Enzymolysis preparation method of low-molecular-weight heparin sodium

Abstract

The invention relates to the technical field of enzymolysis, in particular to an enzymolysis preparation method of low-molecular-weight heparin sodium, which comprises the following steps: S1.1, pretreating heparin sodium, and combining the pretreated heparin sodium with a protective agent to obtain a heparin compound precipitate; s1.2, performing enzymolysis on the heparin compound precipitate for the first time to obtain a supernatant A and a precipitate; s1.3, adjusting the pH value of the precipitate to release a highly sulfated fragment, so as to obtain a supernatant B; s1.4, performing secondary enzymolysis on the supernate B to obtain secondary enzymatic hydrolysate, combining the supernate A and the secondary enzymatic hydrolysate, and filtering to obtain trapped fluid; and S1.5, carrying out post-treatment on the trapped fluid to obtain the low-molecular-weight heparin sodium. A composite protective agent formed by covalent coupling of citric acid modified superparamagnetic Fe3O4 nanoparticles and aminated calixarene is adopted, the enzymolysis path is accurately regulated and controlled, efficient separation is achieved, and the problems of uneven molecular weight, low yield and complex purification in a traditional process are solved.

Description

Technical Field

[0001] The invention relates to the technical field of enzymolysis, in particular to an enzymolysis preparation method of low molecular weight heparin sodium. Background Art

[0002] Low molecular weight heparin sodium is a type of polysaccharide anticoagulant drug that is degraded from ordinary heparin by chemical or enzymatic methods. Compared with ordinary heparin, low molecular weight heparin sodium has more stable pharmacokinetic properties, longer half-life and lower bleeding risk. Its core advantage lies in the optimization of the activity ratio of anticoagulant factor Xa and IIa by controlling the molecular weight distribution (usually 4000-6000Da). Therefore, it is widely used in clinical practice 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 causes steric hindrance and electrostatic repulsion, which hinders 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 preparing 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 an enzymatic preparation method of low molecular weight heparin sodium, comprising: S1.1, pretreating the heparin sodium and combining it with a protective agent to obtain a heparin complex precipitate; S1.2, performing the first enzymatic hydrolysis on the heparin complex precipitate to obtain supernatant A and precipitate; S1.3, adjusting the pH of the precipitate to release the highly sulfated fragments, and obtaining supernatant B; S1.4, performing secondary enzymolysis on the supernatant B to obtain a secondary enzymolysis solution, combining the supernatant A and the secondary enzymolysis solution and filtering to obtain a retentate; S1.5, post-treating the retentate to obtain low molecular weight heparin sodium; Among them, the protective agent is a superparamagnetic Fe surface modified with citric acid. 3 O 4 The nanoparticles were prepared by covalent coupling with aminocalixarene.

[0006] 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); The specific preparation steps of the heparin complex precipitation are: 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, then perform magnetic separation for 10-15 minutes, discard the supernatant, and obtain a heparin complex precipitate.

[0007] Preferably, in S1.2, the mixed heparinase is heparinase I:II=2:1U / mg heparin sodium; the total enzyme amount is 5-10U / mg heparin sodium; The specific steps of the first enzymatic hydrolysis of heparin complex precipitation are: 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, then the temperature was raised to 80-85°C and maintained for 10-15 minutes, and then the solution was quickly cooled to 2-4°C in an ice bath, and finally magnetic separation was performed to collect the supernatant A and the precipitate.

[0008] Preferably, in S1.3, the specific preparation steps of supernatant B are: 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 reserved for later use.

[0009] Preferably, in S1.4, the amount of heparinase III is 3-5 U / mg heparin sodium; The specific preparation steps of the retentate are: The pH of supernatant B was adjusted to 6.8-7.2, heparinase III was added to the supernatant, the reaction was continued at 35-37°C for 3-4h, and the supernatant was inactivated at 80-85°C for 10-15min to obtain a secondary enzymatic hydrolyzate. The supernatant A and the secondary enzymatic hydrolyzate were combined, pre-filtered with a 0.22μm filter membrane, and then centrifuged at 10000-12000rpm for 20-30min with an ultrafiltration membrane with a molecular weight cutoff of 3-5kDa. The dialysate was discarded and the retentate was used for standby use.

[0010] Preferably, in S1.5, the amount of anhydrous ethanol added is 3-4 times the volume of the retentate; The specific steps of post-treatment of the retentate 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 10000-12000 rpm for 10-15 min, and the precipitate was collected. The precipitate was resuspended in deionized water and freeze-dried to obtain low molecular weight heparin sodium.

