfated region creates steric hindrance and electrostatic repulsion, hindering the contact between the active center of the enzyme and the target glycosidic bond, resulting in uneven molecular weight distribution of the enzymatic hydrolysis product. In view of this, we propose an enzymatic hydrolysis method for the preparation of low-molecular-weight heparin sodium. Summary of the Invention

[0004] The object of the present invention is to provide a method for preparing low molecular weight heparin sodium by enzymatic hydrolysis to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides a method for preparing low molecular weight heparin sodium by enzymatic hydrolysis, comprising: S1.1, pretreating the heparin sodium and combining it with a protective agent to obtain a heparin complex precipitate;

[0006] S1.2, performing a first enzymatic hydrolysis on the heparin complex precipitate to obtain supernatant A and precipitate;

[0007] S1.3. Adjust the pH of the precipitate to release the highly sulfated fragments, obtaining supernatant B;

[0008] S1.4. Perform a secondary enzymatic hydrolysis on supernatant B to obtain a secondary enzymatic hydrolyzate. Combine supernatant A and the secondary enzymatic hydrolyzate and filter to obtain a retentate.

[0009] S1.5, post-processing the retentate to obtain low molecular weight heparin sodium;

[0010] Among them, the protective agent is prepared by surface-modifying superparamagnetic Fe3O4 nanoparticles with citric acid and then covalently coupling them with amino-calixarene.

[0011] Preferably, in S1.1, the amount of heparin sodium is 30-50 parts by weight; the amount of the protective agent is 9-15 parts by weight, the molecular weight of heparin sodium is 15-18 kDa, the pH of the phosphate buffer is 7.0-7.2, and the amount of activated carbon added is 0.1-0.5% (w / v);

[0012] The specific preparation steps of heparin complex precipitation are:

[0013] Dissolve heparin sodium in phosphate buffer to obtain a 3-5% (w / v) heparin sodium solution, add activated carbon, stir at 40-45°C for 10-15 minutes, filter through a 0.22μm membrane to obtain a clear solution, then add a protective agent, control the temperature at 25-30°C, stir at 200-300rpm for 50-60 minutes, and then perform magnetic separation for 10-15 minutes. Discard the supernatant to obtain a heparin complex precipitate.

[0014] Preferably, in S1.2, the mixed heparinase is heparinase I:II=2:1 U / mg heparin sodium; the total enzyme amount is 5-10 U / mg heparin sodium;

[0015] The specific steps of the first enzymatic hydrolysis of heparin complex precipitation are:

[0016] The heparin complex precipitate was redispersed in phosphate buffer to obtain a 2-5% (w / v) heparin complex precipitate solution, mixed heparinase was added, and the reaction was carried out at 200-300 rpm and 35-37°C for 6-8 hours. The temperature was raised to 80-85°C and maintained for 10-15 minutes. The solution was quickly cooled to 2-4°C in an ice bath and finally subjected to magnetic separation to collect supernatant A and precipitate.

[0017] Preferably, in S1.3, the specific steps for preparing supernatant B are:

[0018] The precipitate was resuspended in phosphate buffer to obtain a 1-3% (w / v) precipitate resuspended solution, the pH of the solution was adjusted to 9.0-9.5, and the solution was stirred at 200-300 rpm for 10-15 min. The protective agent was removed by magnetic separation, and the supernatant B was set aside.

[0019] Preferably, in S1.4, the amount of heparinase III is 3–5 U / mg heparin sodium;

[0020] The specific preparation steps of the retentate are:

[0021] The pH of supernatant B was adjusted to 6.8-7.2, and heparinase III was added to the supernatant. The reaction was continued at 35-37°C for 3-4 hours, and the supernatant was inactivated at 80-85°C for 10-15 minutes to obtain a secondary enzymatic hydrolysate. The supernatant A and the secondary enzymatic hydrolysate were combined, pre-filtered with a 0.22 μm filter membrane, and then centrifuged at 10,000-12,000 rpm for 20-30 minutes using an ultrafiltration membrane with a molecular weight cutoff of 3-5 kDa. The dialysate was discarded and the retentate was used for standby use.

[0022] Preferably, in S1.5, the amount of anhydrous ethanol added is 3-4 times the volume of the retentate;

[0023] The specific steps of post-treatment of the retentate are:

[0024] Anhydrous ethanol was added dropwise to the retentate, and the mixture was allowed to stand at 2-4°C overnight. The mixture was centrifuged at 10,000-12,000 rpm for 10-15 minutes, and the precipitate was collected. The precipitate was resuspended in deionized water and freeze-dried to obtain low molecular weight heparin sodium.

[0025] Preferably, the steps of preparing the protective agent are specifically as follows:

[0026] S2.1. Ultrasonic dispersion of superparamagnetic Fe3O4 nanoparticles in water, addition of citric acid, adjustment of pH to 4-6, heating to 60-80°C with stirring for 2-4 h, magnetic separation, washing with deionized water and ethanol, and vacuum drying to obtain carboxylated Fe3O4 nanoparticles.

[0027] S2.2. Disperse the carboxylated Fe3O4 nanoparticles in phosphate buffer, sonicate for 20-30 min, magnetically separate, discard the supernatant, and redisperse in phosphate buffer. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and stir at room temperature for 50-60 min to obtain an activated Fe3O4 suspension.

