CN104193849A - Production method of enoxaparin sodium - Google Patents
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- CN104193849A CN104193849A CN201410398891.0A CN201410398891A CN104193849A CN 104193849 A CN104193849 A CN 104193849A CN 201410398891 A CN201410398891 A CN 201410398891A CN 104193849 A CN104193849 A CN 104193849A
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Abstract
The invention discloses a production method of enoxaparin sodium, which comprises the following steps: carrying out enzymolysis on heparin sodium, carrying out HPLC (high performance liquid chromatography) analysis, calculating the contents of various peaks by a peak area normalization process, determining the varieties of disaccharides and tetrasaccharides of the heparin sodium subjected to enzymolysis according to a standard spectrum of disaccharides and tetrasaccharides after heparin sodium enzymolysis, comparing with the contents of disaccharides and tetrasaccharides of the standard spectrum, and selecting the satisfactory heparin sodium raw material to produce the enoxaparin sodium. The method is simple and effective, can ensure the safety and effectiveness of the product, controls the quality of the enoxaparin sodium from the source, can avoid the waste of the active pharmaceutical ingredient heparin sodium, greatly saves the cost and enhances the production efficiency.
Description
Technical field
The present invention relates to biomedicine field, specifically a kind of production method of Enoxaparin Sodium.
Background technology
Heparin is that the disaccharide unit being formed by L-iduronic acid (or glucuronic acid) and N-acetyl-glucosamine (or GLUCOSAMINE) repeats the Sulfated linear polysaccharide of height forming, there is blood coagulation resisting function, be widely used in treating phlebothrombosis and pre-anticoagulant.Low molecular heparin is component or the fragment in unfraction heparin with lower molecular weight, is heparin class antithrombotic reagent of new generation.Heparin just can obtain Low molecular heparin through the mode Partial digestion of enzyme or chemistry.Low molecular heparin series products is because of molecular weight, be difficult for being neutralized by the IV factor, anti-freezing effect and fibrinolysis activity are strengthened, and antiplatelet, affect platelet function, affect blood coagulation, bringing out hemorrhage effect greatly weakens, in addition bioavailability is up to 98%, dose-effect relationship is clear and definite, anti-freezing effect is easy to prediction, plasma half-life is long 2～3 times compared with unfractionated heparin, there is quick and lasting anti-thrombosis function simultaneously, improve haemodynamics, thereby domestic market is with nadroparin calcium, Enoxaparin Sodium is that the Low molecular heparin series products of representative progressively replaces unfractionated heparin product.Enoxaparin Sodium is the novel anticoagulant of a kind of current sales volume maximum developed by French Sanofi-Aventis.
Due to the diversity of low molecular sodium heparin structure, therefore the structure of low molecular sodium heparin is determined extremely important.FDA requires to prove imitated medicine and originate in family's medicine to have biological consistence from structure.Therefore, the analytical procedure of Low molecular heparin fine structure becomes the focus of academia and pharmaceutical industry concern very soon.
Enoxaparin Sodium needs disaccharides that the strict Enoxaparin Sodium of controlling after degradable produces and the content of tetrose, and Enoxaparin Sodium exclusive 1, the content of 6-dehydrogenation ring structure, to ensure validity and the security of product.Patent CN102792158B provides a kind of capillary electrophoresis method of the Enoxaparin Sodium characteristic oligosaccharide structure for quantitative analysis.Patent CN102323355B provides a kind of enzymolysis-HPLC to detect the method for enoxaparin.These two kinds of structure analysis methods that method is all Enoxaparin Sodium, are the quality controls to finished product, cannot the quality to Enoxaparin Sodium control from source.