[0011] Preferably, the steps of preparing the protective agent are specifically as follows: S2.1. Superparamagnetic Fe 3 O 4 The nanoparticles were dispersed in water by ultrasonic treatment, and citric acid was added, and then the pH was adjusted to 4-6, heated to 60-80 ° C and stirred for 2-4 h, and after magnetic separation, washed with deionized water and ethanol, and vacuum dried to obtain carboxylated Fe 3 O 4 Nanoparticles; S2.2, carboxylating Fe 3 O 4 The nanoparticles were dispersed in phosphate buffer and ultrasonically treated for 20-30 min. After magnetic separation, the supernatant was discarded and redispersed in phosphate buffer. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide were added and stirred at room temperature for 50-60 min to obtain activated Fe 3 O 4 Suspension; S2.3, disperse the aminocalixarene in phosphate buffer and add activated Fe 3 O 4 The suspension was protected by nitrogen and stirred for 3-4 hours at room temperature in the dark. The magnetic stirring speed was 300-400 rpm and the pH was controlled at 7.0-7.4. After the reaction, 0.1-0.5% v / v ethanolamine was added and stirring was continued for 1-1.5 hours. After separation by magnet, the mixture was washed with phosphate buffer and deionized water in turn and dried in vacuum to obtain the protective agent.

[0012] Preferably, in S2.1, the superparamagnetic Fe 3 O 4 The molar ratio of the nanoparticles to the citric acid is 1:3-5.

[0013] Preferably, in S2.2, the carboxylated Fe 3 O 4 The mass ratio of the 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.

[0014] Preferably, in S2.3, the carboxylated Fe 3 O 4The molar ratio of the nanoparticles to the aminocalixarene is 1:3.5-4.5.

[0015] Aminated calixarene binds to the highly sulfated region of sodium heparin through electrostatic interaction. At the same time, the cavity structure of calixarene can include the hydrophobic skeleton of sodium heparin through hydrophobic interaction or van der Waals force to avoid excessive enzymatic hydrolysis and temporarily "hide" 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 sodium heparin is not shielded, so 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 an enzymatic hydrolysis product with uniform molecular weight.

[0016] However, the size of the enzymatic hydrolysis product and calixarene is similar, and it is difficult to separate them efficiently by conventional centrifugation / filtration. 3 O 4 Nanoparticles are covalently coupled and rapidly separated using a magnetic field, using superparamagnetic Fe 3 O 4 The nanoparticles were modified with citric acid to introduce carboxyl groups, which transformed the superparamagnetic Fe 3 O 4 The nanoparticles were covalently coupled to the calixarene, and ethanolamine was added to convert the residual carboxyl groups into neutral groups, eliminating the negative charge interference.

[0017] Compared with the prior art, the present invention has the following beneficial effects: In the enzymatic hydrolysis preparation method of low molecular weight heparin sodium, superparamagnetic Fe modified with citric acid is used. 3 O 4 The composite protective agent covalently coupled with the nanoparticles and the aminocalixarene uses the electrostatic effect of the aminocalixarene in the protective agent to preferentially bind to the negatively charged region of the highly sulfated fragment of heparin sodium. Its cavity structure coats the hydrophobic skeleton of heparin through van der Waals force to form a steric hindrance protective layer, so that the heparinase preferentially cuts the low-sulfated region. After the highly sulfated fragment is released by pH regulation, the secondary enzymatic hydrolysis obtains a product with a narrower molecular weight distribution. At the same time, the superparamagnetic Fe 3 O 4 The magnetic core of the nanoparticles enables rapid magnetic separation of the protective agent after enzymatic hydrolysis, achieving precise control of the enzymatic hydrolysis process and facilitating separation, thereby obtaining a product with uniform molecular weight distribution. DETAILED DESCRIPTION

[0018] 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 described embodiments 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 work are within the scope of protection of the present invention.

[0019] A method for preparing low molecular weight heparin sodium by enzymatic hydrolysis of the present invention: S1.1, pretreating heparin sodium and combining it with a protective agent to obtain a heparin complex precipitate; S1.2, performing the first enzymatic hydrolysis on the heparin complex precipitate to obtain supernatant A and precipitate; S1.3, adjusting the pH of the precipitate to release the highly sulfated fragments, and obtaining supernatant B; S1.4, performing secondary enzymolysis on the supernatant B to obtain a secondary enzymolysis solution, combining the supernatant A and the secondary enzymolysis solution and filtering to obtain a retentate; S1.5, post-treating the retentate to obtain low molecular weight heparin sodium; Among them, the protective agent is a superparamagnetic Fe surface modified with citric acid. 3 O 4 The nanoparticles were prepared by covalent coupling with aminocalixarene.

