A stable nadroparin calcium injection and preparation method thereof

Abstract

The invention discloses a stable nadroparin calcium injection and a preparation method thereof, first using crude heparin sodium as raw material, degrading sodium nitrite under pretreatment and magnetic field conditions, obtaining a degradation liquid, performing reduction reaction by sodium borohydride, obtaining a reduction liquid, adding calcium chloride, stirring and mixing, stirring at room temperature under magnetic field conditions to obtain a reaction solution, extracting with n-butanol, ultrafiltration and concentration, obtaining a concentrated solution, alcohol precipitation, obtaining nadroparin calcium; then stirring and mixing Tween 80, soybean lecithin, cholesterol and anhydrous ethanol, transferring to a thin film evaporator, rotating film formation, adding ether to dissolve the film, then adding a PBS solution of nadroparin calcium, ultrasonic oscillation treatment, high-voltage pulse electric field treatment, adding magnetic nitrogen-doped activated carbon, stirring and adsorption, magnetic separation to remove magnetic nitrogen-doped activated carbon, microporous membrane filtration, rotary evaporation, vacuum freeze drying. The injection has good stability and high clinical promotion value.

Description

Technical Field

[0001] The present invention relates to an injection, in particular to a stable nadroparin calcium injection and a preparation method thereof, belonging to the technical field of heparin drugs. Background Art

[0002] Heparin drugs are sulfated glycosaminoglycan compounds extracted from mammalian tissues (such as small intestinal mucosa, lungs, and liver). They have a complex molecular structure and a molecular weight of 3kD to 30kD. They are mainly used for the prevention and treatment of deep vein thrombosis, hemodialysis anticoagulation, as well as for surgical anticoagulation, acute coronary syndrome, ischemic cerebral thrombosis, etc. In addition, they can also be used in obstetrics and gynecology, pediatrics, respiratory medicine, oncology, burns, etc.

[0003] Low molecular weight heparin (LMWH) is mainly obtained from ordinary heparin through physical separation, chemical cleavage, and biological enzyme cleavage, and its average molecular weight generally ranges from 3kD to 6.5kD. The most important production method at present is chemical cleavage, including nitrite cleavage, peroxide cleavage, benzyl ester cleavage, etc. Low molecular weight heparin has a higher anti-FXa / anti-FⅡa potency ratio than ordinary heparin, so it can greatly reduce the bleeding tendency caused by the anti-thrombotic process, and is currently gradually replacing ordinary heparin in clinical practice.

[0004] Nadroparin calcium is a kind of low molecular weight heparin, which is obtained by lysing sodium heparin with sodium nitrite, converting calcium, and precipitating with alcohol. According to the quality standards stipulated in the European Pharmacopoeia, the anti-Xa potency of nadroparin calcium is 95-130 IU / mg, the anti-Xa / anti-Ⅱa ratio is 2.5-4.0, the average molecular weight is 3600-5000D, the molecular weight less than 2000D accounts for less than 15% of the total amount, the molecular weight of 2000D-4000D accounts for 35-55% of the total amount, and the molecular weight of 2000D-8000D accounts for 75-95% of the total amount. Nadroparin calcium has a high anti-FXa / anti-FⅡa potency ratio, strong antithrombotic effect, and weak anticoagulant effect, so it can greatly reduce the bleeding tendency caused by the antithrombotic process, has high bioavailability, long half-life, and is more suitable for clinical use. It has gradually replaced heparin for clinical use.

[0005] Nadroparin calcium is usually made into injection for clinical use. Nadroparin calcium injection needs to be kept in a light-proof and airtight place and should be kept in a dry place away from direct sunlight. If the drug is turbid, precipitated, discolored, etc., it is not recommended for use. During the processing of nadroparin calcium injection, there are various problems such as ethanol residue, free sulfate residue, impurity removal and decolorization leading to potency loss, which affect the stability of nadroparin calcium injection and the quality of the drug cannot be guaranteed.

[0006] Patent application CN109748984A discloses a preparation process of nadroparin calcium, including the steps of preparing degradation solution, preparing reduction solution, precipitation, oxidation, ion exchange, preparing crude nadroparin calcium, refining, freeze drying, etc. This patented technology is mainly aimed at improving the process yield, and the product quality is average. It also does not solve the aforementioned stability problem. The product stability is average, and long-term storage will generate impurities, causing toxic side effects, and there are great hidden dangers in clinical use. Moreover, after the raw material drug is made into an injection solution using injection water, the product stability cannot be guaranteed. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a stable nadroparin calcium injection and a preparation method thereof, which has good stability and high clinical promotion value.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A method for preparing a stable nadroparin calcium injection, the specific steps are as follows:

[0010] (1) First, crude heparin sodium is used as a raw material, and is degraded by sodium nitrite under pretreatment and magnetic field conditions to obtain a degradation solution, and a reduction reaction is carried out by sodium borohydride to obtain a reduction solution, calcium chloride is added, and the mixture is stirred and mixed, and the mixture is stirred at room temperature under magnetic field conditions for 25 to 35 minutes to obtain a reaction solution, and the mixture is extracted with n-butanol, ultrafiltered and concentrated to obtain a concentrated solution, and alcohol precipitation is performed to obtain nadroparin calcium;

[0011] (2) In parts by weight, 0.4-0.5 parts of Tween-80, 1-2 parts of soybean lecithin, 0.4-0.5 parts of cholesterol and 100 parts of anhydrous ethanol are stirred and mixed, transferred to a thin film evaporator, and rotated to form a film, 50-60 parts of ether are added to dissolve the film, and then 18-20 parts of nadroparin calcium PBS solution are added, ultrasonic oscillation treatment is carried out, high-voltage pulse electric field treatment is carried out, magnetic nitrogen-doped activated carbon is added, stirring and adsorption is carried out, magnetic nitrogen-doped activated carbon is removed by magnetic separation, microporous membrane filtration is carried out, rotary evaporation is carried out, and vacuum freeze-drying is carried out to obtain the product.

[0012] Preferably, in step (1), the specific method of pretreatment and sodium nitrite degradation is as follows: first, add 1 part of crude heparin sodium to 8-10 parts of purified water, stir to dissolve, then add 0.01-0.02 parts of sodium chloride, stir at 50-60°C for 2-3 hours, centrifuge to obtain the supernatant, add 0.01-0.02 parts of hydrogen peroxide to the supernatant, use FPA98CL strong alkaline anion exchange resin for adsorption, washing and elution, collect the eluate, adjust the pH to 5.5-6.0, and ultrafilter with a 1000D ultrafiltration membrane to obtain an ultrafiltrate, adjust the pH to 2-3, add 0.02-0.025 parts of sodium nitrite, and stir at 8-10T magnetic field for 1-2 hours at 20-25°C to obtain a degradation solution.

[0013] Preferably, in step (1), the specific method of the reduction reaction is as follows, by weight: add 0.01 to 0.015 parts of sodium borohydride to 10 parts of the degradation solution, stir and react at room temperature for 15 to 17 hours, adjust the pH to 3 to 4, continue stirring and reacting for 20 to 30 minutes, and obtain a reduction solution.

[0014] Preferably, in step (1), the amount of calcium chloride added is 0.8-1% of the mass of the reducing solution.

[0015] Preferably, in step (1), the magnetic field conditions during stirring at room temperature are: the magnetic field pulse frequency is 800-1000 Hz, and the magnetic field strength is 2-3 T.

