CN104193849B - A kind of production method of Enoxaparin Sodium - Google Patents
A kind of production method of Enoxaparin Sodium Download PDFInfo
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- CN104193849B CN104193849B CN201410398891.0A CN201410398891A CN104193849B CN 104193849 B CN104193849 B CN 104193849B CN 201410398891 A CN201410398891 A CN 201410398891A CN 104193849 B CN104193849 B CN 104193849B
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Abstract
The invention discloses the production method of a kind of Enoxaparin Sodium, described method is first by after heparin sodium enzymolysis, carry out HPLC analysis, areas of peak normalization method is used to calculate the content at each peak, according to the disaccharide after heparin sodium enzymolysis and the standard diagram of tetrose, determining disaccharide and the kind of tetrose after heparin sodium enzymolysis to be measured, and compare with the disaccharide of standard diagram and the content of tetrose, the heparin sodium raw material that selector standardization requires carries out the production of Enoxaparin Sodium.The present invention is simply effective, can not only ensure the safety effectiveness of product, and from the quality of Sources controlling Enoxaparin Sodium, it is possible to avoid the waste of heparin sodium crude drug, be greatly saved cost, improve production efficiency.
Description
Technical field
The present invention relates to biomedicine field, the production method of a kind of Enoxaparin Sodium.
Background technology
Heparin is the disaccharide formed by L-iduronic acid (or glucuronic acid) and N-acetyl-glucosamine (or GLUCOSAMINE)
Unit repeats the Sulfated linear polysaccharide of height constituted, and has blood coagulation resisting function, is widely used in treating phlebothrombosis with pre-
Anticoagulant.Low molecular heparin is component or the fragment in unfraction heparin with lower molecular weight, is a new generation's anti-blood of heparin class
Bolt medicine.Heparin is obtained with Low molecular heparin through the mode Partial digestion of enzyme or chemistry.Low molecular heparin series products because of
Molecular weight, is difficult to be neutralized by the iv factor, and anticoagulant effect and fibrinolysis activity are enhanced, and antiplatelet, to affect blood little
Plate function, affecting blood coagulability, induce hemorrhage effect and greatly weaken, bioavailability is up to 98% in addition, dose-effect relationship
Clearly, anticoagulant effect is prone to prediction, plasma half-life relatively unfractionated heparin length 2～3 times, has quick and lasting antithrombotic simultaneously
Formation effect, improves hemodynamics, thus the Low molecular heparin that domestic market is with nadroparin calcium, Enoxaparin Sodium as representative
Series products the most progressively replaces unfractionated heparin product.Enoxaparin Sodium is a kind of pin at present developed by Sanofi-Aventis of France
The novel anticoagulant that the amount of selling is maximum.
Due to the multiformity of low molecular sodium heparin structure, therefore the structure of low molecular sodium heparin determines extremely important.FDA wants
Ask from structure, prove that imitated medicine has biological consistency with original family medicine.Therefore, Low molecular heparin fine structure point
Analysis method is quickly become academia and pharmaceutical industry focus of attention.
The disaccharide of the Enoxaparin Sodium generation after the strict control of Enoxaparin Sodium needs is degradable and the content of tetrose, with
And Enoxaparin Sodium exclusive 1, the content of 6-dehydrogenation ring structure, to ensure effectiveness and the safety of product.Patent
CN102792158B provides the capillary electrophoresis method of a kind of Enoxaparin Sodium characteristic oligosaccharide structure for quantitative analysis.
Patent CN102323355B provides the method for a kind of enzymolysis-HPLC detection Enoxaparin.Both approaches is all according to promise liver
The structure analysis method of element sodium, is the quality control to finished product, it is impossible to be controlled from source to the quality of Enoxaparin Sodium.
Heparin crude drug can be used directly to make standard heparin preparation, or is processed further making Low molecular heparin raw material
Medicine, then make low molecular weight heparin preparations.Heparin sodium is to extract to make from the mucous membrane of small intestine of fresh healthy live pig, due to
The diversity of raw material, causes the diversity of obtained heparin sodium structure.Similarly, poor due to different batches heparin sodium structure
Different so that between the dalteparin sodium batch that processing obtains, to there is also difference.In the production of dalteparin sodium, there is the knot because of heparin sodium
Structure is not suitable for, and causes the non-compliant situation of structure of this batch of dalteparin sodium product, not only loses time, man power and material,
And be unfavorable for producing and economical.