[0020] Example 1: A method for preparing low molecular weight heparin sodium by enzymatic hydrolysis, comprising the following steps: Weigh 50 parts by weight of heparin sodium (molecular weight 15 kDa) and 9 parts by weight of a protective agent; Among them, the protective agent is a superparamagnetic Fe surface modified with citric acid. 3 O 4 Nanoparticles were prepared by covalent coupling with amino calixarene; superparamagnetic Fe 3 O 4 The molar ratio of nanoparticles to citric acid was 1:4; carboxylated Fe 3 O 4 The mass ratio of 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 carboxylated Fe 3 O 4 The molar ratio of nanoparticles to aminocalixarene was 1:3.5; S2.1. Superparamagnetic Fe 3 O 4 The nanoparticles were dispersed in water by ultrasonic treatment, and citric acid was added. The pH was then adjusted to 6, heated to 60 °C and stirred for 4 h. After magnetic separation, they were washed with deionized water and ethanol and dried in vacuo to obtain carboxylated Fe 3O 4 Nanoparticles; S2.2, carboxylating Fe 3 O 4 The nanoparticles were dispersed in phosphate buffer and treated with ultrasound for 30 min. After magnetic separation, the supernatant was discarded and redispersed in phosphate buffer. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide were added and stirred at room temperature for 60 min to obtain activated Fe 3 O 4 Suspension; S2.3, disperse the aminocalixarene in phosphate buffer and add activated Fe 3 O 4 The suspension was protected by nitrogen and stirred at room temperature in the dark for 4 h. The magnetic stirring speed was 300 rpm and the pH was controlled at 7.2. After the reaction, 0.5% v / v ethanolamine was added and stirred for 1 h. After separation by magnet, the mixture was washed with phosphate buffer and deionized water in turn and dried in vacuum to obtain the protective agent. 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 min, filter through a 0.22μm membrane to obtain a clear solution, then add a protective agent, control the temperature at 25°C, stir at 300rpm for 60 min, and then magnetically separate for 15 min, discard the supernatant, and obtain a heparin complex precipitate; 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=2:1U / mg heparin sodium; the total enzyme amount is 5U / mg heparin sodium, 300rpm, 37℃ constant temperature reaction for 8h, heat to 85℃ and maintain for 15min, quickly cool to 4℃ in an ice bath, and finally magnetically separate to collect supernatant A and precipitate; S1.3, resuspend the precipitate in phosphate buffer to obtain a 3% (w / v) precipitate resuspended solution, adjust the solution pH to 9.5, stir at 200 rpm for 10 min, remove the protective agent by magnetic separation, and reserve the supernatant B for later use; S1.4, adjust the pH of supernatant B to 6.8, add heparinase III to the supernatant, wherein the amount of heparinase III is 3U / mg heparin sodium, continue the reaction at 37°C for 4h, inactivate at 85°C for 15min, obtain secondary enzymatic hydrolysate, combine supernatant A and secondary enzymatic hydrolysate, pre-filter with 0.22μm filter membrane, and then centrifuge at 10000rpm for 30min with ultrafiltration membrane with molecular weight cutoff of 3 and 5kDa, discard the dialysate, and use the retentate for later use; S1.5. Add anhydrous ethanol dropwise to the retentate, wherein the amount of anhydrous ethanol added is 3 times the volume of the retentate, stand at 4°C overnight, centrifuge at 10,000 rpm for 15 min, collect the precipitate, resuspend the precipitate with deionized water, and lyophilize to obtain low molecular weight heparin sodium.