[0016] Preferably, in step (1), the specific method of n-butanol extraction is: add 1.5 to 2.5 times the volume of n-butanol to the reaction solution, stir at room temperature for 8 to 10 minutes, let it stand and discard the upper layer.

[0017] Preferably, in step (1), the specific method of ultrafiltration and concentration is: ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 3000D until the conductivity of the ultrafiltrate is ≤450μs / cm, and concentration is performed to 1 / 4 of the volume of the ultrafiltrate to obtain a concentrated solution.

[0018] Preferably, in step (1), the specific method of alcohol precipitation is: take 1 part of the concentrated solution, stir and disperse it evenly with 8 to 10 parts of purified water, adjust the pH to 6.5 to 7.0, add anhydrous ethanol to make the volume concentration of ethanol 50 to 55%, stand at room temperature for 48 to 55 hours, and centrifuge to obtain the precipitate.

[0019] Preferably, in step (2), the temperature of the rotary film formation is 60°C.

[0020] Preferably, in step (2), the PBS solution of nadroparin calcium is obtained by dissolving nadroparin calcium in PBS buffer, wherein the concentration of nadroparin calcium is 0.4-0.5 mg / mL and the pH of the PBS buffer is 7.5.

[0021] Preferably, in step (2), the process conditions of ultrasonic oscillation treatment are: 300-400W ultrasonic oscillation treatment for 20-30 minutes.

[0022] Preferably, in step (2), the process conditions of the high-voltage pulse electric field treatment are: ice water bath, electric field strength of 30-40 kV / cm, electric field frequency of 1000-1500 Hz, and material flow rate of 8-10 mL / min.

[0023] Preferably, in step (2), the amount of the magnetic nitrogen-doped activated carbon is 0.1 g / L, and the stirring adsorption time is 20 to 30 minutes.

[0024] Preferably, in step (2), the magnetic nitrogen-doped activated carbon is prepared by the following method, in parts by weight: first, 0.1-0.2 parts of polyethylene glycol 400, 0.2-0.3 parts of linear phenolic resin and 2-3 parts of diethylene glycol are stirred and mixed to obtain a premix; then, 1 part of 200-300 mesh sepiolite powder is soaked in 6-8 parts of 8-10 mg / mL ammonium ferric oxalate solution for 15-18 hours, centrifuged to obtain a precipitate, the precipitate is mixed and stirred with 4-5 parts of 1 mol / L hydrochloric acid solution for 2-3 hours, centrifuged to obtain an acidified precipitate; then, the acidified precipitate is added to the premix, stirred and mixed, stirred and treated at 60-70° C. for 10-12 hours, 0.1-0.2 parts of urea are added, the stirring is continued for 8-10 hours, vacuum dried, treated at 800-850° C. for 1-2 hours under nitrogen protection, and naturally cooled to room temperature to obtain the product.

[0025] Preferably, in step (2), the microporous membrane filtration comprises: filtering once with a 0.45 μm microporous membrane and filtering once with a 0.22 μm microporous membrane.

[0026] Preferably, in step (2), the process conditions for vacuum freeze drying are: freezing at -40 to -50°C for 3 to 4 hours, evacuating to below 10 Pa, heating to 20 to 30°C, keeping warm and drying for 7 to 8 hours, heating to 35 to 45°C, keeping warm and drying for 5 to 6 hours.

[0027] A stable nadroparin calcium injection is obtained by the above-mentioned preparation method.

[0028] Beneficial effects of the present invention:

[0029] The invention first uses crude heparin sodium as a raw material, degrades sodium nitrite through pretreatment and magnetic field conditions, obtains a degradation solution, performs reduction reaction through sodium borohydride, obtains a reduction solution, adds calcium chloride, stirs and mixes, stirs at room temperature under magnetic field conditions for 25 to 35 minutes, obtains a reaction solution, extracts with n-butanol, ultrafilters and concentrates, obtains a concentrated solution, and precipitates with alcohol to obtain nadroparin calcium; then stirs and mixes Tween-80, soybean lecithin, cholesterol and anhydrous ethanol, transfers to a thin film evaporator, rotates to form a film, adds ether to dissolve the film, then adds a PBS solution of nadroparin calcium, performs ultrasonic oscillation treatment, performs high-voltage pulse electric field treatment, adds magnetic nitrogen-doped activated carbon, stirs and adsorbs, removes the magnetic nitrogen-doped activated carbon through magnetic separation, filters with a microporous filter membrane, rotates and evaporates, and vacuum freeze-dries to obtain a stable nadroparin calcium injection. The injection has good stability, can be used after adding injection water to prepare an injection solution, and has high clinical promotion value.

[0030] First, in the process of converting sodium heparin into nadroparin calcium, the degradation under magnetic field conditions provides a good basis for controlling the molecular weight range of the product and promotes the calcium conversion process. Combined with n-butanol extraction for impurity removal, the purity of nadroparin calcium is improved, which is beneficial to the improvement of product stability.

[0031] Secondly, the present invention prepares the raw material drug nadroparin calcium into liposomes, thereby improving the stability of the product. In addition, residual nitroso compounds and the like are eliminated by high-voltage pulse electric field treatment, the purity is high, which is conducive to further improvement of stability, and plays an activation role, promoting activated carbon impurity removal. The magnetic nitrogen-doped activated carbon used in the present invention has a very good adsorption and impurity removal effect, reduces solvent residues, further improves product purity, and magnetic separation removes activated carbon, the separation is thorough and convenient, does not affect product performance, and ensures stability. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its contents.

[0033] Crude heparin sodium was purchased from Nanjing Bermuda Biotechnology Co., Ltd., with a molecular weight of 10kD to 20kD.

[0034] The room temperature involved in the present invention is 25° C., and 1 mol / L hydrochloric acid solution or 1 mol / L sodium hydroxide solution is used to adjust the pH.

[0035] Embodiment 1:

[0036] A method for preparing a stable nadroparin calcium injection, the specific steps are as follows:

[0037] (1) First, crude heparin sodium is used as a raw material, and is degraded by sodium nitrite after pretreatment and magnetic field conditions to obtain a degradation solution, and reduction reaction is carried out by sodium borohydride to obtain a reduction solution, calcium chloride is added, and the mixture is stirred and mixed, and the mixture is stirred at room temperature for 25 minutes under magnetic field conditions to obtain a reaction solution, and n-butanol extraction, ultrafiltration and concentration are performed to obtain a concentrated solution, and alcohol precipitation is performed to obtain nadroparin calcium; the anti-Xa potency of nadroparin calcium is 128 IU / mg, the anti-Xa / anti-IIa ratio is 3.7, and the weight average molecular weight is 4100D;

[0038] (2) 0.4 kg of Tween-80, 1 kg of soy lecithin, 0.4 kg of cholesterol and 100 kg of anhydrous ethanol are stirred and mixed, and then transferred to a thin film evaporator, and the film is formed by rotation. 50 kg of ether is added to dissolve the film, and then 18 kg of nadroparin calcium PBS solution is added, ultrasonic oscillation treatment is carried out, high-voltage pulse electric field treatment is carried out, magnetic nitrogen-doped activated carbon is added, and adsorption is carried out by stirring. The magnetic nitrogen-doped activated carbon is removed by magnetic separation, and then filtered with a microporous membrane, rotary evaporated, and vacuum freeze-dried to obtain the product.