Summary of the invention
The purpose of the present invention is aiming at above-mentioned the deficiencies in the prior art, it is provided that the production method of a kind of Enoxaparin Sodium,
This law includes the screening to raw material, and the present invention is simply effective, can not only ensure the safety effectiveness of product, and can keep away
Exempt from the waste of heparin sodium raw material, improve production efficiency.
The purpose of the present invention can be reached by following measures:
The production method of a kind of Enoxaparin Sodium, it comprises the steps:
1) heparin sodium enzymolysis: heparin sodium preparation of raw material is become aqueous solution, to its add containing E.C. 3.2.1.19, heparinaseⅡ and
The mixing heparinase solution of heparinase III, carries out enzymolysis 64～72 hours, making heparin sodium enzymolysis solution；Wherein mix heparinase
In solution, the content ratio of each heparinase is for E.C. 3.2.1.19: heparinaseⅡ: heparinase III=1:0.6:0.2；
2) analysis is separated: described heparin sodium enzymolysis solution injection HPLC will detect, and record chromatogram, according to heparin
The disaccharide of sodium and the retention time of tetrose, determine the kind of the disaccharide in heparin sodium to be measured and tetrose, and calculate heparin sodium to be measured
10 disaccharide and the content of 2 tetroses after enzymolysis；Wherein testing conditions is: chromatographic column is quaternary ammonium type strong anion exchange chromatographic
Post, detector is UV-detector, and detection wavelength is 220～240nm, uses mobile phase A and Mobile phase B gradient elution, described
Mobile phase A is sodium dihydrogen phosphate, and described Mobile phase B is the mixed solution of sodium dihydrogen phosphate and sodium perchlorate；
3) selection of crude drugs: after choosing enzymolysis, the content of disaccharide and tetrose meets the heparin sodium to be measured of following requirement as depending on
Promise heparin sodium raw material；Wherein the peak area of disaccharide 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 the range of≤1.0%, 52.4%～66.9%, 1.6%～3.4%；
4) produce Enoxaparin Sodium: with step 3) in the heparin sodium chosen as raw material, through quaternization, be esterified and aoxidize
Reaction, obtains Enoxaparin Sodium.
In step 1) in, hydrolysis temperature is preferably 25 DEG C；In mixing heparinase solution, the content of E.C. 3.2.1.19 is 1.0/3IU/
ML, 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 flowing phase is preferably: flowing is mobile phase A and Mobile phase B mutually, and described mobile phase A is
The sodium dihydrogen phosphate of 0.20～0.30g/L, described Mobile phase B be 0.20～0.30g/L sodium dihydrogen phosphate and 120～
The mixed solution of the sodium perchlorate of 140g/L.During HPLC detects, sample size is 5～20 μ L, and flow velocity is 0.2～1.2mL/
Min, 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 from
Sub-exchange chromatography post, detector used is UV-detector, and detection wavelength is 220～240nm, and sample size is 5～20 μ L, flow velocity
Being 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 to be preferably shown 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, the solution after heparin sodium enzymolysis is injected HPLC, records chromatogram, according to existing heparin sodium
Standard disaccharide and the retention time of tetrose, determine the kind of the disaccharide in heparin sodium to be measured and tetrose, and calculate heparin sodium to be measured
Disaccharide and the content of tetrose after enzymolysis, after heparin sodium enzymolysis, disaccharide has 10, is △ IVA, △ IVSgal, △ IVS, △ respectively
IIA, △ IIIA, △ IISgal, △ IIS, △ IIIS, △ IA, △ IS, tetrose has 2, is △ IIA-IVSglu and △ respectively
IIA-IISglu, the disaccharide of described heparin sodium and the requirement of tetrose see table:
In step 3) in, according to step 2) whether the content of disaccharide and tetrose meets after the heparin sodium enzymolysis to be measured that calculated
With this, content range requirement in standard, judges that heparin sodium raw material is if appropriate for for producing Enoxaparin Sodium.That wherein chooses depends on
After promise heparin sodium raw material enzymolysis, the content requirement of disaccharide and tetrose is shown in Table 2.