[0021] 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; Among them, the protective agent is a superparamagnetic Fe surface modified with citric acid. 3 O 4 Nanoparticles were prepared by covalent coupling with amino calixarene; superparamagnetic Fe 3 O 4 The molar ratio of nanoparticles to citric acid was 1:4; carboxylated Fe 3 O 4 The mass ratio of 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 carboxylated Fe 3 O 4 The molar ratio of nanoparticles to aminocalixarene was 1:3.5; S2.1. Superparamagnetic Fe 3 O 4 The nanoparticles were dispersed in water by ultrasonic treatment, and citric acid was added. The pH was then adjusted to 6, heated to 60 °C and stirred for 4 h. After magnetic separation, they were washed with deionized water and ethanol and dried in vacuo to obtain carboxylated Fe 3 O 4 Nanoparticles; S2.2, carboxylating Fe 3 O 4 The nanoparticles were dispersed in phosphate buffer and treated with ultrasound for 30 min. After magnetic separation, the supernatant was discarded and redispersed in phosphate buffer. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide were added and stirred at room temperature for 60 min to obtain activated Fe 3 O 4 Suspension; S2.3, disperse the aminocalixarene in phosphate buffer and add activated Fe 3 O 4 The suspension was protected by nitrogen and stirred at room temperature in the dark for 4 h. The magnetic stirring speed was 300 rpm and the pH was controlled at 7.2. After the reaction, 0.5% v / v ethanolamine was added and stirred for 1 h. After separation by magnet, the mixture was washed with phosphate buffer and deionized water in turn and dried in vacuum to obtain the protective agent. 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 min, filter through a 0.22μm membrane to obtain a clear solution, then add a protective agent, control the temperature at 25°C, stir at 300rpm for 60 min, and then magnetically separate for 15 min, discard the supernatant, and obtain a heparin complex precipitate; 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=2:1U / mg heparin sodium; the total enzyme amount is 5U / mg heparin sodium, 300rpm, 37℃ constant temperature reaction for 8h, heat to 85℃ and maintain for 15min, quickly cool to 4℃ in an ice bath, and finally magnetically separate to collect supernatant A and precipitate; S1.3, resuspend the precipitate in phosphate buffer to obtain a 3% (w / v) precipitate resuspended solution, adjust the solution pH to 9.5, stir at 200 rpm for 10 min, remove the protective agent by magnetic separation, and reserve the supernatant B for later use; S1.4, adjust the pH of supernatant B to 6.8, add heparinase III to the supernatant, wherein the amount of heparinase III is 3U / mg heparin sodium, continue the reaction at 37°C for 4h, inactivate at 85°C for 15min, obtain secondary enzymatic hydrolysate, combine supernatant A and secondary enzymatic hydrolysate, pre-filter with 0.22μm filter membrane, and then centrifuge at 10000rpm for 30min with ultrafiltration membrane with molecular weight cutoff of 3 and 5kDa, discard the dialysate, and use the retentate for later use; S1.5. Add anhydrous ethanol dropwise to the retentate, wherein the amount of anhydrous ethanol added is 3 times the volume of the retentate, stand at 4°C overnight, centrifuge at 10,000 rpm for 15 min, collect the precipitate, resuspend the precipitate with deionized water, and lyophilize to obtain low molecular weight heparin sodium.