[0039] In step (1), the specific method of pretreatment and sodium nitrite degradation is as follows: first, 1 kg of crude heparin sodium is added to 8 kg of purified water, stirred to dissolve, then added to 0.01 kg of sodium chloride, stirred at 50° C. for 2 hours, centrifuged to obtain the supernatant, 0.01 kg of hydrogen peroxide is added to the supernatant, adsorbed, washed and eluted using FPA98CL strong alkaline anion exchange resin, the eluate is collected, the pH is adjusted to 5.5, ultrafiltration is performed using a 1000D ultrafiltration membrane with a molecular weight cutoff to obtain an ultrafiltrate, the pH is adjusted to 2, 0.02 kg of sodium nitrite is added, and the reaction is stirred at 20° C. for 1 hour under 8T magnetic field conditions to obtain a degradation solution.

[0040] In step (1), the specific method of the reduction reaction is: add 0.01 kg of sodium borohydride to 10 kg of the degradation liquid, stir and react at room temperature for 15 hours, adjust the pH to 3, continue stirring and reacting for 20 minutes, and obtain the reduction liquid.

[0041] In step (1), the amount of calcium chloride added is 0.8% of the mass of the reducing solution.

[0042] In step (1), the magnetic field conditions during stirring at room temperature are: the magnetic field pulse frequency is 800 Hz and the magnetic field intensity is 2 T.

[0043] In step (1), the specific method of n-butanol extraction is: add 1.5 times the volume of n-butanol to the reaction solution, stir at room temperature for 8 minutes, let it stand and discard the upper layer.

[0044] In step (1), the specific method of ultrafiltration and concentration is: ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 3000D until the conductivity of the ultrafiltrate is ≤450μs / cm, and the ultrafiltrate is concentrated to 1 / 4 of the volume of the ultrafiltrate to obtain a concentrated solution.

[0045] In step (1), the specific method of alcohol precipitation is: take 1 kg of concentrated solution, stir and disperse it evenly with 8 kg of purified water, adjust the pH to 6.5, add anhydrous ethanol to make the volume concentration of ethanol 50%, stand at room temperature for 48 hours, and centrifuge to obtain the precipitate.

[0046] In step (2), the temperature of the spin film formation is 60°C.

[0047] In step (2), the PBS solution of nadroparin calcium is obtained by dissolving nadroparin calcium in PBS buffer, wherein the concentration of nadroparin calcium is 0.4 mg / mL and the pH of the PBS buffer is 7.5.

[0048] In step (2), the process conditions of ultrasonic oscillation treatment are: 300W ultrasonic oscillation treatment for 20 minutes.

[0049] Preferably, in step (2), the process conditions of the high-voltage pulse electric field treatment are: ice water bath, electric field strength of 30 kV / cm, electric field frequency of 1000 Hz, and material flow rate of 8 mL / min.

[0050] In step (2), the amount of the magnetic nitrogen-doped activated carbon is 0.1 g / L, and the stirring adsorption time is 20 minutes.

[0051] In step (2), the magnetic nitrogen-doped activated carbon is prepared by the following method: first, 0.1 kg of polyethylene glycol 400, 0.2 kg of linear phenolic resin and 2 kg of diethylene glycol are stirred and mixed to obtain a premix; then, 1 kg of 200-mesh sepiolite powder is soaked in 6 kg of 8 mg / mL ammonium ferric oxalate solution for 15 hours, centrifuged to obtain a precipitate, the precipitate is mixed and stirred with 4 kg of 1 mol / L hydrochloric acid solution for 2 hours, centrifuged to obtain an acidified precipitate; then, the acidified precipitate is added to the premix, stirred and mixed, stirred and treated at 60° C. for 10 hours, 0.1 kg of urea is added, the stirring is continued for 8 hours, vacuum dried, treated at 800° C. for 1 hour under nitrogen protection, and naturally cooled to room temperature to obtain the product.

[0052] In step (2), the microporous membrane filtration includes: filtering once with a 0.45 μm microporous membrane and filtering once with a 0.22 μm microporous membrane.

[0053] In step (2), the process conditions of vacuum freeze drying are: freezing at -40°C for 3 hours, evacuating to below 10 Pa, heating to 20°C, keeping warm and drying for 7 hours, heating to 35°C, keeping warm and drying for 5 hours.

[0054] Embodiment 2:

[0055] A method for preparing a stable nadroparin calcium injection, the specific steps are as follows:

[0056] (1) First, crude heparin sodium is used as a raw material, and is degraded by sodium nitrite after pretreatment and magnetic field conditions to obtain a degradation solution, and reduction reaction is carried out by sodium borohydride to obtain a reduction solution, calcium chloride is added, and the mixture is stirred and mixed, and the mixture is stirred at room temperature for 35 minutes under magnetic field conditions to obtain a reaction solution, and n-butanol extraction, ultrafiltration and concentration are performed to obtain a concentrated solution, and alcohol precipitation is performed to obtain nadroparin calcium; the anti-Xa potency of nadroparin calcium is 127 IU / mg, the anti-Xa / anti-IIa ratio is 3.7, and the weight average molecular weight is 4100D;

[0057] (2) 0.5 kg of Tween-80, 2 kg of soy lecithin, 0.5 kg of cholesterol and 100 kg of anhydrous ethanol are stirred and mixed, and then transferred to a thin film evaporator, and the film is formed by rotation. 60 kg of ether is added to dissolve the film, and then 20 kg of nadroparin calcium PBS solution is added, ultrasonic oscillation treatment is carried out, high-voltage pulse electric field treatment is carried out, magnetic nitrogen-doped activated carbon is added, and adsorption is carried out by stirring. The magnetic nitrogen-doped activated carbon is removed by magnetic separation, and then filtered with a microporous membrane, rotary evaporated, and vacuum freeze-dried to obtain the product.

[0058] In step (1), the specific method of pretreatment and sodium nitrite degradation is as follows: first, 1 kg of crude heparin sodium is added to 10 kg of purified water, stirred to dissolve, then added to 0.02 kg of sodium chloride, stirred at 60° C. for 3 hours, centrifuged to obtain the supernatant, 0.02 kg of hydrogen peroxide is added to the supernatant, adsorbed, washed and eluted using FPA98CL strong alkaline anion exchange resin, the eluate is collected, the pH is adjusted to 6.0, ultrafiltration is performed using a 1000D ultrafiltration membrane with a molecular weight cutoff to obtain an ultrafiltrate, the pH is adjusted to 3, 0.025 kg of sodium nitrite is added, and the reaction is stirred at 25° C. for 2 hours under a 10T magnetic field to obtain a degradation solution.

[0059] In step (1), the specific method of the reduction reaction is: add 0.015 kg of sodium borohydride to 10 kg of the degradation liquid, stir and react at room temperature for 17 hours, adjust the pH to 4, continue stirring and reacting for 30 minutes, and obtain the reduction liquid.

[0060] In step (1), the amount of calcium chloride added is 1% of the mass of the reducing solution.

[0061] In step (1), the magnetic field conditions during stirring at room temperature are: the magnetic field pulse frequency is 1000 Hz and the magnetic field intensity is 3 T.

[0062] In step (1), the specific method of n-butanol extraction is: add 2.5 times the volume of n-butanol to the reaction solution, stir at room temperature for 10 minutes, let it stand and discard the upper layer.

[0063] In step (1), the specific method of ultrafiltration and concentration is: ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 3000D until the conductivity of the ultrafiltrate is ≤450μs / cm, and the ultrafiltrate is concentrated to 1 / 4 of the volume of the ultrafiltrate to obtain a concentrated solution.