Heparin bulk drug can directly be used to make standard heparin preparation, or is further processed into Low molecular heparin bulk drug, then makes low molecular weight heparin preparations.Heparin sodium is to extract and make from the mucous membrane of small intestine of fresh healthy live pig, due to the otherness of raw material, causes the otherness of prepared heparin sodium structure.Similarly, due to different batches heparin sodium textural difference, make also to there are differences between the dalteparin sodium that obtains of processing batch.In the production of dalteparin sodium, exist and be not suitable for because of the structure of heparin sodium, cause the non-compliant situation of structure of this batch of dalteparin sodium product, not only lose time, man power and material, and be unfavorable for producing and economical.
Summary of the invention
Object of the present invention is exactly for above-mentioned the deficiencies in the prior art, a kind of production method of Enoxaparin Sodium is provided, this law comprises the screening to raw material, the present invention is simply effective, not only can ensure the security validity of product, and can avoid the waste of heparin sodium raw material, improve production efficiency.
Object of the present invention can reach by following measures:
A production method for Enoxaparin Sodium, it comprises the steps:
1) heparin sodium enzymolysis: heparin sodium preparation of raw material is become to the aqueous solution, add to it mixing heparinase solution that contains heparinase I, heparinaseⅡ and heparinase III, carry out enzymolysis 64～72 hours, make heparin sodium enzymolysis solution; Wherein mix the content of each heparinase in heparinase solution than being heparinase I: heparinaseⅡ: heparinase III=1:0.6:0.2;
2) compartment analysis: described heparin sodium enzymolysis solution is injected to HPLC and detect, record color atlas, according to the retention time of the disaccharides of heparin sodium and tetrose, determine disaccharides in heparin sodium to be measured and the kind of tetrose, and calculate the content of 10 disaccharides and 2 tetroses after heparin sodium enzymolysis to be measured; Wherein testing conditions is: chromatographic column is quaternary ammonium type reinforcing yin essence ion-exchange chromatography, detector is UV-detector, detection wavelength is 220～240nm, use mobile phase A and Mobile phase B gradient elution, described mobile phase A is sodium dihydrogen phosphate, and described Mobile phase B is the mixing solutions of SODIUM PHOSPHATE, MONOBASIC and sodium perchlorate;
3) selection of crude drugs: choose the content of disaccharides and tetrose meets following requirement after enzymolysis heparin sodium to be measured as enoxaparin sodium raw materials; Wherein the peak area of disaccharides and tetrose chromatographic peak respectively 1.8%～3.7% ,≤1.0%, 1.8%～3.6%, 2.3%～4.4%, 1.1%～2.1% ,≤3.9%, 8.4%～11.9%, 4.0%～8.8%, 1.1%～2.0%, in≤1.0%, 52.4%～66.9%, 1.6%～3.4% scope;
4) produce Enoxaparin Sodium: taking step 3) in the heparin sodium chosen be raw material, through quaternary ammonium salinization, esterification and oxidizing reaction, obtain Enoxaparin Sodium.
In step 1) in, hydrolysis temperature is preferably 25 DEG C; The content that mixes heparinase I in heparinase solution is 1.0/3IU/mL, and the content of heparinaseⅡ is 0.6/3IU/mL, and the content of heparinase III is 0.2/3IU/mL.
In step 2) in, the preparation of moving phase is preferably: moving phase is mobile phase A and Mobile phase B, described mobile phase A is the sodium dihydrogen phosphate of 0.20～0.30g/L, the mixing solutions of the SODIUM PHOSPHATE, MONOBASIC that described Mobile phase B is 0.20～0.30g/L and the sodium perchlorate of 120～140g/L.In HPLC testing process, sample size is 5～20 μ L, and flow velocity is 0.2～1.2mL/min, and column temperature is 50～55 DEG C.
In step 2) in, instrument condition: instrument is high performance liquid chromatograph (HPLC), chromatographic column quaternary ammonium type reinforcing yin essence ion-exchange chromatography, detector used is UV-detector, detection wavelength is 220～240nm, and sample size is 5～20 μ L, and flow velocity is 0.2～1.2mL/min, column temperature is 50～55 DEG C, uses the process of mobile phase A and Mobile phase B gradient elution preferably in table 1.
