CN113004436A - Preparation method of dalteparin sodium and application of method in preparation of low-molecular-weight heparin sodium - Google Patents
Preparation method of dalteparin sodium and application of method in preparation of low-molecular-weight heparin sodium Download PDFInfo
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- CN113004436A CN113004436A CN202110486245.XA CN202110486245A CN113004436A CN 113004436 A CN113004436 A CN 113004436A CN 202110486245 A CN202110486245 A CN 202110486245A CN 113004436 A CN113004436 A CN 113004436A
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- 229920000669 heparin Polymers 0.000 title claims abstract description 111
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 title claims abstract description 90
- 238000000034 method Methods 0.000 title claims abstract description 53
- 229940018872 dalteparin sodium Drugs 0.000 title claims abstract description 50
- 238000002360 preparation method Methods 0.000 title claims abstract description 35
- 239000003055 low molecular weight heparin Substances 0.000 title claims abstract description 25
- 229940127215 low-molecular weight heparin Drugs 0.000 title claims abstract description 14
- 229960001008 heparin sodium Drugs 0.000 claims abstract description 77
- 229920000045 Dermatan sulfate Polymers 0.000 claims abstract description 37
- 229940051593 dermatan sulfate Drugs 0.000 claims abstract description 34
- AVJBPWGFOQAPRH-FWMKGIEWSA-L dermatan sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@H](OS([O-])(=O)=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](C([O-])=O)O1 AVJBPWGFOQAPRH-FWMKGIEWSA-L 0.000 claims abstract description 34
- 238000006243 chemical reaction Methods 0.000 claims abstract description 27
- 238000006731 degradation reaction Methods 0.000 claims abstract description 19
- 230000015556 catabolic process Effects 0.000 claims abstract description 14
- 239000002994 raw material Substances 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims description 120
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 103
- 239000000243 solution Substances 0.000 claims description 83
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 54
- 238000007254 oxidation reaction Methods 0.000 claims description 53
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 52
- 238000003756 stirring Methods 0.000 claims description 48
- 230000003647 oxidation Effects 0.000 claims description 45
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 42
- 239000011347 resin Substances 0.000 claims description 32
- 229920005989 resin Polymers 0.000 claims description 32
- 238000010438 heat treatment Methods 0.000 claims description 31
- 230000008569 process Effects 0.000 claims description 31
- 230000001276 controlling effect Effects 0.000 claims description 30
- 239000002244 precipitate Substances 0.000 claims description 30
- 235000010288 sodium nitrite Nutrition 0.000 claims description 27
- 239000003480 eluent Substances 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- 239000011780 sodium chloride Substances 0.000 claims description 21
- 238000001816 cooling Methods 0.000 claims description 18
- 238000001556 precipitation Methods 0.000 claims description 17
- 238000001914 filtration Methods 0.000 claims description 16
- 239000011229 interlayer Substances 0.000 claims description 15
- 238000005406 washing Methods 0.000 claims description 14
- 238000005303 weighing Methods 0.000 claims description 14
- 239000012043 crude product Substances 0.000 claims description 13
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 108091005658 Basic proteases Proteins 0.000 claims description 11
- 239000000706 filtrate Substances 0.000 claims description 11
- 108010019160 Pancreatin Proteins 0.000 claims description 9
- 229940055695 pancreatin Drugs 0.000 claims description 9
- 230000001376 precipitating effect Effects 0.000 claims description 9
- 239000012266 salt solution Substances 0.000 claims description 8
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 8
- 239000012279 sodium borohydride Substances 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 7
- 238000001179 sorption measurement Methods 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 6
- 108091005804 Peptidases Proteins 0.000 claims description 5
- 239000004365 Protease Substances 0.000 claims description 5
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 5
- 238000005194 fractionation Methods 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 5
- 150000003839 salts Chemical class 0.000 claims description 5
- 238000012545 processing Methods 0.000 claims description 4
- 238000012546 transfer Methods 0.000 claims description 4
- 241000194108 Bacillus licheniformis Species 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000012295 chemical reaction liquid Substances 0.000 claims description 2
- 238000010907 mechanical stirring Methods 0.000 claims description 2
- 230000001590 oxidative effect Effects 0.000 claims description 2
- 150000001450 anions Chemical class 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 19
- 230000002785 anti-thrombosis Effects 0.000 abstract description 6