[0064] In step (1), the specific method of alcohol precipitation is: take 1 kg of concentrated solution, stir and disperse it evenly with 10 kg of purified water, adjust the pH to 7.0, add anhydrous ethanol to make the volume concentration of ethanol 55%, stand at room temperature for 55 hours, and centrifuge to obtain the precipitate.

[0065] In step (2), the temperature of the spin film formation is 60°C.

[0066] In step (2), the PBS solution of nadroparin calcium is obtained by dissolving nadroparin calcium in PBS buffer, wherein the concentration of nadroparin calcium is 0.5 mg / mL and the pH of the PBS buffer is 7.5.

[0067] In step (2), the process conditions of ultrasonic oscillation treatment are: 400W ultrasonic oscillation treatment for 30 minutes.

[0068] Preferably, in step (2), the process conditions of the high-voltage pulse electric field treatment are: ice water bath, electric field strength 40 kV / cm, electric field frequency 1500 Hz, and material flow rate 10 mL / min.

[0069] In step (2), the amount of the magnetic nitrogen-doped activated carbon is 0.1 g / L, and the stirring adsorption time is 30 minutes.

[0070] In step (2), the magnetic nitrogen-doped activated carbon is prepared by the following method: first, 0.2 kg of polyethylene glycol 400, 0.3 kg of linear phenolic resin and 3 kg of diethylene glycol are stirred and mixed to obtain a premix; then, 1 kg of 300-mesh sepiolite powder is soaked in 8 kg of 10 mg / mL ammonium ferric oxalate solution for 18 hours, centrifuged to obtain a precipitate, the precipitate is mixed and stirred with 5 kg of 1 mol / L hydrochloric acid solution for 3 hours, and centrifuged to obtain an acidified precipitate; then, the acidified precipitate is added to the premix, stirred and mixed, stirred and treated at 70° C. for 12 hours, 0.2 kg of urea is added, the stirring is continued for 10 hours, vacuum dried, treated at 850° C. for 2 hours under nitrogen protection, and naturally cooled to room temperature to obtain the product.

[0071] In step (2), the microporous membrane filtration includes: filtering once with a 0.45 μm microporous membrane and filtering once with a 0.22 μm microporous membrane.

[0072] In step (2), the process conditions of vacuum freeze drying are: freezing at -50°C for 4 hours, evacuating to below 10 Pa, heating to 30°C, keeping warm and drying for 8 hours, heating to 45°C, keeping warm and drying for 6 hours.

[0073] Embodiment 3:

[0074] A method for preparing a stable nadroparin calcium injection, the specific steps are as follows:

[0075] (1) First, crude heparin sodium is used as a raw material, and is degraded by sodium nitrite after pretreatment and magnetic field conditions to obtain a degradation solution, and reduction reaction is carried out by sodium borohydride to obtain a reduction solution, calcium chloride is added, and the mixture is stirred and mixed, and the mixture is stirred at room temperature for 25 minutes under magnetic field conditions to obtain a reaction solution, and n-butanol extraction, ultrafiltration and concentration are performed to obtain a concentrated solution, and alcohol precipitation is performed to obtain nadroparin calcium; the anti-Xa potency of nadroparin calcium is 128 IU / mg, the anti-Xa / anti-IIa ratio is 3.8, and the weight average molecular weight is 4080D;

[0076] (2) 0.5 kg of Tween-80, 1 kg of soy lecithin, 0.5 kg of cholesterol and 100 kg of anhydrous ethanol are stirred and mixed, and then transferred to a thin film evaporator, and the film is formed by rotation. 50 kg of ether is added to dissolve the film, and then 20 kg of nadroparin calcium PBS solution is added, ultrasonic oscillation treatment is carried out, high-voltage pulse electric field treatment is carried out, magnetic nitrogen-doped activated carbon is added, and adsorption is carried out by stirring. The magnetic nitrogen-doped activated carbon is removed by magnetic separation, and then filtered with a microporous membrane, rotary evaporated, and vacuum freeze-dried to obtain the product.

[0077] In step (1), the specific method of pretreatment and sodium nitrite degradation is as follows: first, 1 kg of crude heparin sodium is added to 8 kg of purified water, stirred to dissolve, then added to 0.02 kg of sodium chloride, stirred at 50° C. for 3 hours, centrifuged to obtain the supernatant, 0.01 kg of hydrogen peroxide is added to the supernatant, and adsorption, washing and elution are performed using FPA98CL strong alkaline anion exchange resin, the eluate is collected, the pH is adjusted to 6.0, and ultrafiltration is performed using a 1000D ultrafiltration membrane with a molecular weight cutoff to obtain an ultrafiltrate, the pH is adjusted to 2, 0.025 kg of sodium nitrite is added, and the reaction is stirred at 25° C. for 1 hour under 8T magnetic field conditions to obtain a degradation solution.

[0078] In step (1), the specific method of the reduction reaction is: add 0.015 kg of sodium borohydride to 10 kg of the degradation liquid, stir and react at room temperature for 15 hours, adjust the pH to 4, continue stirring and reacting for 20 minutes, and obtain the reduction liquid.

[0079] In step (1), the amount of calcium chloride added is 1% of the mass of the reducing solution.

[0080] In step (1), the magnetic field conditions during stirring at room temperature are: the magnetic field pulse frequency is 800 Hz and the magnetic field intensity is 3 T.

[0081] In step (1), the specific method of n-butanol extraction is: add 1.5 times the volume of n-butanol to the reaction solution, stir at room temperature for 10 minutes, let it stand and discard the upper layer.

[0082] In step (1), the specific method of ultrafiltration and concentration is: ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 3000D until the conductivity of the ultrafiltrate is ≤450μs / cm, and the ultrafiltrate is concentrated to 1 / 4 of the volume of the ultrafiltrate to obtain a concentrated solution.

[0083] In step (1), the specific method of alcohol precipitation is: take 1 kg of concentrated solution, stir and disperse it evenly with 8 kg of purified water, adjust the pH to 7.0, add anhydrous ethanol to make the volume concentration of ethanol 50%, stand at room temperature for 55 hours, and centrifuge to obtain the precipitate.

[0084] In step (2), the temperature of the spin film formation is 60°C.

[0085] In step (2), the PBS solution of nadroparin calcium is obtained by dissolving nadroparin calcium in PBS buffer, wherein the concentration of nadroparin calcium is 0.4 mg / mL and the pH of the PBS buffer is 7.5.

[0086] In step (2), the process conditions of ultrasonic oscillation treatment are: 400W ultrasonic oscillation treatment for 20 minutes.

[0087] Preferably, in step (2), the process conditions of the high-voltage pulse electric field treatment are: ice water bath, electric field strength 40 kV / cm, electric field frequency 1000 Hz, and material flow rate 10 mL / min.

[0088] In step (2), the amount of the magnetic nitrogen-doped activated carbon is 0.1 g / L, and the stirring adsorption time is 20 minutes.

[0089] In step (2), the magnetic nitrogen-doped activated carbon is prepared by the following method: first, 0.2 kg of polyethylene glycol 400, 0.2 kg of linear phenolic resin and 3 kg of diethylene glycol are stirred and mixed to obtain a premix; then, 1 kg of 200 mesh sepiolite powder is soaked in 8 kg of 8 mg / mL ammonium ferric oxalate solution for 18 hours, centrifuged to obtain a precipitate, the precipitate is mixed and stirred with 4 kg of 1 mol / L hydrochloric acid solution for 3 hours, centrifuged to obtain an acidified precipitate; then, the acidified precipitate is added to the premix, stirred and mixed, stirred and treated at 60° C. for 12 hours, 0.1 kg of urea is added, the stirring is continued for 10 hours, vacuum dried, treated at 800° C. for 2 hours under nitrogen protection, and naturally cooled to room temperature to obtain the product.