Table 1
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 97 | 3 |
| 20 | 65 | 35 |
| 50 | 0 | 100 |
| 60 | 0 | 100 |
| 61 | 97 | 3 |
| 79 | 97 | 3 |
In step 2) in, solution after heparin sodium enzymolysis is injected to HPLC, record color atlas, according to the standard disaccharides of existing heparin sodium and the retention time of tetrose, determine disaccharides in heparin sodium to be measured and the kind of tetrose, and calculate the content of disaccharides and tetrose after heparin sodium enzymolysis to be measured, after heparin sodium enzymolysis, disaccharides has 10, respectively △ IVA, △ IVSgal, △ IVS, △ IIA, △ IIIA, △ IISgal, △ IIS, △ IIIS, △ IA, △ IS, tetrose has 2, respectively △ IIA-IVSglu and △ IIA-IISglu, the disaccharides of described heparin sodium and the requirement of tetrose see the following form:
In step 3) in, according to step 2) the whether content range requirement in conformance with standard of content of disaccharides and tetrose after the heparin sodium enzymolysis to be measured that calculates, judge with this whether heparin sodium raw material is suitable for producing Enoxaparin Sodium.After the Enoxaparin Sodium raw material enzymolysis of wherein choosing, the content requirement of disaccharides and tetrose is in table 2.
Table 2
| Peak number | Peak definition | Retention time | Peak area per-cent |
| 1 | △IVA | 7.0～7.5min | 1.8％～3.7％ |
| 2 | △IVSgal | 14.0～15.0min | P～≤1.0％ |
| 3 | △IVS | 14.2～15.2min | 1.8％～3.6％ |
| 4 | △IIA | 16.3～16.8min | 2.3％～4.4％ |
| 5 | △IIIA | 18.0～19.0min | 1.1％～2.1％ |
| 6 | △IISgal | 20.5～21.5min | ≤3.9％ |
| 7 | △IIS | 21.5～23.0min | 8.4％～11.9％ |
| 8 | △IIIS | 23.5～25.0min | 4.0％～8.8％ |
| 9 | △IA | 26.0～28.0min | 1.1％～2.0％ |
| 10 | △IIA-IVSglu | 29.5～31.0min | P～≤1.0％ |
| 11 | △IS | 30.5～32.0min | 52.4％～66.9％ |
| 12 | △IIA-IISglu | 33.0～35.0min | 1.6％～3.4％ |
Wherein " P " represents that this peak must exist.
In step 4) in, process taking heparin sodium as raw material production Enoxaparin Sodium is through quaternary ammonium salinization, esterification and oxidizing reaction, and the separation matching with it and purification step, a kind of production process is: take the heparin sodium raw material filtering out, be dissolved in water and make 8%～15% heparin sodium aqua, at 20～30 DEG C, slowly add Bian rope chlorine ammonium solution, fully mix, centrifugation obtains quaternary ammonium salt heparin, repeat to be dried after washing and centrifugally operated, add Benzyl Chloride to stirring and dissolving after adding methylene dichloride in quaternary ammonium salt heparin, keep 29～31 DEG C of solution temperatures to carry out esterification and obtain esterifying liquid, methanol solution precipitation to esterifying liquid with anhydrous sodium acetate, water and sodium chloride solution with after methanol extraction again, obtain esterification heparin, get after esterification heparin purified water is dissolved and add sodium hydroxide solution reaction, use again acid-conditioning solution pH value to 7.0-7.5 use methanol extraction, after being added to purified water solvent soln, throw out adds hydrogen peroxide, at pH value 10.5-11.0, temperature 25-30 DEG C, carry out oxidizing reaction, regulator solution PH6.1～6.3 after reaction, with membrane filtration and by ethanol precipitation, throw out is dehydrated and obtains Enoxaparin Sodium again.Some optimum conditions in this step are: the add-on of Bian rope oronain solid is 1～5 times of heparin sodium weight, be preferably 2～3 times, more preferably 2.5 times, the mass content of Bian rope chlorine ammonium solution is 5～15%, the drying temperature of described quaternary ammonium salt heparin is 47～53 DEG C, be 60～70h time of drying, and the add-on of the methanol solution of described anhydrous sodium acetate is methylene dichloride and Benzyl Chloride cumulative volume 1.2 times; The weightmeasurement ratio of quaternary ammonium salt heparin and methylene dichloride is 1:3.5 (g/ml), and the weightmeasurement ratio of quaternary ammonium salt heparin and Benzyl Chloride is 1:1 (g/ml), and reaction time of esterification is 24～26h; When esterification heparin reacts with sodium hydroxide solution, the mass ratio of esterification heparin and sodium hydroxide is 10:1.