Table 2
| Peak number | Peak defines | Retention time | Peak area percent |
| 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, with heparin sodium be raw material production Enoxaparin Sodium process through quaternization, be esterified and aoxidize
Reaction, and the separation that matches therewith and purification step, a kind of production process is: weighs the heparin sodium raw material filtered out, adds water
Dissolve the heparin sodium aqua making 8%～15%, be slowly added into Bian rope chlorine ammonium salt solution at 20～30 DEG C, fully mix, centrifugal point
From obtaining quaternary ammonium salt heparin, it is dried after repeating washing and centrifugally operated, stirs after adding dichloromethane in quaternary ammonium salt heparin
Mix dissolving and add benzyl chloride, keep solution temperature 29～31 DEG C to carry out esterification and obtain esterifying liquid, esterifying liquid is used anhydrous second
The methanol solution precipitation of acid sodium, then with water and sodium chloride solution and with after methanol extraction, obtain being esterified heparin；Take esterification heparin with pure
Add sodium hydroxide solution reaction after changing water dissolution, then with acid-conditioning solution pH value to 7.0-7.5 use methanol extraction, will precipitate
Thing adds hydrogen peroxide after adding purified water solvent soln, carries out oxidation reaction at pH value 10.5-11.0, temperature 25-30 DEG C,
Regulate solution PH 6.1～6.3 after reaction, then with membrane filtration and by ethanol precipitation, precipitate dehydrate is obtained according to promise liver
Element sodium.Some optimum conditions in this step are: the addition of Bian rope oronain solid is 1～5 times of heparin sodium weight, are preferably
2～3 times, more preferably 2.5 times, the mass content of Bian rope chlorine ammonium salt solution is 5～15%, the dry temperature of described quaternary ammonium salt heparin
Degree is 47～53 DEG C, and drying time is 60～70h, and the addition of the methanol solution of described anhydrous sodium acetate is dichloromethane and chlorine
Change benzyl cumulative volume 1.2 times；The w/v of quaternary ammonium salt heparin and dichloromethane is 1:3.5 (g/ml), quaternary ammonium salt heparin with
The w/v of benzyl chloride is 1:1 (g/ml), and reaction time of esterification is 24～26h；Esterification heparin is anti-with sodium hydroxide solution
At once, esterification heparin is 10:1 with the mass ratio of sodium hydroxide.
In a kind of preferred version, step 4) process include operating as follows: weigh the heparin sodium raw material filtered out, add water
Dissolve the heparin sodium aqua making 8%～15%, at 20～30 DEG C, be slowly added into the Bian rope chlorine ammonium salt solution that mass content is 10%,
Fully mixing, is centrifugally separating to obtain quaternary ammonium salt heparin, adds heparin sodium volume 35～the purified water of 40 times, agitator treating 30min,
Centrifugal, repeated washing, centrifugally operated 3 times, the quaternary ammonium salt heparin after centrifugal is put in vacuum drying oven dry, by quaternary ammonium salt liver
The 1:3.5 (w/v) of element weight adds dichloromethane, stirs to being completely dissolved, by quaternary ammonium salt heparin weight 1:1 (w/ at 30 ± 1 DEG C
V) add benzyl chloride, keep solution temperature 30 ± 1 DEG C, be esterified 25h ± 30min, obtain esterifying liquid, anhydrous by 10% (w/v)
The methanol solution of sodium acetate is slowly added in esterifying liquid, fully after mixing, centrifugal, and the material after being centrifuged dissolves by purified water, dissolves
Rear volume is about the 4.5-5.0 times amount (w/v) of heparin quaternary ammonium salt weight, adds the sodium chloride of liquor capacity 8% (w/v), stirring
After making sodium chloride dissolve, add the methanol extraction of liquor capacity 2.5 times (v/v), stand 2～4 hours, supernatant discarded, precipitate
Dehydration, centrifugal after, 45 ± 3 DEG C, be dried 30-35h, obtain being esterified heparin, weigh esterification heparin, add purification by 1:20 (w/v)
Water stirring and dissolving obtains being esterified heparin solution, by 10% (w/w) weighing sodium hydroxide of esterification heparin weight, adds by 1:9 (w/v)
Entering purified water makes sodium hydroxide dissolving obtain sodium hydroxide solution, is added by sodium hydroxide solution in esterification heparin solution, 55-60
After minute, with 1moL/L hydrochloric acid conditioning solution PH7.0-7.5, add the methanol extraction 3 of liquor capacity 2.0 times～after 5 hours, add
Entering 0.5-0.6 times of methanol dehydration of liquor capacity, the precipitation obtained adds purified water and is dissolved into the solution of 10～15%, adds solution
30% hydrogen peroxide of volume 0.5%-1.0%, maintains PH10.5-11.0, temperature 25-30 DEG C, aoxidizes 4 hours, and oxidation terminates,
With 1mol/L hydrochloric acid conditioning solution PH6.1～6.3, gained solution 0.45 μm filter membrane and 0.2 μm membrane filtration, molten after filtration
Liquid adds the ethanol precipitation of 2.0 times, and then dehydrate obtains Enoxaparin Sodium crude drug.