[0028] S2.3. Disperse the aminocalixarene in phosphate buffer, add it to the activated Fe3O4 suspension, protect it with nitrogen, stir it in the dark at room temperature for 3-4 hours, stir it magnetically at a speed of 300-400 rpm, and control the pH to 7.0-7.4. After the reaction is completed, add ethanolamine at a concentration of 0.1-0.5% v / v, continue stirring for 1-1.5 hours, separate it with a magnet, wash it with phosphate buffer and deionized water in turn, and dry it in vacuum to obtain the protective agent.

[0029] Preferably, in S2.1, the molar ratio of superparamagnetic Fe3O4 nanoparticles to citric acid is 1:3-5.

[0030] Preferably, in S2.2, the mass ratio of carboxylated Fe3O4 nanoparticles and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:0.17-1.4, and the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 1:1.

[0031] Preferably, in S2.3, the molar ratio of carboxylated Fe3O4 nanoparticles to aminocalixarene is 1:3.5-4.5.

[0032] Aminated calixarene binds to the highly sulfated region of heparin sodium through electrostatic interaction. At the same time, the cavity structure of calixarene can include the hydrophobic skeleton of heparin sodium through hydrophobic interaction or van der Waals force, avoiding excessive enzymatic hydrolysis and temporarily "hiding" the steric hindrance of the sulfated group. Since the charge density of the highly sulfated region is higher, the aminated calixarene can selectively shield the sulfated region. During enzymatic hydrolysis, the low sulfated region of heparin sodium is not shielded, and heparinase can quickly recognize and cut its glycosidic bond. Then, by increasing the pH (7-9), the amino group is deprotonated, the electrostatic interaction is weakened, and the highly sulfated fragment is released. At this time, the highly sulfated fragment is enzymatically hydrolyzed again to effectively obtain enzymatic hydrolysis products with uniform molecular weight.

[0033] However, the size of the enzymatic hydrolysis product is similar to that of calixarene, and traditional centrifugation / filtration is difficult to separate efficiently. Therefore, calixarene is covalently coupled with superparamagnetic Fe3O4 nanoparticles, and a magnetic field is used to achieve rapid separation. Superparamagnetic Fe3O4 nanoparticles are modified with citric acid to introduce carboxyl groups, and the superparamagnetic Fe3O4 nanoparticles are covalently coupled to calixarene. At the same time, ethanolamine is added to convert the residual carboxyl groups into neutral groups to eliminate negative charge interference.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] In the enzymatic hydrolysis preparation method of low-molecular-weight heparin sodium, a composite protective agent comprising citric acid-modified superparamagnetic Fe3O4 nanoparticles covalently coupled with aminocalixarene is used. The electrostatic effect of the aminocalixarene in the protective agent is utilized to preferentially bind to the negatively charged region of the highly sulfated fragment of the heparin sodium. The cavity structure of the protective agent coats the heparin hydrophobic backbone through van der Waals forces to form a steric hindrance protective layer, enabling heparinase to preferentially cleave the low-sulfated region. After the highly sulfated fragment is released through pH control, a product with a narrower molecular weight distribution is obtained through secondary enzymatic hydrolysis. Simultaneously, the magnetic core of the superparamagnetic Fe3O4 nanoparticles is utilized to achieve rapid magnetic separation of the protective agent after enzymatic hydrolysis, thereby achieving precise control of the enzymatic hydrolysis process and facilitating separation, thereby obtaining a product with a uniform molecular weight distribution. DETAILED DESCRIPTION

[0036] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0037] The enzymatic hydrolysis preparation method of low molecular weight heparin sodium of the present invention:

[0038] S1.1. Pretreating heparin sodium and combining it with a protective agent to obtain a heparin complex precipitate;

[0039] S1.2, performing a first enzymatic hydrolysis on the heparin complex precipitate to obtain supernatant A and precipitate;

[0040] S1.3. Adjust the pH of the precipitate to release the highly sulfated fragments, obtaining supernatant B;

[0041] S1.4. Perform a secondary enzymatic hydrolysis on supernatant B to obtain a secondary enzymatic hydrolyzate. Combine supernatant A and the secondary enzymatic hydrolyzate and filter to obtain a retentate.

[0042] S1.5, post-processing the retentate to obtain low molecular weight heparin sodium;

[0043] Among them, the protective agent is prepared by surface-modifying superparamagnetic Fe3O4 nanoparticles with citric acid and then covalently coupling them with amino-calixarene.

[0044] Example 1: A method for preparing low molecular weight heparin sodium by enzymatic hydrolysis, comprising the following steps:

[0045] Weigh 50 parts by weight of heparin sodium (molecular weight 15 kDa) and 9 parts by weight of a protective agent;

[0046] The protective agent was prepared by surface-modifying superparamagnetic Fe3O4 nanoparticles with citric acid and then covalently coupling them with aminocalixarene. The molar ratio of superparamagnetic Fe3O4 nanoparticles to citric acid was 1:4. The mass ratio of carboxylated Fe3O4 nanoparticles to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was 1:1.0, and the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide was 1:1. The molar ratio of carboxylated Fe3O4 nanoparticles to aminocalixarene was 1:3.5.