In a kind of preferred version, step 4) process comprise following operation: take the heparin sodium raw material filtering out, be dissolved in water and make 8%～15% heparin sodium aqua, at 20～30 DEG C, slowly adding mass content is 10% Bian rope chlorine ammonium solution, fully mix, centrifugation obtains quaternary ammonium salt heparin, add the purified water of 35～40 times of heparin sodium volumes, agitator treating 30min, centrifugal, repeated washing, centrifugally operated 3 times, quaternary ammonium salt heparin after centrifugal is put into vacuum drying oven dry, add methylene dichloride by the 1:3.5 of quaternary ammonium salt heparin weight (w/v), at 30 ± 1 DEG C, be stirred to completely and dissolve, add Benzyl Chloride by quaternary ammonium salt heparin weight 1:1 (w/v), keep 30 ± 1 DEG C of solution temperatures, esterification 25h ± 30min, obtain esterifying liquid, the methanol solution of the anhydrous sodium acetate of 10% (w/v) is slowly added in esterifying liquid, after fully mixing, centrifugal, material after centrifugal dissolves by purified water, the 4.5-5.0 that the rear volume of dissolving is about heparin quaternary ammonium salt weight doubly measures (w/v), add the sodium-chlor of liquor capacity 8% (w/v), after stirring is dissolved sodium-chlor, add the methanol extraction of liquor capacity 2.5 times (v/v), leave standstill 2～4 hours, supernatant discarded, throw out dehydration, after centrifugal, 45 ± 3 DEG C, dry 30-35h, obtain esterification heparin, take esterification heparin, add purified water stirring and dissolving to obtain esterification heparin solution by 1:20 (w/v), by 10% (w/w) weighing sodium hydroxide of esterification heparin weight, add purified water to make dissolution of sodium hydroxide obtain sodium hydroxide solution by 1:9 (w/v), sodium hydroxide solution is added in esterification heparin solution, after 55-60 minute, with 1moL/L hydrochloric acid conditioning solution PH7.0-7.5, add the methanol extraction of 2.0 times of liquor capacities after 3～5 hours, add liquor capacity 0.5-0.6 times methanol dehydration, the precipitation obtaining adds purified water to be dissolved into 10～15% solution, add 30% hydrogen peroxide of liquor capacity 0.5%-1.0%, maintain PH10.5-11.0, temperature 25-30 DEG C, be oxidized 4 hours, oxidation finishes, with 1mol/L hydrochloric acid conditioning solution PH6.1～6.3, 0.45 μ m filter membrane and 0.2 μ m membrane filtration for gained solution, solution after filtration adds the ethanol precipitation of 2.0 times, then dehydrate and obtain Enoxaparin Sodium bulk drug.
Beneficial effect of the present invention: present method can avoid producing from source the non-compliant Enoxaparin Sodium of structure, causes the waste of heparin sodium bulk drug.This law is measured the structure of heparin sodium bulk drug, filter out the heparin sodium bulk drug that is applicable to producing Enoxaparin Sodium, effectively avoid the Enoxaparin Sodium product that causes because of heparin sodium bulk drug discomfort defective, control the quality of Enoxaparin Sodium from source, greatly save cost, improved production efficiency.