Beneficial effects of the present invention: this method can be avoided producing the non-compliant Enoxaparin of structure from source
Sodium, causes the waste of heparin sodium crude drug.The structure of heparin sodium crude drug is measured by this law, filters out and is suitable for producing according to promise
The heparin sodium crude drug of heparin sodium, efficiently avoid the Enoxaparin Sodium product caused because of heparin sodium crude drug discomfort and does not conforms to
Lattice, from the quality of Sources controlling Enoxaparin Sodium, are greatly saved cost, improve production efficiency.
Accompanying drawing explanation
Fig. 1 is the HPLC standard diagram of the disaccharide after heparin sodium enzymolysis and tetrose.
Fig. 2 be satisfactory 3 batches of heparin sodium crude drug in embodiment 1 (50114232 batches, 50114236 batches,
50114238 batches) HPLC collection of illustrative plates after enzymolysis.
Fig. 3 be undesirable 3 batches of heparin sodium crude drug in embodiment 1 (50114231 batches, 50114233 batches,
50114235 batches) HPLC collection of illustrative plates after enzymolysis.
Detailed description of the invention
Embodiment 1:
1) heparin sodium aqua preparation: precision weighs heparin sodium 20mg, puts in the centrifuge tube of 2mL, adds the ultra-pure water of 1mL, whirlpool
Rotation is dissolved, and shakes up, obtains the heparin sodium aqua of 20mg/mL；
2) phosphate buffer solution preparation: precision weighs potassium dihydrogen phosphate 68mg, adds bovine serum albumin 10mg, puts 50mL and holds
In measuring bottle, add 30mL ultra-pure water and dissolve, measure pH, if needed with the potassium hydroxide regulation pH7.0 of 1M, be diluted to ultra-pure water
Scale, shakes up.Use front syringe to draw certain volume every time, use with after 0.45 μm membrane filtration；
3) Heparinase I, the mixed liquor of II, III: according to the labelled amount of three kinds of heparinases, calculate the most required volume, point
Not drawing this volume to be dissolved in a certain amount of phosphate buffer solution, preparation is containing Heparinase I, and II, III content is respectively 1.0/
The heparinase mixed solution of 3IU/mL, 0.6/3IU/mL, 0.2/3IU/mL.
4) heparin sodium enzymolysis: add mixing heparinase solution, the water-bath of 25 DEG C in the aqueous solution of the heparin sodium of 20mg/mL
Middle enzymolysis 64～72 hours, make heparin sodium enzymolysis solution.Choosing the heparin sodium of 8 batches, parallel preparation heparin sodium enzymolysis is molten
Liquid.
5) flowing preparation mutually: mobile phase A: precision weighs the sodium dihydrogen phosphate of 0.280g, adds after 950mL ultra-pure water dissolves and uses
Phosphorus acid for adjusting pH, to 3.0, is diluted to 1000mL, after the filtering with microporous membrane by 0.22 μm or 0.45 μm, and ultrasonic degassing, to obtain final product.
Mobile phase B: precision weighs sodium dihydrogen phosphate and the 140g sodium perchlorate of 0.280g, adds after 950mL ultra-pure water dissolves and adjusts with phosphoric acid
Joint pH to 3.0, is diluted to 1000mL, after the filtering with microporous membrane by 0.22 μm or 0.45 μm, and ultrasonic degassing, to obtain final product.