[0090] In step (2), the microporous membrane filtration includes: filtering once with a 0.45 μm microporous membrane and filtering once with a 0.22 μm microporous membrane.

[0091] In step (2), the process conditions of vacuum freeze drying are: freezing at -40°C for 4 hours, evacuating to below 10 Pa, heating to 20°C, keeping warm and drying for 8 hours, heating to 35°C, keeping warm and drying for 6 hours.

[0092] Embodiment 4:

[0093] A method for preparing a stable nadroparin calcium injection, the specific steps are as follows:

[0094] (1) First, crude heparin sodium is used as a raw material, and is degraded by sodium nitrite after pretreatment and magnetic field conditions to obtain a degradation solution, and reduction reaction is carried out by sodium borohydride to obtain a reduction solution, calcium chloride is added, and the mixture is stirred and mixed, and the mixture is stirred at room temperature for 30 minutes under magnetic field conditions to obtain a reaction solution, and n-butanol extraction, ultrafiltration and concentration are performed to obtain a concentrated solution, and alcohol precipitation is performed to obtain nadroparin calcium; the anti-Xa potency of nadroparin calcium is 129 IU / mg, the anti-Xa / anti-IIa ratio is 3.8, and the weight average molecular weight is 4050D;

[0095] (2) 0.45 kg of Tween-80, 1.5 kg of soy lecithin, 0.45 kg of cholesterol and 100 kg of anhydrous ethanol are stirred and mixed, and then transferred to a thin film evaporator, and the film is formed by rotation. 55 kg of ether is added to dissolve the film, and then 19 kg of nadroparin calcium PBS solution is added, ultrasonic oscillation treatment is carried out, high-voltage pulse electric field treatment is carried out, magnetic nitrogen-doped activated carbon is added, and adsorption is carried out by stirring. The magnetic nitrogen-doped activated carbon is removed by magnetic separation, and then filtered with a microporous membrane, rotary evaporated, and vacuum freeze-dried to obtain the product.

[0096] In step (1), the specific method of pretreatment and sodium nitrite degradation is as follows: first, 1 kg of crude heparin sodium is added to 9 kg of purified water, stirred to dissolve, then added to 0.015 kg of sodium chloride, stirred at 55° C. for 2.5 hours, centrifuged to obtain the supernatant, 0.015 kg of hydrogen peroxide is added to the supernatant, adsorbed, washed and eluted using FPA98CL strong alkaline anion exchange resin, the eluate is collected, the pH is adjusted to 6.0, ultrafiltration is performed using a 1000D ultrafiltration membrane with a molecular weight cutoff to obtain an ultrafiltrate, the pH is adjusted to 2, 0.025 kg of sodium nitrite is added, and the reaction is stirred at 22° C. for 1.5 hours under 9T magnetic field conditions to obtain a degradation solution.

[0097] In step (1), the specific method of the reduction reaction is: add 0.01 kg of sodium borohydride to 10 kg of the degradation liquid, stir and react at room temperature for 16 hours, adjust the pH to 4, continue stirring and reacting for 25 minutes, and obtain the reduction liquid.

[0098] In step (1), the amount of calcium chloride added is 0.9% of the mass of the reducing solution.

[0099] In step (1), the magnetic field conditions during stirring at room temperature are: the magnetic field pulse frequency is 900 Hz and the magnetic field intensity is 3 T.

[0100] In step (1), the specific method of n-butanol extraction is: add 2 volumes of n-butanol to the reaction solution, stir at room temperature for 9 minutes, let it stand and discard the upper layer.

[0101] In step (1), the specific method of ultrafiltration and concentration is: ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 3000D until the conductivity of the ultrafiltrate is ≤450μs / cm, and the ultrafiltrate is concentrated to 1 / 4 of the volume of the ultrafiltrate to obtain a concentrated solution.

[0102] In step (1), the specific method of alcohol precipitation is: take 1 kg of concentrated solution, stir and disperse it evenly with 9 kg of purified water, adjust the pH to 7.0, add anhydrous ethanol to make the volume concentration of ethanol 52%, stand at room temperature for 50 hours, and centrifuge to obtain the precipitate.

[0103] In step (2), the temperature of the spin film formation is 60°C.

[0104] In step (2), the PBS solution of nadroparin calcium is obtained by dissolving nadroparin calcium in PBS buffer, wherein the concentration of nadroparin calcium is 0.45 mg / mL and the pH of the PBS buffer is 7.5.

[0105] In step (2), the process conditions of ultrasonic oscillation treatment are: 400W ultrasonic oscillation treatment for 25 minutes.

[0106] Preferably, in step (2), the process conditions of the high-voltage pulse electric field treatment are: ice water bath, electric field strength 35 kV / cm, electric field frequency 1200 Hz, and material flow rate 9 mL / min.

[0107] In step (2), the amount of the magnetic nitrogen-doped activated carbon is 0.1 g / L, and the stirring adsorption time is 25 minutes.

[0108] In step (2), the magnetic nitrogen-doped activated carbon is prepared by the following method: first, 0.15 kg of polyethylene glycol 400, 0.25 kg of linear phenolic resin and 2.5 kg of diethylene glycol are stirred and mixed to obtain a premix; then, 1 kg of 300-mesh sepiolite powder is soaked in 7 kg of 9 mg / mL ammonium ferric oxalate solution for 16 hours, centrifuged to obtain a precipitate, the precipitate is mixed and stirred with 4.5 kg of 1 mol / L hydrochloric acid solution for 2.5 hours, centrifuged to obtain an acidified precipitate; then, the acidified precipitate is added to the premix, stirred and mixed, stirred and treated at 65° C. for 11 hours, 0.15 kg of urea is added, the stirring is continued for 9 hours, vacuum dried, treated at 820° C. for 1.5 hours under nitrogen protection, and naturally cooled to room temperature to obtain the product.

[0109] In step (2), the microporous membrane filtration includes: filtering once with a 0.45 μm microporous membrane and filtering once with a 0.22 μm microporous membrane.

[0110] In step (2), the process conditions of vacuum freeze drying are: freezing at -45°C for 3.5 hours, evacuating to below 10 Pa, heating to 25°C, keeping warm and drying for 7.5 hours, heating to 40°C, keeping warm and drying for 5.5 hours.

[0111] Comparative Example 1

[0112] A method for preparing nadroparin calcium injection, the specific steps are as follows:

[0113] (1) First, crude heparin sodium is used as a raw material, pretreated and degraded with sodium nitrite to obtain a degradation solution, and reduction reaction is carried out with sodium borohydride to obtain a reduction solution, calcium chloride is added, stirred and mixed, and stirred at room temperature for 25 minutes to obtain a reaction solution, extracted with n-butanol, ultrafiltered and concentrated to obtain a concentrated solution, and precipitated with alcohol to obtain nadroparin calcium; the anti-Xa potency of nadroparin calcium is 115 IU / mg, the anti-Xa / anti-IIa ratio is 3.2, and the weight average molecular weight is 4600D;

[0114] (2) 0.4 kg of Tween-80, 1 kg of soy lecithin, 0.4 kg of cholesterol and 100 kg of anhydrous ethanol are stirred and mixed, and then transferred to a thin film evaporator, and the film is formed by rotation. 50 kg of ether is added to dissolve the film, and then 18 kg of nadroparin calcium PBS solution is added, ultrasonic oscillation treatment is carried out, high-voltage pulse electric field treatment is carried out, magnetic nitrogen-doped activated carbon is added, and adsorption is carried out by stirring. The magnetic nitrogen-doped activated carbon is removed by magnetic separation, and then filtered with a microporous membrane, rotary evaporated, and vacuum freeze-dried to obtain the product.