[0047] S2.1. Ultrasonic dispersion of superparamagnetic Fe3O4 nanoparticles in water, addition of citric acid, adjustment of pH to 6, heating to 60°C with stirring for 4 h, magnetic separation, washing with deionized water and ethanol, and vacuum drying to obtain carboxylated Fe3O4 nanoparticles.

[0048] S2.2. Disperse the carboxylated Fe3O4 nanoparticles in phosphate buffer, sonicate for 30 min, magnetically separate, discard the supernatant, and redisperse in phosphate buffer. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and stir at room temperature for 60 min to obtain an activated Fe3O4 suspension.

[0049] S2.3. Disperse the aminocalixarene in phosphate buffer, add it to the activated Fe3O4 suspension, purge with nitrogen, and stir at room temperature in the dark for 4 h with magnetic stirring at 300 rpm. Control the pH at 7.2. After the reaction, add 0.5% v / v ethanolamine and continue stirring for 1 h. After separation with a magnet, wash with phosphate buffer and deionized water, and dry in vacuo to obtain the protective agent.

[0050] S1.1. Dissolve heparin sodium in phosphate buffer (pH 7.0) to obtain a 5% (w / v) heparin sodium solution. Add 0.1% (w / v) activated carbon, stir at 40°C for 15 minutes, and filter through a 0.22 μm membrane to obtain a clear solution. Then, add a protective agent, control the temperature to 25°C, stir at 300 rpm for 60 minutes, and perform magnetic separation for 15 minutes. Discard the supernatant to obtain a heparin complex precipitate.

[0051] S1.2. Redisperse the heparin complex precipitate in phosphate buffer to obtain a 3% (w / v) heparin complex precipitate solution, add mixed heparinase, wherein the mixed heparinase is heparinase I:II = 2:1 U / mg heparin sodium; the total enzyme amount is 5 U / mg heparin sodium. Incubate at 37°C at 300 rpm for 8 hours, then raise the temperature to 85°C and maintain for 15 minutes. Cool rapidly to 4°C in an ice bath, and finally perform magnetic separation to collect supernatant A and precipitate.

[0052] S1.3. Resuspend the pellet in phosphate buffer to obtain a 3% (w / v) pellet suspension. Adjust the pH of the solution to 9.5 and stir at 200 rpm for 10 min. Remove the protective agent by magnetic separation and reserve the supernatant B for later use.

[0053] S1.4. Adjust the pH of supernatant B to 6.8, add heparinase III to the supernatant at a concentration of 3 U / mg heparin sodium, continue the reaction at 37°C for 4 h, and inactivate at 85°C for 15 min to obtain a secondary enzymatic hydrolyzate. Combine supernatant A and the secondary enzymatic hydrolyzate, pre-filter through a 0.22 μm filter membrane, and centrifuge at 10,000 rpm for 30 min using ultrafiltration membranes with molecular weight cutoffs of 3 and 5 kDa. Discard the dialysate and reserve the retentate for later use.

[0054] S1.5. Add anhydrous ethanol dropwise to the retentate, wherein the amount of anhydrous ethanol added is 3 times the volume of the retentate. Let it stand at 4°C overnight, centrifuge at 10,000 rpm for 15 minutes, collect the precipitate, resuspend the precipitate in deionized water, and lyophilize to obtain low molecular weight heparin sodium.

[0055] Example 2: A method for preparing low molecular weight heparin sodium by enzymatic hydrolysis, comprising the following steps: weighing 50 parts by weight of heparin sodium (molecular weight 15 kDa) and 13 parts by weight of a protective agent;

[0056] The protective agent was prepared by surface-modifying superparamagnetic Fe3O4 nanoparticles with citric acid and then covalently coupling them with aminocalixarene. The molar ratio of superparamagnetic Fe3O4 nanoparticles to citric acid was 1:4. The mass ratio of carboxylated Fe3O4 nanoparticles to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was 1:1.0, and the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide was 1:1. The molar ratio of carboxylated Fe3O4 nanoparticles to aminocalixarene was 1:3.5.

[0057] S2.1. Ultrasonic dispersion of superparamagnetic Fe3O4 nanoparticles in water, addition of citric acid, adjustment of pH to 6, heating to 60°C with stirring for 4 h, magnetic separation, washing with deionized water and ethanol, and vacuum drying to obtain carboxylated Fe3O4 nanoparticles.

[0058] S2.2. Disperse the carboxylated Fe3O4 nanoparticles in phosphate buffer, sonicate for 30 min, magnetically separate, discard the supernatant, and redisperse in phosphate buffer. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and stir at room temperature for 60 min to obtain an activated Fe3O4 suspension.

[0059] S2.3. Disperse the aminocalixarene in phosphate buffer, add it to the activated Fe3O4 suspension, purge with nitrogen, and stir at room temperature in the dark for 4 h with magnetic stirring at 300 rpm. Control the pH at 7.2. After the reaction, add 0.5% v / v ethanolamine and continue stirring for 1 h. After separation with a magnet, wash with phosphate buffer and deionized water, and dry in vacuo to obtain the protective agent.

[0060] S1.1. Dissolve heparin sodium in phosphate buffer (pH 7.0) to obtain a 5% (w / v) heparin sodium solution. Add 0.1% (w / v) activated carbon, stir at 40°C for 15 minutes, and filter through a 0.22 μm membrane to obtain a clear solution. Then, add a protective agent, control the temperature to 25°C, stir at 300 rpm for 60 minutes, and perform magnetic separation for 15 minutes. Discard the supernatant to obtain a heparin complex precipitate.