6) instrument condition: instrument is high performance liquid chromatograph (HPLC), chromatographic column quaternary ammonium type strong anion exchange color
Spectrum post, detector used is UV-detector, and detection wavelength is 234nm, and sample size is 10 μ L, and flow velocity is 1.0mL/min, column temperature
Being 50 DEG C, use mobile phase A and Mobile phase B gradient elution, gradient elution is shown 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) analysis is separated: the heparin sodium enzymolysis solution of 6 batches injects HPLC respectively, and record chromatogram is (see Fig. 2, figure
3)。
8) according to standard disaccharide and the retention time of tetrose of existing heparin sodium, determine in heparin sodium enzymolysis solution to be measured
Disaccharide and the kind of tetrose, the standard disaccharide of heparin sodium and the requirement of tetrose see table 4.
Table 4
| Peak number | Peak defines | Retention time | Peak area percent accept standard |
| 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 the disaccharide in heparin sodium enzymolysis solution to be measured and the kind of tetrose, use areas of peak normalization method to calculate and treat
Survey disaccharide and the peak area percent content of tetrose after heparin sodium enzymolysis.
10) heparin sodium selection of crude drugs: choose six batches of heparin sodium raw materials (respectively 50114231 of different batches respectively
Batch, 50114232 batches, 50114233 batches, 50114235 batches, 50114236 batches, 50114238 batches) by above-mentioned steps 1)-9) carry out
Enzymolysis and HPLC detection, calculate disaccharide and the peak area percent content of tetrose after enzymolysis, it is judged that after heparin sodium enzymolysis to be measured two
Content range requirement in the content of sugar and tetrose whether conformance with standard.Statistical result is shown in Table 5.
Table 5
Visible, 50114232,50114236,50114238 these 3 batches of heparin sodium crude drug meet the requirements, and may be used for producing
Enoxaparin Sodium.And 50114231,50114233,50114235 these 3 batches of heparin sodium crude drug are undesirable, be not suitable for
Produce Enoxaparin Sodium.
Produce Enoxaparin Sodium: weigh above-mentioned each batch of heparin sodium raw material, be dissolved in water make 10% heparin sodium aqua, 20
～at 30 DEG C, it is slowly added into the Bian rope chlorine ammonium salt solution of 10% (w/w), fully mix, be centrifugally separating to obtain quaternary ammonium salt heparin, add
The purified water that heparin sodium volume is 37 times, agitator treating 30min, centrifugal, repeated washing, centrifugally operated 3 times, the quaternary ammonium after being centrifuged
Salt heparin put in vacuum drying oven be dried, by quaternary ammonium salt heparin weight 1:3.5 (w/v, g/ml) add dichloromethane, 30 ±
Stir at 1 DEG C to being completely dissolved, add benzyl chloride by quaternary ammonium salt heparin weight 1:1 (w/v, g/ml), keep solution temperature 30 ± 1
DEG C, it is esterified 25h ± 30min, obtains esterifying liquid, the methanol solution of the anhydrous sodium acetate that quality volume content is 10% (g/ml) is delayed
Delaying and add in esterifying liquid, fully after mixing, centrifugal, the material after being centrifuged dissolves by purified water, and after dissolving, volume is about heparin season
5.0 times amount (w/v, g/ml) of ammonium salt weight, add the sodium chloride of liquor capacity 8% (g/ml), after stirring makes sodium chloride dissolve,
Add liquor capacity 2.5 times (v/v) methanol extraction, stands 3 hours, supernatant discarded, precipitate dehydration, be centrifuged after, 45 ± 3
DEG C, it is dried 35h, obtains being esterified heparin, weigh esterification heparin, add purified water stirring and dissolving by 1:20 (w/v, g/ml) and obtain ester
Change heparin solution, by 10% (w/w) weighing sodium hydroxide of esterification heparin weight, add purified water by 1:9 (w/v, g/ml) and make
Sodium hydroxide dissolves and obtains sodium hydroxide solution, is added by sodium hydroxide solution in esterification heparin solution, after 60 minutes, uses
1moL/L hydrochloric acid conditioning solution PH7.0, the methanol extraction of addition liquor capacity 2.0 times, after 3 hours, adds liquor capacity 0.6 times
Methanol dehydration, the precipitation obtained adds purified water and is dissolved into the solution of 10%, adds 30% peroxidating of liquor capacity 0.8%
Hydrogen, maintains PH10.5, temperature 25-30 DEG C, aoxidizes 4 hours, and oxidation terminates, and with 1mol/L hydrochloric acid conditioning solution PH6.3, gained is molten
Liquid 0.45 μm filter membrane and 0.2 μm membrane filtration, the solution after filtration adds the ethanol precipitation of 2.0 times, and then dehydrate obtains
To Enoxaparin Sodium crude drug.