Brief description of the drawings
Fig. 1 is disaccharides after heparin sodium enzymolysis and the HPLC standard diagram of tetrose.
Fig. 2 is the HPLC collection of illustrative plates of satisfactory 3 batches of heparin sodium bulk drugs (50114232 batches, 50114236 batches, 50114238 batches) after enzymolysis in embodiment 1.
Fig. 3 is the HPLC collection of illustrative plates of undesirable 3 batches of heparin sodium bulk drugs (50114231 batches, 50114233 batches, 50114235 batches) after enzymolysis in embodiment 1.
Embodiment
Embodiment 1:
1) heparin sodium aqua preparation: precision takes heparin sodium 20mg, puts in the centrifuge tube of 2mL, adds the ultrapure water of 1mL, vortex dissolves, and shakes up, and obtains the heparin sodium aqua of 20mg/mL;
2) phosphate buffer solution preparation: precision takes potassium primary phosphate 68mg, adds bovine serum albumin 10mg, puts in 50mL volumetric flask, add 30mL ultrapure water and dissolve, measure pH, regulate pH7.0 with the potassium hydroxide of 1M if needed, be diluted to scale with ultrapure water, shake up.Before each use, draw certain volume with syringe, with using after 0.45 μ m membrane filtration;
3) Heparinase I, II, the mixed solution of III: according to the labelled amount of three kinds of heparinases, calculate required separately volume, drawing respectively this volume is dissolved in a certain amount of phosphate buffer solution, preparation contains Heparinase I, II, and III content is respectively the heparinase mixing solutions of 1.0/3IU/mL, 0.6/3IU/mL, 0.2/3IU/mL.
4) heparin sodium enzymolysis: add and mix heparinase solution in the aqueous solution of the heparin sodium of 20mg/mL, enzymolysis 64～72 hours in the water-bath of 25 DEG C, makes heparin sodium enzymolysis solution.Choose the heparin sodium of 8 batches, parallel preparation heparin sodium enzymolysis solution.
5) moving phase preparation: mobile phase A: precision takes the SODIUM PHOSPHATE, MONOBASIC of 0.280g, add after 950mL ultrapure water dissolves and use phosphorus acid for adjusting pH to 3.0, be diluted to 1000mL, after the filtering with microporous membrane with 0.22 μ m or 0.45 μ m, ultrasonic degas, to obtain final product.Mobile phase B: precision takes SODIUM PHOSPHATE, MONOBASIC and the 140g sodium perchlorate of 0.280g, adds after 950mL ultrapure water dissolves and uses phosphorus acid for adjusting pH to 3.0, is diluted to 1000mL, and after the filtering with microporous membrane with 0.22 μ m or 0.45 μ m, ultrasonic degas, to obtain final product.
6) instrument condition: instrument is high performance liquid chromatograph (HPLC), chromatographic column quaternary ammonium type reinforcing yin essence ion-exchange chromatography, detector used is UV-detector, detection wavelength is 234nm, sample size is 10 μ L, and flow velocity is 1.0mL/min, and column temperature is 50 DEG C, use mobile phase A and Mobile phase B gradient elution, gradient elution is in table 3.
Table 3
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 97 | 3 |
| 20 | 65 | 35 |
| 50 | 0 | 100 |
| 60 | 0 | 100 |
| 61 | 97 | 3 |
| 79 | 97 | 3 |
7) compartment analysis: respectively the heparin sodium enzymolysis solution of 6 batches is injected to HPLC, record color atlas (seeing Fig. 2, Fig. 3).
8) according to the standard disaccharides of existing heparin sodium and the retention time of tetrose, determine disaccharides in heparin sodium enzymolysis solution to be measured and the kind of tetrose, the standard disaccharides of heparin sodium and the requirement of tetrose see the following form 4.