[0061] S1.2. Redisperse the heparin complex precipitate in phosphate buffer to obtain a 3% (w / v) heparin complex precipitate solution, add mixed heparinase, wherein the mixed heparinase is heparinase I:II = 2:1 U / mg heparin sodium; the total enzyme amount is 5 U / mg heparin sodium. Incubate at 37°C at 300 rpm for 8 hours, then raise the temperature to 85°C and maintain for 15 minutes. Cool rapidly to 4°C in an ice bath, and finally perform magnetic separation to collect supernatant A and precipitate.

[0062] S1.3. Resuspend the pellet in phosphate buffer to obtain a 3% (w / v) pellet suspension. Adjust the pH of the solution to 9.5 and stir at 200 rpm for 10 min. Remove the protective agent by magnetic separation and reserve the supernatant B for later use.

[0063] S1.4. Adjust the pH of supernatant B to 6.8, add heparinase III to the supernatant at a concentration of 3 U / mg heparin sodium, continue the reaction at 37°C for 4 h, and inactivate at 85°C for 15 min to obtain a secondary enzymatic hydrolyzate. Combine supernatant A and the secondary enzymatic hydrolyzate, pre-filter through a 0.22 μm filter membrane, and centrifuge at 10,000 rpm for 30 min using ultrafiltration membranes with molecular weight cutoffs of 3 and 5 kDa. Discard the dialysate and reserve the retentate for later use.

[0064] S1.5. Add anhydrous ethanol dropwise to the retentate, wherein the amount of anhydrous ethanol added is 3 times the volume of the retentate. Let it stand at 4°C overnight, centrifuge at 10,000 rpm for 15 minutes, collect the precipitate, resuspend the precipitate in deionized water, and lyophilize to obtain low molecular weight heparin sodium.

[0065] Example 3: A method for preparing low molecular weight heparin sodium by enzymatic hydrolysis, comprising the following steps:

[0066] Weigh 50 parts by weight of heparin sodium (molecular weight 15 kDa) and 15 parts by weight of a protective agent;

[0067] The protective agent was prepared by surface-modifying superparamagnetic Fe3O4 nanoparticles with citric acid and then covalently coupling them with aminocalixarene. The molar ratio of superparamagnetic Fe3O4 nanoparticles to citric acid was 1:4. The mass ratio of carboxylated Fe3O4 nanoparticles to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was 1:1.0, and the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide was 1:1. The molar ratio of carboxylated Fe3O4 nanoparticles to aminocalixarene was 1:3.5.

[0068] S2.1. Ultrasonic dispersion of superparamagnetic Fe3O4 nanoparticles in water, addition of citric acid, adjustment of pH to 6, heating to 60°C with stirring for 4 h, magnetic separation, washing with deionized water and ethanol, and vacuum drying to obtain carboxylated Fe3O4 nanoparticles.

[0069] S2.2. Disperse the carboxylated Fe3O4 nanoparticles in phosphate buffer, sonicate for 30 min, magnetically separate, discard the supernatant, and redisperse in phosphate buffer. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and stir at room temperature for 60 min to obtain an activated Fe3O4 suspension.

[0070] S2.3. Disperse the aminocalixarene in phosphate buffer, add it to the activated Fe3O4 suspension, purge with nitrogen, and stir at room temperature in the dark for 4 h with magnetic stirring at 300 rpm. Control the pH at 7.2. After the reaction, add 0.5% v / v ethanolamine and continue stirring for 1 h. After separation with a magnet, wash with phosphate buffer and deionized water, and dry in vacuo to obtain the protective agent.

[0071] S1.1. Dissolve heparin sodium in phosphate buffer (pH 7.0) to obtain a 5% (w / v) heparin sodium solution. Add 0.1% (w / v) activated carbon, stir at 40°C for 15 minutes, and filter through a 0.22 μm membrane to obtain a clear solution. Then, add a protective agent, control the temperature to 25°C, stir at 300 rpm for 60 minutes, and perform magnetic separation for 15 minutes. Discard the supernatant to obtain a heparin complex precipitate.

[0072] S1.2. Redisperse the heparin complex precipitate in phosphate buffer to obtain a 3% (w / v) heparin complex precipitate solution, add mixed heparinase, wherein the mixed heparinase is heparinase I:II = 2:1 U / mg heparin sodium; the total enzyme amount is 5 U / mg heparin sodium. Incubate at 37°C at 300 rpm for 8 hours, then raise the temperature to 85°C and maintain for 15 minutes. Cool rapidly to 4°C in an ice bath, and finally perform magnetic separation to collect supernatant A and precipitate.

[0073] S1.3. Resuspend the pellet in phosphate buffer to obtain a 3% (w / v) pellet suspension. Adjust the pH of the solution to 9.5 and stir at 200 rpm for 10 min. Remove the protective agent by magnetic separation and reserve the supernatant B for later use.