With this 6 batches of heparin sodiums Enoxaparin Sodium as raw material production after heparinase enzymolysis, carry out HPLC analysis, with face, peak
Long-pending normalization method adds up the peak area percent of its polysaccharide, marks with existing Enoxaparin Sodium polyoses content (peak area percent)
Standard compares, and statistical result is shown in Table 6 and table 7.
Table 6
Table 7
Wherein, with 50114232,50114236,50114238 these 3 batches of heparin sodiums for raw material production Enoxaparin Sodium, obtain
Structure all conformance with standard of Enoxaparin Sodium, and with 50114231,50114233,50114235 these 3 batches of heparin sodiums as raw material
Producing Enoxaparin Sodium, the structure of the Enoxaparin Sodium obtained does not complys with standard.
Claims (10)
1. the production method of an Enoxaparin Sodium, it is characterised in that it comprises the steps:
1) heparin sodium enzymolysis: heparin sodium preparation of raw material is become aqueous solution, adds containing E.C. 3.2.1.19, heparinaseⅡ and heparin to it
The mixing heparinase solution of enzyme III, carries out enzymolysis 64～72 hours, making heparin sodium enzymolysis solution；Wherein mixing heparinase solution
In the content ratio of each heparinase for E.C. 3.2.1.19: heparinaseⅡ: heparinase III=1:0.6:0.2；
2) analysis is separated: described heparin sodium enzymolysis solution injection HPLC will detect, and record chromatogram, according to heparin sodium
Disaccharide and the retention time of tetrose, determine the kind of the disaccharide in heparin sodium to be measured and tetrose, and calculate heparin sodium enzymolysis to be measured
Rear 10 disaccharide and the content of 2 tetroses；Wherein testing conditions is: chromatographic column is quaternary ammonium type strong anion exchange chromatographic post, inspection
Survey device is UV-detector, and detection wavelength is 220～240nm, uses mobile phase A and Mobile phase B gradient elution, described flowing phase
A is sodium dihydrogen phosphate, and described Mobile phase B is the mixed solution of sodium dihydrogen phosphate and sodium perchlorate；
3) selection of crude drugs: after choosing enzymolysis, the content of disaccharide and tetrose meets the heparin sodium to be measured of following requirement as according to promise liver
Element sodium raw materials；Wherein disaccharide is respectively △ IVA, △ IVSgal, △ IVS, △ IIA, △ IIIA, △ IISgal, △ IIS, △
IIIS, △ IA and △ IS, the peak area of disaccharide chromatographic peak successively 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 the range of 52.4%～66.9%；Tetrose is respectively △ IIA-IVSglu and △ IIA-IISglu, the peak area of tetrose chromatographic peak
Successively in the range of≤1.0%, 1.6%～3.4%；
4) produce Enoxaparin Sodium: with step 3) in the heparin sodium chosen as raw material, through quaternization, be esterified and aoxidize anti-
Should, obtain Enoxaparin Sodium.
The production method of Enoxaparin Sodium the most according to claim 1, it is characterised in that in step 1) in, hydrolysis temperature is
25℃；In mixing heparinase solution, the content of E.C. 3.2.1.19 is 1.0/3IU/mL, and the content of heparinaseⅡ is 0.6/3IU/mL, liver
The content of element enzyme III is 0.2/3IU/mL.