[0022] Example 3: A method for preparing low molecular weight heparin sodium by enzymatic hydrolysis, comprising the following steps: Weigh 50 parts by weight of heparin sodium (molecular weight 15 kDa) and 15 parts by weight of a protective agent; Among them, the protective agent is a superparamagnetic Fe surface modified with citric acid. 3 O 4 Nanoparticles were prepared by covalent coupling with amino calixarene; superparamagnetic Fe 3 O 4 The molar ratio of nanoparticles to citric acid was 1:4; carboxylated Fe 3 O 4The mass ratio of 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 carboxylated Fe 3 O 4 The molar ratio of nanoparticles to aminocalixarene was 1:3.5; S2.1. Superparamagnetic Fe 3 O 4 The nanoparticles were dispersed in water by ultrasonic treatment, and citric acid was added. The pH was then adjusted to 6, heated to 60 °C and stirred for 4 h. After magnetic separation, they were washed with deionized water and ethanol and dried in vacuo to obtain carboxylated Fe 3 O 4 Nanoparticles; S2.2, carboxylating Fe 3 O 4 The nanoparticles were dispersed in phosphate buffer and treated with ultrasound for 30 min. After magnetic separation, the supernatant was discarded and redispersed in phosphate buffer. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide were added and stirred at room temperature for 60 min to obtain activated Fe 3 O 4 Suspension; S2.3, disperse the aminocalixarene in phosphate buffer and add activated Fe 3 O 4 The suspension was protected by nitrogen and stirred at room temperature in the dark for 4 h. The magnetic stirring speed was 300 rpm and the pH was controlled at 7.2. After the reaction, 0.5% v / v ethanolamine was added and stirred for 1 h. After separation by magnet, the mixture was washed with phosphate buffer and deionized water in turn and dried in vacuum to obtain the protective agent. 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 min, filter through a 0.22μm membrane to obtain a clear solution, then add a protective agent, control the temperature at 25°C, stir at 300rpm for 60 min, and then magnetically separate for 15 min, discard the supernatant, and obtain a heparin complex precipitate; 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=2:1U / mg heparin sodium; the total enzyme amount is 5U / mg heparin sodium, 300rpm, 37℃ constant temperature reaction for 8h, heat to 85℃ and maintain for 15min, quickly cool to 4℃ in an ice bath, and finally magnetically separate to collect supernatant A and precipitate; S1.3, resuspend the precipitate in phosphate buffer to obtain a 3% (w / v) precipitate resuspended solution, adjust the solution pH to 9.5, stir at 200 rpm for 10 min, remove the protective agent by magnetic separation, and reserve the supernatant B for later use; S1.4, adjust the pH of supernatant B to 6.8, add heparinase III to the supernatant, wherein the amount of heparinase III is 3U / mg heparin sodium, continue the reaction at 37°C for 4h, inactivate at 85°C for 15min, obtain secondary enzymatic hydrolysate, combine supernatant A and secondary enzymatic hydrolysate, pre-filter with 0.22μm filter membrane, and then centrifuge at 10000rpm for 30min with ultrafiltration membrane with molecular weight cutoff of 3 and 5kDa, discard the dialysate, and use the retentate for later use; S1.5. Add anhydrous ethanol dropwise to the retentate, wherein the amount of anhydrous ethanol added is 3 times the volume of the retentate, stand at 4°C overnight, centrifuge at 10,000 rpm for 15 min, collect the precipitate, resuspend the precipitate with deionized water, and lyophilize to obtain low molecular weight heparin sodium.

[0023] Example 4: A method for preparing low molecular weight heparin sodium by enzymatic hydrolysis, comprising the following steps: Weigh 50 parts by weight of heparin sodium (molecular weight 15 kDa) and 15 parts by weight of a protective agent; Among them, the protective agent is a superparamagnetic Fe surface modified with citric acid. 3 O 4 Nanoparticles were prepared by covalent coupling with amino calixarene; superparamagnetic Fe 3 O 4 The molar ratio of nanoparticles to citric acid was 1:4; carboxylated Fe 3 O 4 The mass ratio of 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 carboxylated Fe 3 O 4 The molar ratio of nanoparticles to aminocalixarene was 1:4.0; S2.1. Superparamagnetic Fe 3 O 4 The nanoparticles were dispersed in water by ultrasonic treatment, and citric acid was added. The pH was then adjusted to 6, heated to 60 °C and stirred for 4 h. After magnetic separation, they were washed with deionized water and ethanol and dried in vacuo to obtain carboxylated Fe 3 O 4 Nanoparticles; S2.2, carboxylating Fe 3 O 4The nanoparticles were dispersed in phosphate buffer and treated with ultrasound for 30 min. After magnetic separation, the supernatant was discarded and redispersed in phosphate buffer. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide were added and stirred at room temperature for 60 min to obtain activated Fe 3 O 4 Suspension; S2.3, disperse the aminocalixarene in phosphate buffer and add activated Fe 3 O 4 The suspension was protected by nitrogen and stirred at room temperature in the dark for 4 h. The magnetic stirring speed was 300 rpm and the pH was controlled at 7.2. After the reaction, 0.5% v / v ethanolamine was added and stirred for 1 h. After separation by magnet, the mixture was washed with phosphate buffer and deionized water in turn and dried in vacuum to obtain the protective agent. 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 min, filter through a 0.22μm membrane to obtain a clear solution, then add a protective agent, control the temperature at 25°C, stir at 300rpm for 60 min, and then magnetically separate for 15 min, discard the supernatant, and obtain a heparin complex precipitate; 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=2:1U / mg heparin sodium; the total enzyme amount is 5U / mg heparin sodium, 300rpm, 37℃ constant temperature reaction for 8h, heat to 85℃ and maintain for 15min, quickly cool to 4℃ in an ice bath, and finally magnetically separate to collect supernatant A and precipitate; S1.3, resuspend the precipitate in phosphate buffer to obtain a 3% (w / v) precipitate resuspended solution, adjust the solution pH to 9.5, stir at 200 rpm for 10 min, remove the protective agent by magnetic separation, and reserve the supernatant B for later use; S1.4, adjust the pH of supernatant B to 6.8, add heparinase III to the supernatant, wherein the amount of heparinase III is 3U / mg heparin sodium, continue the reaction at 37°C for 4h, inactivate at 85°C for 15min, obtain secondary enzymatic hydrolysate, combine supernatant A and secondary enzymatic hydrolysate, pre-filter with 0.22μm filter membrane, and then centrifuge at 10000rpm for 30min with ultrafiltration membrane with molecular weight cutoff of 3 and 5kDa, discard the dialysate, and use the retentate for later use; S1.5. Add anhydrous ethanol dropwise to the retentate, wherein the amount of anhydrous ethanol added is 3 times the volume of the retentate, stand at 4°C overnight, centrifuge at 10,000 rpm for 15 min, collect the precipitate, resuspend the precipitate with deionized water, and lyophilize to obtain low molecular weight heparin sodium.