[0074] S1.4. Adjust the pH of supernatant B to 6.8, add heparinase III to the supernatant at a concentration of 3 U / mg heparin sodium, continue the reaction at 37°C for 4 h, and inactivate at 85°C for 15 min to obtain a secondary enzymatic hydrolyzate. Combine supernatant A and the secondary enzymatic hydrolyzate, pre-filter through a 0.22 μm filter membrane, and centrifuge at 10,000 rpm for 30 min using ultrafiltration membranes with molecular weight cutoffs of 3 and 5 kDa. Discard the dialysate and reserve the retentate for later use.

[0075] S1.5. Add anhydrous ethanol dropwise to the retentate, wherein the amount of anhydrous ethanol added is 3 times the volume of the retentate. Let it stand at 4°C overnight, centrifuge at 10,000 rpm for 15 minutes, collect the precipitate, resuspend the precipitate in deionized water, and lyophilize to obtain low molecular weight heparin sodium.

[0076] Example 4: A method for preparing low molecular weight heparin sodium by enzymatic hydrolysis, comprising the following steps:

[0077] Weigh 50 parts by weight of heparin sodium (molecular weight 15 kDa) and 15 parts by weight of a protective agent;

[0078] The protective agent was prepared by surface-modifying superparamagnetic Fe3O4 nanoparticles with citric acid and then covalently coupling them with aminocalixarene. The molar ratio of superparamagnetic Fe3O4 nanoparticles to citric acid was 1:4. The mass ratio of carboxylated Fe3O4 nanoparticles to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was 1:1.0, and the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide was 1:1. The molar ratio of carboxylated Fe3O4 nanoparticles to aminocalixarene was 1:4.0.

[0079] S2.1. Ultrasonic dispersion of superparamagnetic Fe3O4 nanoparticles in water, addition of citric acid, adjustment of pH to 6, heating to 60°C with stirring for 4 h, magnetic separation, washing with deionized water and ethanol, and vacuum drying to obtain carboxylated Fe3O4 nanoparticles.

[0080] S2.2. Disperse the carboxylated Fe3O4 nanoparticles in phosphate buffer, sonicate for 30 min, magnetically separate, discard the supernatant, and redisperse in phosphate buffer. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and stir at room temperature for 60 min to obtain an activated Fe3O4 suspension.

[0081] S2.3. Disperse the aminocalixarene in phosphate buffer, add it to the activated Fe3O4 suspension, purge with nitrogen, and stir at room temperature in the dark for 4 h with magnetic stirring at 300 rpm. Control the pH at 7.2. After the reaction, add 0.5% v / v ethanolamine and continue stirring for 1 h. After separation with a magnet, wash with phosphate buffer and deionized water, and dry in vacuo to obtain the protective agent.

[0082] S1.1. Dissolve heparin sodium in phosphate buffer (pH 7.0) to obtain a 5% (w / v) heparin sodium solution. Add 0.1% (w / v) activated carbon, stir at 40°C for 15 minutes, and filter through a 0.22 μm membrane to obtain a clear solution. Then, add a protective agent, control the temperature to 25°C, stir at 300 rpm for 60 minutes, and perform magnetic separation for 15 minutes. Discard the supernatant to obtain a heparin complex precipitate.

[0083] S1.2. Redisperse the heparin complex precipitate in phosphate buffer to obtain a 3% (w / v) heparin complex precipitate solution, add mixed heparinase, wherein the mixed heparinase is heparinase I:II = 2:1 U / mg heparin sodium; the total enzyme amount is 5 U / mg heparin sodium. Incubate at 37°C at 300 rpm for 8 hours, then raise the temperature to 85°C and maintain for 15 minutes. Cool rapidly to 4°C in an ice bath, and finally perform magnetic separation to collect supernatant A and precipitate.

[0084] S1.3. Resuspend the pellet in phosphate buffer to obtain a 3% (w / v) pellet suspension. Adjust the pH of the solution to 9.5 and stir at 200 rpm for 10 min. Remove the protective agent by magnetic separation and reserve the supernatant B for later use.

[0085] S1.4. Adjust the pH of supernatant B to 6.8, add heparinase III to the supernatant at a concentration of 3 U / mg heparin sodium, continue the reaction at 37°C for 4 h, and inactivate at 85°C for 15 min to obtain a secondary enzymatic hydrolyzate. Combine supernatant A and the secondary enzymatic hydrolyzate, pre-filter through a 0.22 μm filter membrane, and centrifuge at 10,000 rpm for 30 min using ultrafiltration membranes with molecular weight cutoffs of 3 and 5 kDa. Discard the dialysate and reserve the retentate for later use.

[0086] S1.5. Add anhydrous ethanol dropwise to the retentate, wherein the amount of anhydrous ethanol added is 3 times the volume of the retentate. Let it stand at 4°C overnight, centrifuge at 10,000 rpm for 15 minutes, collect the precipitate, resuspend the precipitate in deionized water, and lyophilize to obtain low molecular weight heparin sodium.

[0087] Example 5: A method for preparing low molecular weight heparin sodium by enzymatic hydrolysis, comprising the following steps:

[0088] Weigh 50 parts by weight of heparin sodium (molecular weight 15 kDa) and 15 parts by weight of a protective agent;

[0089] The protective agent was prepared by surface-modifying superparamagnetic Fe3O4 nanoparticles with citric acid and then covalently coupling them with aminocalixarene. The molar ratio of superparamagnetic Fe3O4 nanoparticles to citric acid was 1:4. The mass ratio of carboxylated Fe3O4 nanoparticles to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was 1:1.0, and the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide was 1:1. The molar ratio of carboxylated Fe3O4 nanoparticles to aminocalixarene was 1:4.5.