- 239000003146 anticoagulant agent Substances 0.000 abstract description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 72
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 30
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 20
- 229960002897 heparin Drugs 0.000 description 20
- 238000001514 detection method Methods 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- PGWTYMLATMNCCZ-UHFFFAOYSA-M azure A Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 PGWTYMLATMNCCZ-UHFFFAOYSA-M 0.000 description 9
- 239000010410 layer Substances 0.000 description 9
- 239000008213 purified water Substances 0.000 description 9
- 239000000203 mixture Substances 0.000 description 7
- 238000001035 drying Methods 0.000 description 6
- 239000012467 final product Substances 0.000 description 6
- 238000005342 ion exchange Methods 0.000 description 6
- 238000001223 reverse osmosis Methods 0.000 description 6
- 238000005070 sampling Methods 0.000 description 6
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 4
- 230000000593 degrading effect Effects 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 4
- 238000012869 ethanol precipitation Methods 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- 239000012466 permeate Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 229910052708 sodium Inorganic materials 0.000 description 4
- OEANUJAFZLQYOD-CXAZCLJRSA-N (2r,3s,4r,5r,6r)-6-[(2r,3r,4r,5r,6r)-5-acetamido-3-hydroxy-2-(hydroxymethyl)-6-methoxyoxan-4-yl]oxy-4,5-dihydroxy-3-methoxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](OC)O[C@H](CO)[C@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](OC)[C@H](C(O)=O)O1 OEANUJAFZLQYOD-CXAZCLJRSA-N 0.000 description 3
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 description 3
- 238000001802 infusion Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000012452 mother liquor Substances 0.000 description 3
- 238000001728 nano-filtration Methods 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 238000006722 reduction reaction Methods 0.000 description 3
- 238000007670 refining Methods 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 229910052719 titanium Inorganic materials 0.000 description 3
- 239000010936 titanium Substances 0.000 description 3
- 230000007306 turnover Effects 0.000 description 3
- 208000032843 Hemorrhage Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000010100 anticoagulation Effects 0.000 description 2
- 208000034158 bleeding Diseases 0.000 description 2
- 230000000740 bleeding effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000020764 fibrinolysis Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 108010074860 Factor Xa Proteins 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 229960004676 antithrombotic agent Drugs 0.000 description 1
- 229940127217 antithrombotic drug Drugs 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 229960005153 enoxaparin sodium Drugs 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000000004 hemodynamic effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 229950003543 nadroparin calcium Drugs 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- CIJQTPFWFXOSEO-NDMITSJXSA-J tetrasodium;(2r,3r,4s)-2-[(2r,3s,4r,5r,6s)-5-acetamido-6-[(1r,2r,3r,4r)-4-[(2r,3s,4r,5r,6r)-5-acetamido-6-[(4r,5r,6r)-2-carboxylato-4,5-dihydroxy-6-[[(1r,3r,4r,5r)-3-hydroxy-4-(sulfonatoamino)-6,8-dioxabicyclo[3.2.1]octan-2-yl]oxy]oxan-3-yl]oxy-2-(hydroxy Chemical compound [Na+].[Na+].[Na+].[Na+].O([C@@H]1[C@@H](COS(O)(=O)=O)O[C@@H]([C@@H]([C@H]1O)NC(C)=O)O[C@@H]1C(C[C@H]([C@@H]([C@H]1O)O)O[C@@H]1[C@@H](CO)O[C@H](OC2C(O[C@@H](OC3[C@@H]([C@@H](NS([O-])(=O)=O)[C@@H]4OC[C@H]3O4)O)[C@H](O)[C@H]2O)C([O-])=O)[C@H](NC(C)=O)[C@H]1C)C([O-])=O)[C@@H]1OC(C([O-])=O)=C[C@H](O)[C@H]1O CIJQTPFWFXOSEO-NDMITSJXSA-J 0.000 description 1
- 229940064689 tinzaparin sodium Drugs 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
- C08B37/0078—Degradation products
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Sustainable Development (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention particularly relates to a preparation method of dalteparin sodium and application of the method in preparation of low-molecular heparin sodium. The dalteparin sodium as a low molecular weight heparin sodium has good bioavailability and is a new generation of antithrombotic active medicine. The existing preparation method of dalteparin sodium takes refined heparin sodium as a raw material for degradation, and the refined heparin sodium is obtained by purifying crude heparin sodium, so that the existing preparation process of dalteparin sodium is complex, and the damage degree of heparin sodium in the preparation engineering is large. The invention provides a method for preparing dalteparin sodium by taking crude heparin sodium as a raw material, wherein the preparation method is optimized for the enzymolysis mode of the crude heparin sodium and the treatment mode of dermatan sulfate, so that the process steps from the crude heparin sodium to the dalteparin sodium are effectively shortened, and the reaction yield is improved.