Table 4
| Peak number | Peak definition | Retention time | The Acceptable criterion of peak area per-cent |
| 1 | △IVA | 7.0～7.5min | 1.8％～3.7％ |
| 2 | △IVSgal | 14.0～15.0min | P～≤1.0％ |
| 3 | △IVS | 14.2～15.2min | 1.8％～3.6％ |
| 4 | △IIA | 16.3～16.8min | 2.3％～4.4％ |
| 5 | △IIIA | 18.0～19.0min | 1.1％～2.1％ |
| 6 | △IISgal | 20.5～21.5min | ≤3.9 |
| 7 | △IIS | 21.5～23.0min | 8.4％～11.9％ |
| 8 | △IIIS | 23.5～25.0min | 4.0％～8.8％ |
| 9 | △IA | 26.0～28.0min | 1.1％～2.0％ |
| 10 | △IIA-IVSglu | 29.5～31.0min | P～≤1.0％ |
| 11 | △IS | 30.5～32.0min | 52.4％～66.9％ |
| 12 | △IIA-IISglu | 33.0～35.0min | 1.6％～3.4％ |
Wherein " P " represents that this peak must exist.
9) determine disaccharides in heparin sodium enzymolysis solution to be measured and the kind of tetrose, adopt areas of peak normalization method to calculate the peak area degree of disaccharides and tetrose after heparin sodium enzymolysis to be measured.
10) heparin sodium selection of crude drugs: choose respectively six batches of heparin sodium raw materials (being respectively 50114231 batches, 50114232 batches, 50114233 batches, 50114235 batches, 50114236 batches, 50114238 batches) of different batches by above-mentioned steps 1)-9) carry out enzymolysis and HPLC and detect, calculate the peak area degree of disaccharides and tetrose after enzymolysis, whether content range requirement in conformance with standard of the content that judges disaccharides and tetrose after heparin sodium enzymolysis to be measured.Statistics is in table 5.
Table 5
Visible, 50114232,50114236,50114238 these 3 batches of heparin sodium bulk drugs meet the requirements, can be for the production of Enoxaparin Sodium.And 50114231,50114233,50114235 these 3 batches of heparin sodium bulk drugs are undesirable, be not suitable for producing Enoxaparin Sodium.
Produce Enoxaparin Sodium: take above-mentioned each batch of heparin sodium raw material, be dissolved in water and make 10% heparin sodium aqua, at 20～30 DEG C, slowly add the Bian rope chlorine ammonium solution of 10% (w/w), fully mix, centrifugation obtains quaternary ammonium salt heparin, add the purified water of 37 times of heparin sodium volumes, agitator treating 30min, centrifugal, repeated washing, centrifugally operated 3 times, quaternary ammonium salt heparin after centrifugal is put into vacuum drying oven dry, press the 1:3.5 (w/v of quaternary ammonium salt heparin weight, g/ml) add methylene dichloride, at 30 ± 1 DEG C, be stirred to completely and dissolve, press quaternary ammonium salt heparin weight 1:1 (w/v, g/ml) add Benzyl Chloride, keep 30 ± 1 DEG C of solution temperatures, esterification 25h ± 30min, obtain esterifying liquid, the methanol solution that by quality volume content is the anhydrous sodium acetate of 10% (g/ml) slowly adds in esterifying liquid, after fully mixing, centrifugal, material after centrifugal dissolves by purified water, after dissolving, volume is about 5.0 times of amount (w/v of heparin quaternary ammonium salt weight, g/ml), add the sodium-chlor of liquor capacity 8% (g/ml), after stirring is dissolved sodium-chlor, add the methanol extraction of liquor capacity 2.5 times (v/v), leave standstill 3 hours, supernatant discarded, throw out dehydration, after centrifugal, 45 ± 3 DEG C, dry 35h, obtain esterification heparin, take esterification heparin, press 1:20 (w/v, g/ml) add purified water stirring and dissolving to obtain esterification heparin solution, by 10% (w/w) weighing sodium hydroxide of esterification heparin weight, press 1:9 (w/v, g/ml) add purified water to make dissolution of sodium hydroxide obtain sodium hydroxide solution, sodium hydroxide solution is added in esterification heparin solution, after 60 minutes, with 1moL/L hydrochloric acid conditioning solution PH7.0, add the methanol extraction of 2.0 times of liquor capacities after 3 hours, add 0.6 times of methanol dehydration of liquor capacity, the precipitation obtaining adds purified water to be dissolved into 10% solution, add 30% hydrogen peroxide of liquor capacity 0.8%, maintain PH10.5, temperature 25-30 DEG C, be oxidized 4 hours, oxidation finishes, with 1mol/L hydrochloric acid conditioning solution PH6.3, 0.45 μ m filter membrane and 0.2 μ m membrane filtration for gained solution, solution after filtration adds the ethanol precipitation of 2.0 times, then dehydrate and obtain Enoxaparin Sodium bulk drug.