[0024] Example 5: A method for preparing low molecular weight heparin sodium by enzymatic hydrolysis, comprising the following steps: Weigh 50 parts by weight of heparin sodium (molecular weight 15 kDa) and 15 parts by weight of a protective agent; Among them, the protective agent is a superparamagnetic Fe surface modified with citric acid. 3 O 4 Nanoparticles were prepared by covalent coupling with amino calixarene; superparamagnetic Fe 3 O 4 The molar ratio of nanoparticles to citric acid was 1:4; carboxylated Fe 3 O 4 The mass ratio of 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 carboxylated Fe 3 O 4 The molar ratio of nanoparticles to aminocalixarene was 1:4.5; S2.1. Superparamagnetic Fe 3 O 4 The nanoparticles were dispersed in water by ultrasonic treatment, and citric acid was added. The pH was then adjusted to 6, heated to 60 °C and stirred for 4 h. After magnetic separation, they were washed with deionized water and ethanol and dried in vacuo to obtain carboxylated Fe 3 O 4 Nanoparticles; S2.2, carboxylating Fe 3 O 4 The nanoparticles were dispersed in phosphate buffer and treated with ultrasound for 30 min. After magnetic separation, the supernatant was discarded and redispersed in phosphate buffer. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide were added and stirred at room temperature for 60 min to obtain activated Fe 3 O 4 Suspension; S2.3, disperse the aminocalixarene in phosphate buffer and add activated Fe 3 O 4 The suspension was protected by nitrogen and stirred at room temperature in the dark for 4 h. The magnetic stirring speed was 300 rpm and the pH was controlled at 7.2. After the reaction, 0.5% v / v ethanolamine was added and stirred for 1 h. After separation by magnet, the mixture was washed with phosphate buffer and deionized water in turn and dried in vacuum to obtain the protective agent. 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 min, filter through a 0.22μm membrane to obtain a clear solution, then add a protective agent, control the temperature at 25°C, stir at 300rpm for 60 min, and then magnetically separate for 15 min, discard the supernatant, and obtain a heparin complex precipitate; 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=2:1U / mg heparin sodium; the total enzyme amount is 5U / mg heparin sodium, 300rpm, 37℃ constant temperature reaction for 8h, heat to 85℃ and maintain for 15min, quickly cool to 4℃ in an ice bath, and finally magnetically separate to collect supernatant A and precipitate; S1.3, resuspend the precipitate in phosphate buffer to obtain a 3% (w / v) precipitate resuspended solution, adjust the solution pH to 9.5, stir at 200 rpm for 10 min, remove the protective agent by magnetic separation, and reserve the supernatant B for later use; S1.4, adjust the pH of supernatant B to 6.8, add heparinase III to the supernatant, wherein the amount of heparinase III is 3U / mg heparin sodium, continue the reaction at 37°C for 4h, inactivate at 85°C for 15min, obtain secondary enzymatic hydrolysate, combine supernatant A and secondary enzymatic hydrolysate, pre-filter with 0.22μm filter membrane, and then centrifuge at 10000rpm for 30min with ultrafiltration membrane with molecular weight cutoff of 3 and 5kDa, discard the dialysate, and use the retentate for later use; S1.5. Add anhydrous ethanol dropwise to the retentate, wherein the amount of anhydrous ethanol added is 3 times the volume of the retentate, stand at 4°C overnight, centrifuge at 10,000 rpm for 15 min, collect the precipitate, resuspend the precipitate with deionized water, and lyophilize to obtain low molecular weight heparin sodium.

[0025] Comparative Example 1: The method of Example 5 was adopted without adding a protective agent.