Description
Technical Field
The invention belongs to the technical field of preparation of dalteparin sodium, and particularly relates to a preparation method of dalteparin sodium and application of the preparation method in preparation of low-molecular-weight heparin sodium.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The low molecular heparin is a fragment generated after subdivision or cracking and separated from common heparin (SH), has low molecular weight and simple structure, and is a new generation of heparin anticoagulant drug developed in the last decade. The low molecular heparin product is small in molecular weight, not easy to be neutralized by the factor IV, the anticoagulation effect and the fibrinolysis effect are enhanced, the platelet resistance is improved, the platelet function is influenced, the blood coagulability is influenced, the effect of inducing bleeding is greatly weakened, the bioavailability is up to 98%, the dose-effect relationship is clear, the anticoagulation effect is easy to predict, the half-life period of plasma is 2-3 times longer than that of common heparin, the placenta barrier is not easy to permeate, the low molecular heparin product can be applied to pregnant women, the administration is convenient, the low molecular heparin product has quick and continuous antithrombotic effect, the hemodynamics is improved, and therefore the low molecular heparin product is favored clinically. The low molecular weight heparin used in clinic includes dalteparin sodium, nadroparin calcium, enoxaparin sodium, sertoxepin sodium, tinzaparin sodium, and heparitin sodium. The dalteparin sodium is low-component heparin sodium of porcine mucosa, has the average molecular weight of 4000-6000, is a new generation antithrombotic drug, has an obvious antithrombotic function, and particularly has a strong function of resisting the coagulation factor Xa. Compared with the conventional heparin, the dalteparin sodium has high bioavailability (90%), long action time (24h) and stable antithrombotic effect. Dalteparin sodium is obtained by depolymerizing or enzymolyzing common heparin, and has advantages that common heparin does not have. The research results in recent years show that the pharmacological action of the dalteparin sodium is far greater than that of the common heparin. Therefore, the antithrombotic agent has the characteristics of strong antithrombotic action and low bleeding risk. In addition, the dalteparin sodium can promote fibrinolysis, protect endothelial cells and enhance antithrombotic effect by combining with vascular endothelial cells, and has smaller influence on platelet function and lipid metabolism than common heparin.
At present, dalteparin sodium in the market is mostly obtained by chemically degrading refined heparin sodium, and the refined heparin sodium is obtained by treating crude heparin sodium obtained from small intestines of pigs through a plurality of steps, so that a large amount of materials are lost in the process, the yield is greatly reduced, the loss of bioavailability groups of the heparin sodium is caused, and the bioavailability is reduced. In addition, the refined heparin sodium takes crude heparin sodium as a raw material, the refining process comprises enzymolysis, oxidation and purification, and the loss of the heparin sodium cannot be avoided in the refining process. The quality heparin sodium also brings inevitable quality loss in the molecular weight grading process.
Disclosure of Invention
Aiming at the problems recorded in the background art, the invention aims to provide a preparation method of dalteparin sodium, which directly prepares the dalteparin sodium by taking crude heparin sodium as a raw material, reduces the process flow by optimizing the preparation process, reduces the energy consumption and improves the production efficiency.
The invention provides a preparation method of dalteparin sodium, which comprises enzymolysis, resin separation, oxidation, sodium nitrite degradation and molecular weight grading treatment.
The preparation method of the invention is characterized in that the heparin sodium is refined from crude heparin sodium, and the process of removing the dermatan is required, and the ethanol precipitation method is adopted, because the precipitation point of the dermatan sulfate is close to that of the heparin sodium, a certain amount of heparin sodium must be lost while the dermatan sulfate is precipitated. The technical improvement of the invention is that the crude heparin sodium is adopted to directly produce the heparin sodium, and when the sodium nitrite is used for degrading the heparin sodium in the process, the dermatan sulfate can not be degraded by the sodium nitrite, so that the difference of precipitation points of the heparin sodium and the dermatan sulfate is enlarged, the dermatan sulfate is removed after the sodium nitrite is degraded, and when the dermatan sulfate is precipitated, the low molecular heparin sodium is not precipitated, thereby avoiding the loss of active products.
The improvement of the technical elements effectively shortens the preparation period of the existing dalteparin sodium, simplifies the production flow, and the prepared product has good purity and potency. At present, the preparation idea of other types of low molecular weight heparin sodium is similar to that of dalteparin sodium, and the improvement of the technical process can be further applied to the preparation of other types of low molecular weight heparin sodium. Therefore, in the second aspect of the present invention, the application of the preparation method of dalteparin sodium in the first aspect in preparing low molecular heparin sodium is provided.