Enoxaparin Sodium taking these 6 batches of heparin sodiums as raw material production is after heparinase enzymolysis, carry out HPLC analysis, add up the peak area per-cent of its polysaccharide with areas of peak normalization method, compare with existing Enoxaparin Sodium polysaccharide content (peak area per-cent) standard, statistics is in table 6 and table 7.
Table 6
Table 7
Wherein, taking 50114232,50114236,50114238 these 3 batches of heparin sodiums as raw material production Enoxaparin Sodium, all conformance with standard of the structure of the Enoxaparin Sodium obtaining, and taking 50114231,50114233,50114235 these 3 batches of heparin sodiums as raw material production Enoxaparin Sodium, the structure of the Enoxaparin Sodium obtaining does not all meet standard.
Claims (9)
1. a production method for Enoxaparin Sodium, is characterized in that it comprises the steps:
1) heparin sodium enzymolysis: heparin sodium preparation of raw material is become to the aqueous solution, add to it mixing heparinase solution that contains heparinase I, heparinaseⅡ and heparinase III, carry out enzymolysis 64～72 hours, make heparin sodium enzymolysis solution; Wherein mix the content of each heparinase in heparinase solution than being heparinase I: heparinaseⅡ: heparinase III=1:0.6:0.2;
2) compartment analysis: described heparin sodium enzymolysis solution is injected to HPLC and detect, record color atlas, according to the retention time of the disaccharides of heparin sodium and tetrose, determine disaccharides in heparin sodium to be measured and the kind of tetrose, and calculate the content of 10 disaccharides and 2 tetroses after heparin sodium enzymolysis to be measured; Wherein testing conditions is: chromatographic column is quaternary ammonium type reinforcing yin essence ion-exchange chromatography, detector is UV-detector, detection wavelength is 220～240nm, use mobile phase A and Mobile phase B gradient elution, described mobile phase A is sodium dihydrogen phosphate, and described Mobile phase B is the mixing solutions of SODIUM PHOSPHATE, MONOBASIC and sodium perchlorate;
3) selection of crude drugs: choose the content of disaccharides and tetrose meets following requirement after enzymolysis heparin sodium to be measured as enoxaparin sodium raw materials; Wherein the peak area of disaccharides and tetrose chromatographic peak respectively 1.8%～3.7% ,≤1.0%, 1.8%～3.6%, 2.3%～4.4%, 1.1%～2.1% ,≤3.9%, 8.4%～11.9%, 4.0%～8.8%, 1.1%～2.0%, in≤1.0%, 52.4%～66.9%, 1.6%～3.4% scope;
4) produce Enoxaparin Sodium: taking step 3) in the heparin sodium chosen be raw material, through quaternary ammonium salinization, esterification and oxidizing reaction, obtain Enoxaparin Sodium.