[0090] S2.1. Ultrasonic dispersion of superparamagnetic Fe3O4 nanoparticles in water, addition of citric acid, adjustment of pH to 6, heating to 60°C with stirring for 4 h, magnetic separation, washing with deionized water and ethanol, and vacuum drying to obtain carboxylated Fe3O4 nanoparticles.

[0091] S2.2. Disperse the carboxylated Fe3O4 nanoparticles in phosphate buffer, sonicate for 30 min, magnetically separate, discard the supernatant, and redisperse in phosphate buffer. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and stir at room temperature for 60 min to obtain an activated Fe3O4 suspension.

[0092] S2.3. Disperse the aminocalixarene in phosphate buffer, add it to the activated Fe3O4 suspension, purge with nitrogen, and stir at room temperature in the dark for 4 h with magnetic stirring at 300 rpm. Control the pH at 7.2. After the reaction, add 0.5% v / v ethanolamine and continue stirring for 1 h. After separation with a magnet, wash with phosphate buffer and deionized water, and dry in vacuo to obtain the protective agent.

[0093] S1.1. Dissolve heparin sodium in phosphate buffer (pH 7.0) to obtain a 5% (w / v) heparin sodium solution. Add 0.1% (w / v) activated carbon, stir at 40°C for 15 minutes, and filter through a 0.22 μm membrane to obtain a clear solution. Then, add a protective agent, control the temperature to 25°C, stir at 300 rpm for 60 minutes, and perform magnetic separation for 15 minutes. Discard the supernatant to obtain a heparin complex precipitate.

[0094] S1.2. Redisperse the heparin complex precipitate in phosphate buffer to obtain a 3% (w / v) heparin complex precipitate solution, add mixed heparinase, wherein the mixed heparinase is heparinase I:II = 2:1 U / mg heparin sodium; the total enzyme amount is 5 U / mg heparin sodium. Incubate at 37°C at 300 rpm for 8 hours, then raise the temperature to 85°C and maintain for 15 minutes. Cool rapidly to 4°C in an ice bath, and finally perform magnetic separation to collect supernatant A and precipitate.

[0095] S1.3. Resuspend the pellet in phosphate buffer to obtain a 3% (w / v) pellet suspension. Adjust the pH of the solution to 9.5 and stir at 200 rpm for 10 min. Remove the protective agent by magnetic separation and reserve the supernatant B for later use.

[0096] S1.4. Adjust the pH of supernatant B to 6.8, add heparinase III to the supernatant at a concentration of 3 U / mg heparin sodium, continue the reaction at 37°C for 4 h, and inactivate at 85°C for 15 min to obtain a secondary enzymatic hydrolyzate. Combine supernatant A and the secondary enzymatic hydrolyzate, pre-filter through a 0.22 μm filter membrane, and centrifuge at 10,000 rpm for 30 min using ultrafiltration membranes with molecular weight cutoffs of 3 and 5 kDa. Discard the dialysate and reserve the retentate for later use.

[0097] S1.5. Add anhydrous ethanol dropwise to the retentate, wherein the amount of anhydrous ethanol added is 3 times the volume of the retentate. Let it stand at 4°C overnight, centrifuge at 10,000 rpm for 15 minutes, collect the precipitate, resuspend the precipitate in deionized water, and lyophilize to obtain low molecular weight heparin sodium.

[0098] Comparative Example 1: The method of Example 5 was adopted without adding a protective agent.

[0099] Comparative Example 2: The method of Example 5 was adopted, and the aminocalixarene was directly used to modify the aminocalixarene without using carboxylated Fe3O4 nanoparticles.

[0100] The present invention uses a protective agent to prepare low molecular weight heparin sodium, wherein the performance index test items and test standards of the low molecular weight heparin sodium are as follows:

[0101] The yield of heparin sodium was tested according to the detection method and detection indicators for the yield of heparin sodium in the "Chinese Pharmacopoeia 2015". The substances before and after the reaction were weighed, and the yield was calculated, that is, yield = freeze-dried weight / initial heparin sodium input amount × 100%. The higher the yield, the greater the target molecular weight, which indirectly indicates that heparinase can effectively and specifically cut and the enzymatic hydrolysis efficiency is improved.

[0102] The secondary enzymatic hydrolysate and supernatant A of the experimental process were combined to obtain 10 mL, and 30%, 50%, and 70% ethanol (v / v) were added in sequence. After standing for 1 hour each time, the mixture was centrifuged (10,000 rpm, 10 min). The components were freeze-dried and weighed. According to the low molecular weight component (3-5 kDa): 30% ethanol precipitation; medium molecular weight component (5-8 kDa): 50% ethanol precipitation; high molecular weight component (>8 kDa): 70% ethanol precipitation, the product molecular weight concentration was evaluated.

[0103] The purity of the product low molecular weight heparin sodium was evaluated by measuring the absorbance at 260 nm and 280 nm using a UV-visible spectrophotometer. The smaller the absorbance value, the higher the purity of the product.