In the prior art, the low molecular weight heparin sodium usually takes refined heparin sodium as a raw material to obtain a heparin sodium product with a corresponding low molecular weight through degradation. The preparation method of the dalteparin sodium provided by the invention is also suitable for the preparation of the low molecular weight heparin sodium, and the difference is only that the degradation mode of the sodium nitrite is different, and the degradation steps can be adjusted by persons skilled in the art according to the preparation purpose.
The beneficial effects of one or more technical schemes are as follows:
(1) the method for preparing the dalteparin sodium from the crude product heparin sodium removes the process of refining the crude product heparin sodium into the refined product heparin sodium, greatly reduces the process steps, shortens the process period, and improves the yield and the titer of the product dalteparin sodium.
(2) The invention adds a process for removing dermatan sulfate impurities after degradation of heparan, greatly reduces the residue of dermatan sulfate impurities in the finished product of dalteparin sodium, and ensures the safety of the finished product.
(3) The method is simple, convenient to operate, high in practicability and suitable for industrial production.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As described in the background art, in the prior art, the preparation of the dalteparin sodium usually uses the refined heparin sodium as the raw material, the production period from the preparation of the crude heparin sodium to the preparation of the dalteparin sodium is long, and the raw material waste is accompanied. In order to solve the technical problems, the invention provides a method for preparing dalteparin sodium by using crude heparin sodium as a raw material.
The invention provides a preparation method of dalteparin sodium, which comprises enzymolysis, resin separation, oxidation, sodium nitrite degradation and molecular weight grading treatment.
Preferably, the enzymolysis process adopts pancreatin and alkaline protease to carry out enzymolysis in sequence.
Further, the enzymolysis process comprises the following specific steps: adjusting the pH value of the crude heparin sodium solution to 8.5-9.0, adding pancreatin, and controlling the temperature to be 49-52 ℃ to react for 4-6 h; adjusting pH to 6.5 +/-0.5 after enzymolysis, heating for a period of time, and cooling to remove impure protein; regulating the pH value to 8.5-9.0 again, adding alkaline protease, controlling the temperature to 49-52 ℃, reacting for 4-6 h, carrying out secondary enzymolysis, regulating the pH value to 6.5 +/-0.5 after the second enzymolysis is finished, heating for a period of time, and cooling to remove foreign proteins; adding ethanol solution into the material liquid obtained by centrifugation, standing and precipitating to obtain a precipitate part.
Further, the alkaline protease is produced by bacillus licheniformis; in a more effective embodiment of the present invention, the alkaline protease is 2709 protease.
Furthermore, the concentration of the crude heparin sodium solution is 14-16%, in a specific embodiment, the concentration of the crude heparin sodium is 14%, 15% or 16%, and in a best embodiment, the concentration of the crude heparin sodium is 15%.
Furthermore, the pancreatin or the alkaline protease accounts for 0.4-0.6% of the weight of the crude heparin sodium; specifically 0.4%, 0.5% or 0.6%, and in a more effective embodiment 0.5%.
Furthermore, the enzymatic hydrolysis reaction process also comprises a stirring step, and the stirring mode is not limited to a mechanical stirring mode or a manual stirring mode.
Furthermore, the specific steps of heating for a period of time and cooling to remove impure proteins are as follows: after the enzymolysis is finished, adjusting the pH value of a reaction system to 6.5 +/-0.5, heating to 85-90 ℃, preserving heat for 18-22 min, then cooling, and reducing the temperature of the reaction system to 48-52 ℃ within 8-12 min.
In the process, the heating mode can adopt a direct heating reaction system or a heat transfer mode for heating, in one embodiment provided by the invention, steam is adopted for heat transfer, further, steam is introduced into an interlayer of the reaction device, and the heating mode can realize rapid heating without contacting with a reaction solution; meanwhile, the cooling process can adopt a mode of introducing cold water into the interlayer of the reaction device to realize rapid cooling.
Furthermore, the concentration of the ethanol solution is 92% -93%, and the volume of the ethanol solution is 1-3 times of that of the feed liquid part.
Furthermore, the standing and precipitating time is 3-5 h.
Preferably, the resin separation method employs a strong anionic resin.
Further, the resin separation mode is as follows: adjusting the pH of the feed liquid to 8.5 +/-0.5, adding the feed liquid into resin for adsorption, washing by using a salt solution after adsorption is finished to obtain an eluent, and adding ethanol into the eluent for precipitation for a period of time to obtain a precipitate part.