[0115] In step (1), the specific method of pretreatment and sodium nitrite degradation is as follows: first, 1 kg of crude heparin sodium is added to 8 kg of purified water, stirred to dissolve, then added to 0.01 kg of sodium chloride, stirred at 50° C. for 2 hours, centrifuged to obtain the supernatant, 0.01 kg of hydrogen peroxide is added to the supernatant, and adsorption, washing and elution are performed using FPA98CL strong alkaline anion exchange resin, the eluate is collected, the pH is adjusted to 5.5, and ultrafiltration is performed using a 1000D ultrafiltration membrane with a molecular weight cutoff to obtain an ultrafiltrate, the pH is adjusted to 2, 0.02 kg of sodium nitrite is added, and the reaction is stirred at 20° C. for 1 hour to obtain a degradation solution.

[0116] In step (1), the specific method of the reduction reaction is: add 0.01 kg of sodium borohydride to 10 kg of the degradation liquid, stir and react at room temperature for 15 hours, adjust the pH to 3, continue stirring and reacting for 20 minutes, and obtain the reduction liquid.

[0117] In step (1), the amount of calcium chloride added is 0.8% of the mass of the reducing solution.

[0118] In step (1), the specific method of n-butanol extraction is: add 1.5 times the volume of n-butanol to the reaction solution, stir at room temperature for 8 minutes, let it stand and discard the upper layer.

[0119] In step (1), the specific method of ultrafiltration and concentration is: ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 3000D until the conductivity of the ultrafiltrate is ≤450μs / cm, and the ultrafiltrate is concentrated to 1 / 4 of the volume of the ultrafiltrate to obtain a concentrated solution.

[0120] In step (1), the specific method of alcohol precipitation is: take 1 kg of concentrated solution, stir and disperse it evenly with 8 kg of purified water, adjust the pH to 6.5, add anhydrous ethanol to make the volume concentration of ethanol 50%, stand at room temperature for 48 hours, and centrifuge to obtain the precipitate.

[0121] In step (2), the temperature of the spin film formation is 60°C.

[0122] In step (2), the PBS solution of nadroparin calcium is obtained by dissolving nadroparin calcium in PBS buffer, wherein the concentration of nadroparin calcium is 0.4 mg / mL and the pH of the PBS buffer is 7.5.

[0123] In step (2), the process conditions of ultrasonic oscillation treatment are: 300W ultrasonic oscillation treatment for 20 minutes.

[0124] Preferably, in step (2), the process conditions of the high-voltage pulse electric field treatment are: ice water bath, electric field strength of 30 kV / cm, electric field frequency of 1000 Hz, and material flow rate of 8 mL / min.

[0125] In step (2), the amount of the magnetic nitrogen-doped activated carbon is 0.1 g / L, and the stirring adsorption time is 20 minutes.

[0126] In step (2), the magnetic nitrogen-doped activated carbon is prepared by the following method: first, 0.1 kg of polyethylene glycol 400, 0.2 kg of linear phenolic resin and 2 kg of diethylene glycol are stirred and mixed to obtain a premix; then, 1 kg of 200-mesh sepiolite powder is soaked in 6 kg of 8 mg / mL ammonium ferric oxalate solution for 15 hours, centrifuged to obtain a precipitate, the precipitate is mixed and stirred with 4 kg of 1 mol / L hydrochloric acid solution for 2 hours, centrifuged to obtain an acidified precipitate; then, the acidified precipitate is added to the premix, stirred and mixed, stirred and treated at 60° C. for 10 hours, 0.1 kg of urea is added, the stirring is continued for 8 hours, vacuum dried, treated at 800° C. for 1 hour under nitrogen protection, and naturally cooled to room temperature to obtain the product.

[0127] In step (2), the microporous membrane filtration includes: filtering once with a 0.45 μm microporous membrane and filtering once with a 0.22 μm microporous membrane.

[0128] In step (2), the process conditions of vacuum freeze drying are: freezing at -40°C for 3 hours, evacuating to below 10 Pa, heating to 20°C, keeping warm and drying for 7 hours, heating to 35°C, keeping warm and drying for 5 hours.

[0129] Comparative Example 2

[0130] A method for preparing nadroparin calcium injection, the specific steps are as follows:

[0131] (1) First, crude heparin sodium is used as a raw material, and is degraded by sodium nitrite after pretreatment and magnetic field conditions to obtain a degradation solution, and reduction reaction is carried out by sodium borohydride to obtain a reduction solution, calcium chloride is added, and the mixture is stirred and mixed, and the mixture is stirred at room temperature for 25 minutes under magnetic field conditions to obtain a reaction solution, and n-butanol extraction, ultrafiltration and concentration are performed to obtain a concentrated solution, and alcohol precipitation is performed to obtain nadroparin calcium; the anti-Xa potency of nadroparin calcium is 128 IU / mg, the anti-Xa / anti-IIa ratio is 3.7, and the weight average molecular weight is 4100D;

[0132] (2) 0.4 kg of Tween-80, 1 kg of soy lecithin, 0.4 kg of cholesterol and 100 kg of anhydrous ethanol are stirred and mixed, and then transferred to a thin film evaporator, and the film is formed by rotation. 50 kg of ether is added to dissolve the film, and then 18 kg of nadroparin calcium PBS solution is added, and ultrasonic oscillation is performed. Magnetic nitrogen-doped activated carbon is added, and adsorption is stirred. The magnetic nitrogen-doped activated carbon is removed by magnetic separation, and then filtered with a microporous membrane, rotary evaporated, and vacuum freeze-dried to obtain the product.

[0133] In step (1), the specific method of pretreatment and sodium nitrite degradation is as follows: first, 1 kg of crude heparin sodium is added to 8 kg of purified water, stirred to dissolve, then added to 0.01 kg of sodium chloride, stirred at 50° C. for 2 hours, centrifuged to obtain the supernatant, 0.01 kg of hydrogen peroxide is added to the supernatant, adsorbed, washed and eluted using FPA98CL strong alkaline anion exchange resin, the eluate is collected, the pH is adjusted to 5.5, ultrafiltration is performed using a 1000D ultrafiltration membrane with a molecular weight cutoff to obtain an ultrafiltrate, the pH is adjusted to 2, 0.02 kg of sodium nitrite is added, and the reaction is stirred at 20° C. for 1 hour under 8T magnetic field conditions to obtain a degradation solution.

[0134] In step (1), the specific method of the reduction reaction is: add 0.01 kg of sodium borohydride to 10 kg of the degradation liquid, stir and react at room temperature for 15 hours, adjust the pH to 3, continue stirring and reacting for 20 minutes, and obtain the reduction liquid.

[0135] In step (1), the amount of calcium chloride added is 0.8% of the mass of the reducing solution.

[0136] In step (1), the magnetic field conditions during stirring at room temperature are: the magnetic field pulse frequency is 800 Hz and the magnetic field intensity is 2 T.