[0026] Comparative Example 2: Using the method of Example 5, the amino calixarene was directly used without carboxylation of Fe 3 O 4 The nanoparticles were used to modify the aminocalixarene.

[0027] The present invention adopts the low molecular weight heparin sodium prepared by using the protective agent, wherein the performance index inspection items and inspection standards of the low molecular weight heparin sodium are as follows: The test was performed according to the test method and test 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 = weight after lyophilization / initial heparin sodium input × 100%. The higher the yield, the more target molecular weight there is, which indirectly shows that heparinase can effectively and specifically cut and the enzymatic hydrolysis efficiency is improved.

[0028] 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. The low molecular weight component (3-5 kDa): 30% ethanol precipitation; the medium molecular weight component (5-8 kDa): 50% ethanol precipitation; the high molecular weight component (>8 kDa): 70% ethanol precipitation was used to evaluate the molecular weight concentration of the product.

[0029] 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.

[0030] The low molecular weight heparin sodium prepared in the above examples 1-5 and comparative examples 1-2 was tested according to the above standards, and the obtained data are shown in Table 1: Table 1 Performance data of Examples 1-5 and Comparative Examples 1-2 The above data fully show that compared with Comparative Examples 1-2, Examples 1-5 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.

[0031] Since the present invention adopts a protective agent to prepare an enzymatic hydrolysis preparation method of low molecular weight heparin sodium, the protective agent effectively improves the molecular weight distribution, yield and purity of low molecular weight heparin sodium, as follows: 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 high sulfated regions of the heparin sodium through electrostatic action. Therefore, when the amount of the protective agent increases, more high 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 action to form a physical barrier, avoiding random cutting of the high sulfated regions by the enzyme, thereby further inhibiting excessive degradation.

[0032] It can be seen from Examples 3-5 that as the carboxylation of Fe 3 O 4As the molar ratio of nanoparticles to aminocalixarene increases, the molecular weight distribution, yield and purity of enzymatically hydrolyzed low molecular weight heparin sodium change continuously, which means that the unit Fe 3 O 4 The number of calixarene molecules coupled to the nanoparticle surface increases, forming denser electrostatic binding sites, enhancing the shielding ability of the highly sulfated area, and indirectly reducing Fe 3 O 4 The negative charge on the surface of the nanoparticles electrostatically repels heparinase, thereby significantly improving enzymatic selectivity and product uniformity.

[0033] According to the above test experiments, the enzymatic preparation method of low molecular weight heparin sodium prepared according to Example 5 has the best performance, so Example 5 is taken as the best example; By comparing Example 5 with Comparative Examples 1-2, it can be seen that: In Comparative Example 1, no protective agent was added, and the molecular weight distribution, yield and purity of the enzymatically hydrolyzed low molecular weight heparin sodium 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 macromolecular fragments, resulting in a loss of enzymatic hydrolysis selectivity, a wide distribution of products, many impurities and a low yield.

[0034] Comparative Example 2: Direct use of amino calixarene without carboxylation of Fe 3 O 4 Nanoparticles were used to modify aminocalixarene. The molecular weight distribution, yield and purity of low molecular weight heparin sodium hydrolyzed by enzyme were poor. 3 O 4 The calixarene in the nanoparticles cannot be quickly recovered by a magnetic field, and the residual protective agent is mixed with the product, requiring an additional purification step, which increases product losses.

[0035] In summary, the superparamagnetic Fe 3 O 4 The composite protective agent covalently coupled the nanoparticles with amino calixarene utilizes the amino calixarene to preferentially bind to the highly sulfated region of heparin sodium through electrostatic interaction, shielding its steric hindrance and negative charge, forcing heparinase to preferentially cut the glycosidic bonds in the low-sulfated region, reducing random degradation, and coating the heparin skeleton through the hydrophobic cavity of the calixarene through van der Waals force to avoid nonspecific cutting of the highly sulfated region by the enzyme. At the same time, through pH regulation, the shielded fragments are released for secondary enzymolysis, achieving step-by-step controllable degradation. Finally, due to Fe 3 O 4 The superparamagnetism of the nanoparticles enables rapid separation of the protective agent and the product. Combined with ethanolamine to cap the unreacted carboxyl groups, charge interference is eliminated, effectively solving the problems of uneven molecular weight, low yield and complex purification in traditional processes.