[0104] The low molecular weight heparin sodium prepared in Examples 1-5 and Comparative Examples 1-2 was tested using the above standards, and the obtained data are shown in Table 1:

[0105] Table 1 Performance data of Examples 1-5 and Comparative Examples 1-2

[0106]

[0107] The above data fully demonstrate that Examples 1-5, compared with Comparative Examples 1-2, can fully show the effect of the protective agent on the molecular weight distribution, yield and purity of the enzymatic hydrolysis preparation method of low molecular weight heparin sodium.

[0108] Since the present invention adopts a protective agent to prepare a method for preparing low molecular weight heparin sodium by enzymatic hydrolysis, the protective agent effectively improves the molecular weight distribution, yield and purity of the low molecular weight heparin sodium, as follows:

[0109] It can be seen from Examples 1-3 that as the content of the protective agent continues to increase, the molecular weight distribution, yield and purity of the enzymatically hydrolyzed low molecular weight heparin sodium continue to change. The amino-calixarene in the protective agent preferentially binds to the highly sulfated regions of the heparin sodium through electrostatic interaction. Therefore, when the amount of the protective agent increases, more highly sulfated sites are shielded, reducing the nonspecific binding of the enzyme to these regions, forcing the heparinase to preferentially cut the low sulfated regions. At the same time, the cavity structure of the calixarene coats the heparin skeleton through hydrophobic interaction, forming a physical barrier to prevent the enzyme from randomly cutting the highly sulfated regions, thereby further inhibiting excessive degradation.

[0110] It can be seen from Examples 3-5 that as the molar ratio of carboxylated Fe3O4 nanoparticles to amino-calixarene continues to increase, the molecular weight distribution, yield and purity of the enzymatically hydrolyzed low molecular weight heparin sodium continue to change, which means that the number of calixarene molecules coupled to the surface of a unit Fe3O4 nanoparticle increases, forming a denser electrostatic binding site, enhancing the shielding ability for highly sulfated areas, and indirectly reducing the negative charge on the surface of the Fe3O4 nanoparticles, electrostatically repelling heparinase, thereby significantly improving the enzymatic hydrolysis selectivity and product uniformity.

[0111] According to the above test experiments, the enzymatic hydrolysis preparation method of a low molecular weight heparin sodium prepared according to Example 5 has the best performance, so Example 5 is regarded as the best example;

[0112] By comparing Example 5 with Comparative Examples 1-2, it can be seen that:

[0113] In Comparative Example 1, no protective agent was added, and the molecular weight distribution, yield and purity of the low molecular weight heparin sodium hydrolyzed by enzymatic hydrolysis were poor. Since heparinase could not distinguish between high / low sulfated regions, it randomly cut glycosidic bonds, resulting in a diffuse molecular weight distribution. The high sulfated region had a large steric hindrance and a slow enzymatic hydrolysis rate, so it was easy to generate large molecular fragments, resulting in a loss of enzymatic hydrolysis selectivity, a wide product distribution, many impurities, and a low yield.

[0114] In Comparative Example 2, amino-calixarene was directly used without modifying the amino-calixarene through carboxylated Fe3O4 nanoparticles. The molecular weight distribution, yield and purity of the enzymatically hydrolyzed low molecular weight heparin sodium were poor. The calixarene without modified Fe3O4 nanoparticles could not be quickly recovered by the magnetic field. The residual protective agent was mixed with the product, requiring an additional purification step, which increased the product loss.

[0115] In summary, a composite protective agent was prepared by covalently coupling citric acid-modified superparamagnetic Fe3O4 nanoparticles with aminocalixarene. The aminocalixarene preferentially binds to the highly sulfated regions of heparin sodium through electrostatic interaction, shielding its steric hindrance and negative charge, forcing heparinase to preferentially cleave the glycosidic bonds in the low-sulfated regions, reducing random degradation. The hydrophobic cavity of the calixarene coats the heparin skeleton through van der Waals forces, avoiding nonspecific cleavage of the highly sulfated regions by the enzyme. At the same time, by pH regulation, the shielded fragments are released for secondary enzymatic hydrolysis, achieving step-by-step controllable degradation. Finally, the superparamagnetism of the Fe3O4 nanoparticles enables rapid separation of the protective agent and the product. Combined with the capping of unreacted carboxyl groups with ethanolamine, charge interference is eliminated, effectively solving the problems of uneven molecular weight, low yield, and complex purification in the traditional process.

[0116] The basic principles, main features, and advantages of the present invention are shown and described above. It should be understood by those skilled in the art that the present invention is not limited to the above-described embodiments. The above-described embodiments and descriptions are merely preferred examples of the present invention and are not intended to limit the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention claimed.

Claims

1. A method for preparing heparin sodium by enzymatic hydrolysis, characterized in that: S1.

1. Pretreating heparin sodium and combining it with a protective agent to obtain a heparin complex precipitate; S1.2, performing a first enzymatic hydrolysis on the heparin complex precipitate to obtain supernatant A and precipitate; S1.