[0137] In step (1), the specific method of n-butanol extraction is: add 1.5 times the volume of n-butanol to the reaction solution, stir at room temperature for 8 minutes, let it stand and discard the upper layer.

[0138] In step (1), the specific method of ultrafiltration and concentration is: ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 3000D until the conductivity of the ultrafiltrate is ≤450μs / cm, and the ultrafiltrate is concentrated to 1 / 4 of the volume of the ultrafiltrate to obtain a concentrated solution.

[0139] In step (1), the specific method of alcohol precipitation is: take 1 kg of concentrated solution, stir and disperse it evenly with 8 kg of purified water, adjust the pH to 6.5, add anhydrous ethanol to make the volume concentration of ethanol 50%, stand at room temperature for 48 hours, and centrifuge to obtain the precipitate.

[0140] In step (2), the temperature of the spin film formation is 60°C.

[0141] In step (2), the PBS solution of nadroparin calcium is obtained by dissolving nadroparin calcium in PBS buffer, wherein the concentration of nadroparin calcium is 0.4 mg / mL and the pH of the PBS buffer is 7.5.

[0142] In step (2), the process conditions of ultrasonic oscillation treatment are: 300W ultrasonic oscillation treatment for 20 minutes.

[0143] In step (2), the amount of the magnetic nitrogen-doped activated carbon is 0.1 g / L, and the stirring adsorption time is 20 minutes.

[0144] In step (2), the magnetic nitrogen-doped activated carbon is prepared by the following method: first, 0.1 kg of polyethylene glycol 400, 0.2 kg of linear phenolic resin and 2 kg of diethylene glycol are stirred and mixed to obtain a premix; then, 1 kg of 200-mesh sepiolite powder is soaked in 6 kg of 8 mg / mL ammonium ferric oxalate solution for 15 hours, centrifuged to obtain a precipitate, the precipitate is mixed and stirred with 4 kg of 1 mol / L hydrochloric acid solution for 2 hours, centrifuged to obtain an acidified precipitate; then, the acidified precipitate is added to the premix, stirred and mixed, stirred and treated at 60° C. for 10 hours, 0.1 kg of urea is added, the stirring is continued for 8 hours, vacuum dried, treated at 800° C. for 1 hour under nitrogen protection, and naturally cooled to room temperature to obtain the product.

[0145] In step (2), the microporous membrane filtration includes: filtering once with a 0.45 μm microporous membrane and filtering once with a 0.22 μm microporous membrane.

[0146] In step (2), the process conditions of vacuum freeze drying are: freezing at -40°C for 3 hours, evacuating to below 10 Pa, heating to 20°C, keeping warm and drying for 7 hours, heating to 35°C, keeping warm and drying for 5 hours.

[0147] Comparative Example 3

[0148] A method for preparing nadroparin calcium injection, the specific steps are as follows:

[0149] (1) First, crude heparin sodium is used as a raw material, and is degraded by sodium nitrite after pretreatment and magnetic field conditions to obtain a degradation solution, and reduction reaction is carried out by sodium borohydride to obtain a reduction solution, calcium chloride is added, and the mixture is stirred and mixed, and the mixture is stirred at room temperature for 25 minutes under magnetic field conditions to obtain a reaction solution, and n-butanol extraction, ultrafiltration and concentration are performed to obtain a concentrated solution, and alcohol precipitation is performed to obtain nadroparin calcium; the anti-Xa potency of nadroparin calcium is 128 IU / mg, the anti-Xa / anti-IIa ratio is 3.7, and the weight average molecular weight is 4100D;

[0150] (2) 0.4 kg of Tween-80, 1 kg of soy lecithin, 0.4 kg of cholesterol and 100 kg of anhydrous ethanol were stirred and mixed, and then transferred to a thin film evaporator and rotated to form a film. 50 kg of ether was added to dissolve the film, and then 18 kg of nadroparin calcium PBS solution was added. The mixture was treated with ultrasonic oscillation and high-voltage pulse electric field. Activated carbon (purchased from Xi'an Tianzheng Pharmaceutical Excipients Co., Ltd.) was added, stirred for adsorption, and the activated carbon was removed by centrifugation. The supernatant was collected, filtered with a microporous membrane, rotary evaporated, and vacuum freeze-dried to obtain the product.

[0151] In step (1), the specific method of pretreatment and sodium nitrite degradation is as follows: first, 1 kg of crude heparin sodium is added to 8 kg of purified water, stirred to dissolve, then added to 0.01 kg of sodium chloride, stirred at 50° C. for 2 hours, centrifuged to obtain the supernatant, 0.01 kg of hydrogen peroxide is added to the supernatant, adsorbed, washed and eluted using FPA98CL strong alkaline anion exchange resin, the eluate is collected, the pH is adjusted to 5.5, ultrafiltration is performed using a 1000D ultrafiltration membrane with a molecular weight cutoff to obtain an ultrafiltrate, the pH is adjusted to 2, 0.02 kg of sodium nitrite is added, and the reaction is stirred at 20° C. for 1 hour under 8T magnetic field conditions to obtain a degradation solution.

[0152] In step (1), the specific method of the reduction reaction is: add 0.01 kg of sodium borohydride to 10 kg of the degradation liquid, stir and react at room temperature for 15 hours, adjust the pH to 3, continue stirring and reacting for 20 minutes, and obtain the reduction liquid.

[0153] In step (1), the amount of calcium chloride added is 0.8% of the mass of the reducing solution.

[0154] In step (1), the magnetic field conditions during stirring at room temperature are: the magnetic field pulse frequency is 800 Hz and the magnetic field intensity is 2 T.

[0155] In step (1), the specific method of n-butanol extraction is: add 1.5 times the volume of n-butanol to the reaction solution, stir at room temperature for 8 minutes, let it stand and discard the upper layer.

[0156] In step (1), the specific method of ultrafiltration and concentration is: ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 3000D until the conductivity of the ultrafiltrate is ≤450μs / cm, and the ultrafiltrate is concentrated to 1 / 4 of the volume of the ultrafiltrate to obtain a concentrated solution.

[0157] In step (1), the specific method of alcohol precipitation is: take 1 kg of concentrated solution, stir and disperse it evenly with 8 kg of purified water, adjust the pH to 6.5, add anhydrous ethanol to make the volume concentration of ethanol 50%, stand at room temperature for 48 hours, and centrifuge to obtain the precipitate.

[0158] In step (2), the temperature of the spin film formation is 60°C.

[0159] In step (2), the PBS solution of nadroparin calcium is obtained by dissolving nadroparin calcium in PBS buffer, wherein the concentration of nadroparin calcium is 0.4 mg / mL and the pH of the PBS buffer is 7.5.

[0160] In step (2), the process conditions of ultrasonic oscillation treatment are: 300W ultrasonic oscillation treatment for 20 minutes.

[0161] Preferably, in step (2), the process conditions of the high-voltage pulse electric field treatment are: ice water bath, electric field strength of 30 kV / cm, electric field frequency of 1000 Hz, and material flow rate of 8 mL / min.

[0162] In step (2), the amount of activated carbon used is 0.1 g / L, and the stirring adsorption time is 20 minutes.

[0163] In step (2), the microporous membrane filtration includes: filtering once with a 0.45 μm microporous membrane and filtering once with a 0.22 μm microporous membrane.