2. the production method of Enoxaparin Sodium according to claim 1, is characterized in that in step 1) in, hydrolysis temperature is 25 DEG C; The content that mixes heparinase I in heparinase solution is 1.0/3IU/mL, and the content of heparinaseⅡ is 0.6/3IU/mL, and the content of heparinase III is 0.2/3IU/mL.
3. the production method of Enoxaparin Sodium according to claim 1, it is characterized in that in step 2) in, described mobile phase A is the sodium dihydrogen phosphate of 0.20～0.30g/L, the mixing solutions of the SODIUM PHOSPHATE, MONOBASIC that described Mobile phase B is 0.20～0.30g/L and the sodium perchlorate of 120～140g/L.
4. the production method of Enoxaparin Sodium according to claim 1, is characterized in that in step 2) in, in HPLC testing process, sample size is 5～20 μ L, and flow velocity is 0.2～1.2mL/min, and column temperature is 50～55 DEG C.
5. the production method of Enoxaparin Sodium according to claim 1, it is characterized in that in step 2) in, 10 disaccharides are respectively △ IVA, △ IVSgal, △ IVS, △ IIA, △ IIIA, △ IISgal, △ IIS, △ IIIS, △ IA and △ IS, and 2 tetroses are respectively △ IIA-IVSglu and △ IIA-IISglu.
6. the production method of Enoxaparin Sodium according to claim 1 or 5, is characterized in that in step 3) in, after the Enoxaparin Sodium raw material enzymolysis of choosing, the content requirement of disaccharides and tetrose is:
Wherein " P " represents that this peak must exist.
7. the production method of Enoxaparin Sodium according to claim 1, is characterized in that in step 2) in, the process that uses mobile phase A and Mobile phase B to carry out gradient elution is:
。
8. the production method of Enoxaparin Sodium according to claim 1, it is characterized in that in step 4) in, take the heparin sodium raw material filtering out, be dissolved in water and make 8%～15% heparin sodium aqua, at 20～30 DEG C, slowly add Bian rope chlorine ammonium solution, fully mix, centrifugation obtains quaternary ammonium salt heparin, repeat to be dried after washing and centrifugally operated, add Benzyl Chloride to stirring and dissolving after adding methylene dichloride in quaternary ammonium salt heparin, keep 29～31 DEG C of solution temperatures to carry out esterification and obtain esterifying liquid, methanol solution precipitation to esterifying liquid with anhydrous sodium acetate, water and sodium chloride solution with after methanol extraction again, obtain esterification heparin, get after esterification heparin purified water is dissolved and add sodium hydroxide solution reaction, use again acid-conditioning solution pH value to 7.0-7.5 use methanol extraction, after being added to purified water solvent soln, throw out adds hydrogen peroxide, at pH value 10.5-11.0, temperature 25-30 DEG C, carry out oxidizing reaction, regulator solution PH6.1～6.3 after reaction, with membrane filtration and by ethanol precipitation, throw out is dehydrated and obtains Enoxaparin Sodium again.
9. the production method of Enoxaparin Sodium according to claim 8, it is characterized in that in step 4) in, the add-on of Bian rope oronain solid is 1～5 times of heparin sodium weight, be preferably 2～3 times, more preferably 2.5 times, the mass content of Bian rope chlorine ammonium solution is 5～15%, and the drying temperature of described quaternary ammonium salt heparin is 47～53 DEG C, be 60～70h time of drying, and the add-on of the methanol solution of described anhydrous sodium acetate is methylene dichloride and Benzyl Chloride cumulative volume 1.2 times; The weightmeasurement ratio of quaternary ammonium salt heparin and methylene dichloride is 1:3.5 (g/ml), and the weightmeasurement ratio of quaternary ammonium salt heparin and Benzyl Chloride is 1:1 (g/ml), and reaction time of esterification is 24～26h; When esterification heparin reacts with sodium hydroxide solution, the mass ratio of esterification heparin and sodium hydroxide is 10:1.
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