3. Adjust the pH of the precipitate to release the highly sulfated fragments, obtaining supernatant B; S1.

4. Perform a secondary enzymatic hydrolysis on supernatant B to obtain a secondary enzymatic hydrolyzate. Combine supernatant A and the secondary enzymatic hydrolyzate and filter to obtain a retentate. S1.5, post-processing the retentate to obtain low molecular weight heparin sodium; The protective agent is prepared by surface-modifying superparamagnetic Fe3O4 nanoparticles with citric acid and then covalently coupling them with aminocalixarene. The steps of preparing the protective agent are specifically as follows: S2.

1. Ultrasonic dispersion of superparamagnetic Fe3O4 nanoparticles in water, addition of citric acid, adjustment of pH to 4-6, heating to 60-80°C with stirring for 2-4 h, magnetic separation, washing with deionized water and ethanol, and vacuum drying to obtain carboxylated Fe3O4 nanoparticles. S2.

2. Disperse the carboxylated Fe3O4 nanoparticles in phosphate buffer, sonicate for 20-30 min, magnetically separate, discard the supernatant, and redisperse in phosphate buffer. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and stir at room temperature for 50-60 min to obtain an activated Fe3O4 suspension. S2.

3. Disperse the aminocalixarene in phosphate buffer, add the solution to the activated Fe3O4 suspension, purge with nitrogen, and react in the dark at room temperature with stirring for 3-4 hours. Stir magnetically at 300-400 rpm and control the pH to 7.0-7.

4. After the reaction, add 0.1-0.5% v / v ethanolamine and continue stirring for 1-1.5 hours. After separation with a magnet, wash with phosphate buffer and deionized water, and dry in vacuo to obtain the protective agent. The specific steps of the first enzymatic hydrolysis of the heparin complex precipitation are: The heparin complex precipitate is redispersed in phosphate buffer to obtain a 2-5% (w / v) heparin complex precipitate solution, mixed heparinase is added, and the mixture is reacted at 200-300 rpm, 35-37°C for 6-8 hours, then the temperature is raised to 80-85°C and maintained for 10-15 minutes, and then rapidly cooled to 2-4°C in an ice bath. Finally, the mixture is magnetically separated, and the supernatant A and the precipitate are collected; wherein the mixed heparinase is heparinase I:II = 2:1 U / mg heparin sodium; and the total enzyme amount is 5-10 U / mg heparin sodium; In S1.3, the specific steps for preparing supernatant B are: Resuspend the precipitate in phosphate buffer to obtain a 1-3% (w / v) precipitate resuspended solution, adjust the solution pH to 9.0-9.5, stir at 200-300 rpm for 10-15 min, remove the protective agent by magnetic separation, and reserve the supernatant B for later use; The specific preparation steps of the retentate are: The pH of supernatant B was adjusted to 6.8-7.2, and heparinase III was added to the supernatant. The reaction was continued at 35-37°C for 3-4 hours, and the solution was inactivated at 80-85°C for 10-15 minutes to obtain a secondary enzymatic hydrolysate. The supernatant A and the secondary enzymatic hydrolysate were combined, pre-filtered with a 0.22 μm filter membrane, and then centrifuged at 10,000-12,000 rpm for 20-30 minutes using an ultrafiltration membrane with a molecular weight cutoff of 3-5 kDa. The dialysate was discarded and the retentate was used for later use; the amount of heparinase III was 3-5 U / mg heparin sodium.

2. The method for preparing heparin sodium by enzymatic hydrolysis according to claim 1, wherein: The specific preparation steps of the heparin complex precipitation are: Sodium heparin is dissolved in phosphate buffer to obtain a 3-5% (w / v) sodium heparin solution, activated carbon is added, and the solution is stirred at 40-45°C for 10-15 minutes. The solution is filtered through a 0.22 μm membrane to obtain a clear solution. A protective agent is then added, the temperature is controlled at 25-30°C, and the solution is stirred at 200-300 rpm for 50-60 minutes. The solution is then magnetically separated for 10-15 minutes, and the supernatant is discarded to obtain a heparin complex precipitate. The heparin sodium is 30-50 parts by weight, the protective agent is 9-15 parts by weight, the molecular weight of the heparin sodium is 15-18 kDa, the pH of the phosphate buffer is 7.0-7.2, and the amount of activated carbon added is 0.1-0.5% (w / v).

3. The enzymatic hydrolysis preparation method of heparin sodium according to claim 1, characterized in that: The specific steps of the retentate post-treatment are: Anhydrous ethanol was added dropwise to the retentate, and the mixture was allowed to stand overnight at 2-4°C. The mixture was centrifuged at 10,000-12,000 rpm for 10-15 minutes, and the precipitate was collected. The precipitate was resuspended in deionized water and freeze-dried to obtain low molecular weight heparin sodium. The amount of anhydrous ethanol added was 3-4 times the volume of the retentate.

4. The method for preparing heparin sodium by enzymatic hydrolysis according to claim 1, wherein: In the above-mentioned S2.1, the molar ratio of superparamagnetic Fe3O4 nanoparticles to citric acid is 1:3-5.

5. The method for preparing heparin sodium by enzymatic hydrolysis according to claim 1, wherein: In the S2.2, the mass ratio of carboxylated Fe3O4 nanoparticles to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:0.17-1.4, and the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:

1.

6. The method for preparing heparin sodium by enzymatic hydrolysis according to claim 1, wherein: In the above-mentioned S2.3, the molar ratio of carboxylated Fe3O4 nanoparticles to aminocalixarene is 1:3.5-4.5.

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