[0164] In step (2), the process conditions of vacuum freeze drying are: freezing at -40°C for 3 hours, evacuating to below 10 Pa, heating to 20°C, keeping warm and drying for 7 hours, heating to 35°C, keeping warm and drying for 5 hours.

[0165] Test example

[0166] The nadroparin calcium injections obtained in Examples 1 to 4 and Comparative Examples 1 to 3 were dissolved in injection water to prepare nadroparin calcium injections with a specification of 0.6 ml: 6150AXaIU. Accelerated tests (temperature 40° C., humidity 75%) and long-term tests (temperature 25° C., humidity 60%) were performed. The test results are shown in Tables 1 and 2.

[0167] Table 1. Accelerated test results

[0168]

[0169]

[0170] Table 2. Long-term test results

[0171]

[0172]

[0173] It can be seen from Table 1 and Table 2 that the nadroparin calcium injection obtained in Examples 1 to 4 has good stability; in Comparative Example 1, no magnetic field conditions were applied during the degradation of sodium nitrite and the calcium conversion by stirring at room temperature, Comparative Example 2 omitted the high-voltage pulse electric field treatment, and Comparative Example 3 replaced the magnetic nitrogen-doped activated carbon with commercially available activated carbon, and the stability was significantly deteriorated, especially under high temperature conditions, indicating that the synergistic effects of degradation, control of the degree of calcium conversion, and impurity removal treatment in the preparation process improve the product stability.

[0174] Although the above describes the specific implementation methods of the present invention, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A method for preparing a stable nadroparin calcium injection, characterized in that: The specific steps are as follows: (1) First, crude heparin sodium is used as a raw material, and is degraded by sodium nitrite under pretreatment and magnetic field conditions to obtain a degradation solution, and a reduction reaction is carried out by sodium borohydride to obtain a reduction solution, calcium chloride is added, and the mixture is stirred and mixed, and the mixture is stirred at room temperature under magnetic field conditions for 25 to 35 minutes to obtain a reaction solution, and the mixture is extracted with n-butanol, ultrafiltered and concentrated to obtain a concentrated solution, and alcohol precipitation is performed to obtain nadroparin calcium; (2) In parts by weight, 0.4-0.5 parts of Tween-80, 1-2 parts of soybean lecithin, 0.4-0.5 parts of cholesterol and 100 parts of anhydrous ethanol are stirred and mixed, transferred to a thin film evaporator, and rotated to form a film, 50-60 parts of ether are added to dissolve the film, and then 18-20 parts of nadroparin calcium PBS solution are added, ultrasonic oscillation treatment, high-voltage pulse electric field treatment, magnetic nitrogen-doped activated carbon is added, stirred for adsorption, magnetic nitrogen-doped activated carbon is removed by magnetic separation, filtered with a microporous membrane, rotary evaporated, and vacuum freeze-dried to obtain; In step (1), the specific method of pretreatment and degradation of sodium nitrite under magnetic field conditions is as follows: first, add 1 part of crude heparin sodium to 8-10 parts of purified water, stir to dissolve, then add 0.01-0.02 parts of sodium chloride, stir at 50-60° C. for 2-3 hours, centrifuge to obtain supernatant, add 0.01-0.02 parts of hydrogen peroxide to the supernatant, use FPA98CL strong alkaline anion exchange resin for adsorption, washing and elution, collect the eluate, adjust the pH to 5.5-6.0, and perform ultrafiltration with a 1000D ultrafiltration membrane to obtain an ultrafiltrate, adjust the pH to 2-3, add 0.02-0.025 parts of sodium nitrite, and react at 8-10T magnetic field at 20-25° C. for 1-2 hours to obtain a degradation solution; In step (2), the process conditions of high-voltage pulse electric field treatment are: ice water bath, electric field strength 30-40 kV / cm, electric field frequency 1000-1500 Hz, material flow rate 8-10 mL / min; The dosage of the magnetic nitrogen-doped activated carbon is 0.1 g / L, and the stirring adsorption time is 20 to 30 minutes; The magnetic nitrogen-doped activated carbon is prepared by the following method, in parts by weight: first, 0.1-0.2 parts of polyethylene glycol 400, 0.2-0.3 parts of linear phenolic resin and 2-3 parts of diethylene glycol are stirred and mixed to obtain a premixed solution; then, 1 part of 200-300 mesh sepiolite powder is soaked in 6-8 parts of 8-10 mg / mL ammonium ferric oxalate solution for 15-18 hours, centrifuged to obtain a precipitate, the precipitate is mixed and stirred with 4-5 parts of 1 mol / L hydrochloric acid solution for 2-3 hours, centrifuged to obtain an acidified precipitate; then, the acidified precipitate is added to the premixed solution, stirred and mixed, stirred and treated at 60-70°C for 10-12 hours, 0.1-0.2 parts of urea are added, the stirring is continued for 8-10 hours, vacuum dried, treated at 800-850°C for 1-2 hours under nitrogen protection, and naturally cooled to room temperature to obtain the obtained product.

2. The preparation method according to claim 1, characterized in that: In step (1), the specific method of the reduction reaction is as follows: add 0.01 to 0.015 parts of sodium borohydride to 10 parts of the degradation solution, stir and react at room temperature for 15 to 17 hours, adjust the pH to 3 to 4, continue stirring and reacting for 20 to 30 minutes, and obtain a reduction solution.

3. The preparation method according to claim 1, characterized in that: In step (1), the amount of calcium chloride added is 0.8-1% of the mass of the reducing solution; The magnetic field conditions during stirring at room temperature are: magnetic field pulse frequency is 800-1000 Hz, magnetic field intensity is 2-3 T; The specific method of n-butanol extraction is: add 1.5 to 2.5 times the volume of n-butanol to the reaction solution, stir at room temperature for 8 to 10 minutes, and discard the upper layer after standing; The specific method of ultrafiltration and concentration is: ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 3000D until the conductivity of the ultrafiltrate is ≤450μs / cm, and the ultrafiltrate is concentrated to 1 / 4 of the volume of the ultrafiltrate to obtain a concentrated solution; The specific method of alcohol precipitation is: take 1 part of the concentrated solution, stir and disperse it evenly with 8-10 parts of purified water, adjust the pH to 6.5-7.0, add anhydrous ethanol to make the volume concentration of ethanol 50-55%, stand at room temperature for 48-55 hours, and centrifuge to obtain the precipitate.

4. The preparation method according to claim 1, characterized in that: In step (2), the temperature of the spin film formation is 60°C.

5. The preparation method according to claim 1, characterized in that: In step (2), the PBS solution of nadroparin calcium is obtained by dissolving nadroparin calcium in PBS buffer, wherein the concentration of nadroparin calcium is 0.4-0.5 mg / mL, and the pH of the PBS buffer is 7.

5.

6. The preparation method according to claim 1, characterized in that: In step (2), the process conditions of ultrasonic oscillation treatment are: 300-400W ultrasonic oscillation treatment for 20-30 minutes.

7. The preparation method according to claim 1, characterized in that: In step (2), the microporous membrane filtration comprises: filtering once with a 0.45 μm microporous membrane and filtering once with a 0.22 μm microporous membrane; The process conditions of vacuum freeze drying are: freezing at -40 to -50°C for 3 to 4 hours, evacuating to below 10Pa, heating to 20 to 30°C, keeping warm and drying for 7 to 8 hours, heating to 35 to 45°C, keeping warm and drying for 5 to 6 hours.

8. A stable nadroparin calcium injection, characterized in that: The invention is obtained by the preparation method according to any one of claims 1 to 7.

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