[0036] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and the above embodiments and descriptions are only preferred examples of the present invention, and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for preparing low molecular weight 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 the first enzymatic hydrolysis on the heparin complex precipitate to obtain supernatant A and precipitate; S1.3, adjusting the pH of the precipitate to release the highly sulfated fragments, and obtaining supernatant B; S1.4, performing secondary enzymolysis on the supernatant B to obtain a secondary enzymolysis solution, combining the supernatant A and the secondary enzymolysis solution and filtering to obtain a retentate; S1.5, post-treating 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 amino-calixarene.

2. The method for preparing low molecular weight heparin sodium by enzymatic hydrolysis according to claim 1, characterized in that: In the S1.1, 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); The specific preparation steps of the heparin complex precipitation are: 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, then perform magnetic separation for 10-15 minutes, discard the supernatant, and obtain a heparin complex precipitate.

3. The method for preparing low molecular weight heparin sodium by enzymatic hydrolysis according to claim 1, characterized in that: In S1.2, the mixed heparinase is heparinase I:II=2:1U / mg heparin sodium; the total enzyme amount is 5-10U / mg heparin sodium; The specific steps of the first enzymatic hydrolysis of heparin complex precipitation are: 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, then the temperature was raised to 80-85°C and maintained for 10-15 minutes, and then the solution was quickly cooled to 2-4°C in an ice bath, and finally magnetic separation was performed to collect the supernatant A and the precipitate.

4. The method for preparing low molecular weight heparin sodium by enzymatic hydrolysis according to claim 1, characterized in that: In S1.3, the specific preparation steps of supernatant B are: 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 reserved for later use.

5. The method for preparing low molecular weight heparin sodium by enzymatic hydrolysis according to claim 1, characterized in that: In S1.4, the amount of heparinase III is 3–5 U / mg heparin sodium; The specific preparation steps of the retentate are: The pH of supernatant B was adjusted to 6.8-7.2, heparinase III was added to the supernatant, the reaction was continued at 35-37°C for 3-4h, and the supernatant was inactivated at 80-85°C for 10-15min to obtain a secondary enzymatic hydrolyzate. The supernatant A and the secondary enzymatic hydrolyzate were combined, pre-filtered with a 0.22μm filter membrane, and then centrifuged at 10000-12000rpm for 20-30min with an ultrafiltration membrane with a molecular weight cutoff of 3-5kDa. The dialysate was discarded and the retentate was used for standby use.

6. The method for preparing low molecular weight heparin sodium by enzymatic hydrolysis according to claim 1, characterized in that: In S1.5, the amount of anhydrous ethanol added is 3-4 times the volume of the retentate; The specific steps of post-treatment of the retentate 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 10000-12000 rpm for 10-15 min, and the precipitate was collected. The precipitate was resuspended in deionized water and freeze-dried to obtain low molecular weight heparin sodium.

7. The method for preparing low molecular weight heparin sodium by enzymatic hydrolysis according to claim 1, characterized in that: The steps of preparing the protective agent are specifically as follows: S2.1, dispersing superparamagnetic Fe3O4 nanoparticles in water by ultrasonic treatment, adding citric acid, adjusting the pH to 4-6, heating to 60-80°C and stirring for 2-4h, washing with deionized water and ethanol after magnetic separation, and vacuum drying to obtain carboxylated Fe3O4 nanoparticles; S2.2, dispersing the carboxylated Fe3O4 nanoparticles in phosphate buffer, ultrasonically treating for 20-30 min, discarding the supernatant after magnetic separation, and re-dispersing in phosphate buffer, adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, stirring at room temperature for 50-60 min, to obtain an activated Fe3O4 suspension; S2.

3. Disperse the aminocalixarene in phosphate buffer, add it to the activated Fe3O4 suspension, protect it with nitrogen, stir and react for 3-4 hours at room temperature in the dark, stir magnetically at 300-400rpm, control the pH to 7.0-7.4, after the reaction, add 0.1-0.5% v / v ethanolamine, 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.

8. The method for preparing low molecular weight heparin sodium by enzymatic hydrolysis according to claim 7, characterized in that: In S2.1, the molar ratio of superparamagnetic Fe3O4 nanoparticles to citric acid is 1:3-5.

9. The method for preparing low molecular weight heparin sodium by enzymatic hydrolysis according to claim 7, characterized in that: In 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.

10. The method for preparing low molecular weight heparin sodium by enzymatic hydrolysis according to claim 7, characterized in that: In S2.3, the molar ratio of carboxylated Fe3O4 nanoparticles to aminocalixarene is 1:3.5-4.5.

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