The production method of Enoxaparin Sodium the most according to claim 1, it is characterised in that in step 2) in, described flowing phase
A is the sodium dihydrogen phosphate of 0.20～0.30g/L, and described Mobile phase B is the mixed solution of sodium dihydrogen phosphate and sodium perchlorate,
In Mobile phase B, the concentration of sodium dihydrogen phosphate is 0.20～0.30g/L, and the concentration of sodium perchlorate is 120～140g/L.
The production method of Enoxaparin Sodium the most according to claim 1, it is characterised in that in step 2) in, detect at HPLC
During, sample size is 5～20 μ L, and flow velocity is 0.2～1.2mL/min, and column temperature is 50～55 DEG C.
The production method of Enoxaparin Sodium the most according to claim 1, it is characterised in that in step 3) in, choose according to promise
After heparin sodium raw material enzymolysis, the content requirement of disaccharide and tetrose is:
Wherein " P " represents that this peak must exist.
The production method of Enoxaparin Sodium the most according to claim 1, it is characterised in that in step 2) in, use flowing phase
A and Mobile phase B carry out the process of gradient elution:
。
The production method of Enoxaparin Sodium the most according to claim 1, it is characterised in that in step 4) in, weigh and filter out
Heparin sodium raw material, be dissolved in water and make the heparin sodium aqua of 8%～15%, be slowly added into Bian rope oronain at 20～30 DEG C molten
Liquid, fully mixes, and is centrifugally separating to obtain quaternary ammonium salt heparin, is dried, to quaternary ammonium salt heparin after repeating washing and centrifugally operated
After middle addition dichloromethane, stirring and dissolving adds benzyl chloride, keeps solution temperature 29～31 DEG C to carry out esterification and be esterified
Liquid, precipitates the methanol solution of esterifying liquid anhydrous sodium acetate, then dissolves and with after methanol extraction with water and sodium chloride, is esterified
Heparin；Take after esterification heparin purified water is dissolved and add sodium hydroxide solution reaction, then with acid-conditioning solution pH value to 7.0-7.5
Afterwards with methanol extraction, after precipitate is added purified water solvent soln, add hydrogen peroxide, in pH value 10.5-11.0, temperature 25-
Carry out oxidation reaction at 30 DEG C, after reaction, regulate pH value of solution 6.1～6.3, then with membrane filtration and by ethanol precipitation, by precipitate
Dehydrate obtains Enoxaparin Sodium.
The production method of Enoxaparin Sodium the most according to claim 7, it is characterised in that in step 4) in, Bian rope oronain
Addition is 1～5 times of heparin sodium weight, and in the Bian rope chlorine ammonium salt solution added, the mass content of Bian rope oronain is 5～15%,
The baking temperature of described quaternary ammonium salt heparin is 47～53 DEG C, and drying time is 60～70h, the methanol of described anhydrous sodium acetate
The addition of liquid is 1.2 times of dichloromethane and benzyl chloride cumulative volume；Quaternary ammonium salt heparin with the w/v of dichloromethane is
1:3.5g/ml, quaternary ammonium salt heparin is 1:1g/ml with the w/v of benzyl chloride, and reaction time of esterification is 24～26h；Esterification
When heparin reacts with sodium hydroxide solution, esterification heparin is 10:1 with the mass ratio of sodium hydroxide.
The production method of Enoxaparin Sodium the most according to claim 8, it is characterised in that in step 4) in, Bian rope oronain
Addition is 2～3 times of heparin sodium weight.
The production method of Enoxaparin Sodium the most according to claim 9, it is characterised in that in step 4) in, Bian rope oronain
Addition be 2.5 times of heparin sodium weight.
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| CN106932494A (en) * | 2015-12-30 | 2017-07-07 | 深圳市海普瑞药业股份有限公司 | A kind of method that enzymolysis-HPLC detects Sulodexide |
| CN105628849B (en) * | 2015-12-31 | 2018-01-12 | 成都百裕制药股份有限公司 | A kind of detection method of Enoxaparin Sodium |
| CN109613157A (en) * | 2018-11-27 | 2019-04-12 | 山东万邦赛诺康生化制药股份有限公司 | A kind of raw material heparin sodium for producing Enoxaparin Sodium and using |
| CN111239321A (en) * | 2020-03-13 | 2020-06-05 | 北京赛升药业股份有限公司 | Method for detecting nadroparin calcium disaccharide spectrum |
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