Preferably, the oxidation is performed by using hydrogen peroxide, the oxidation time is one or two, and in a more preferred scheme, two oxidations are required.
Further, the oxidation treatment comprises the following specific steps: adjusting the pH value of the solution to 9.5 +/-0.5, controlling the reaction temperature to 28 +/-5 ℃, adding 0.5-1.5% of hydrogen peroxide solution into the reaction liquid, oxidizing for 2-4 hours, and filtering to obtain a liquid part; repeating the above operation until the two times of oxidation and co-oxidation are carried out for 7-9 hours.
Furthermore, in the oxidation treatment process, the hydrogen peroxide solution is added in a divided manner, in some specific embodiments, the hydrogen peroxide solution is added in 3-4 times, and the interval time is 20-30 min.
Further, after the oxidation is finished, adjusting the pH value of the feed liquid to 6.5 +/-0.5, adding salt into the reaction feed liquid until the concentration is 1-2%, filtering, adding ethanol into the obtained filtrate for precipitation, and obtaining a precipitate.
Preferably, the step of degrading the sodium nitrite is as follows: preparing the oxidized heparin sodium into a solution, adjusting the pH to 2.5-3.0, adding a sodium nitrite solution, and continuously reacting for 1-2 hours at the temperature of 20-25 ℃; after the reaction is finished, adjusting the pH value to 9.5-10.5%, adding sodium borohydride accounting for 0.8-1.2% of the weight of the heparin sodium, and reacting for 10-14 hours.
Preferably, the dermatan sulfate is treated as follows: obtaining solution after degradation reaction of sodium nitrite, adjusting pH to 6.0-7.0, weighing sodium chloride, adding into filtrate, adding ethanol solution, standing for precipitation, and obtaining liquid part, wherein the precipitation part is dermatan sulfate.
Further, the concentration of the sodium chloride added into the filtrate is 1-2% (w/v).
Further, the concentration of the ethanol solution is 90-98%, and the adding amount is 0.2-0.5 time of the volume of the filtrate.
Further, the obtained liquid part is obtained by continuously adding ethanol with the volume of 1-2 times of that of the liquid part for precipitation, and a precipitate part is obtained, namely the low-molecular heparin sodium.
Preferably, the molecular weight fractionation is performed in the following manner: obtaining low-molecular-weight heparin sodium treated by dermatan sulfate, preparing feed liquid with the concentration of 4-6%, and adding sodium chloride until the concentration of sodium chloride in the feed liquid is 1-2%; adjusting the pH value of the solution to 6.0-7.0, adding ethanol, and standing.
Further, the ethanol is absolute ethanol, and the volume of the ethanol added is 0.8-1.2 of the volume of the feed liquid.
Further, the step of molecular weight classification also comprises a step of detection, and if the molecular weight detection is unqualified, the treatment mode is repeated.
In a second aspect of the present invention, there is provided an application of the method for preparing dalteparin sodium of the first aspect in preparing low molecular heparin sodium.
In the technical scheme of the second aspect, the low molecular weight heparin sodium represents unfractionated low molecular weight heparin sodium and is obtained by degrading sodium nitrite.
In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the technical solution of the present invention will be described in detail below with reference to specific examples and comparative examples.
Example 1
In the embodiment, a method for preparing dalteparin sodium by using crude heparin sodium as a raw material is provided, and the preparation method comprises the following steps:
(1) dissolving a crude product: dissolving crude heparin sodium at 15% (w/v), adding 3% (w/v) enteric salt, heating to 52 deg.C, and stirring to dissolve.
(2) Primary enzymolysis of a crude product: the solution from (1) is adjusted to pH8.5 with 40% sodium hydroxide solution. Pancreatin, 0.5% of the weight of the crude heparin, was then added to start the enzymatic hydrolysis. Controlling the pH value within 8.5-9.0 during enzymolysis, controlling the temperature at 49 ℃, stirring once every 20min, stirring 10min every time, rotating speed at 60-70rpm, and carrying out enzymolysis for 5h, and recording the temperature and the pH value once every 30 min.
(3) Heating to remove foreign proteins: after the enzymolysis is finished, adjusting the pH value to 6.5 +/-0.5 by using 4mol/L hydrochloric acid, introducing steam into the interlayer, quickly heating to 85 ℃, keeping the temperature for 20min, introducing cold water into the interlayer, and cooling to 50 ℃ within about 10 min.
(4) Secondary enzymolysis of the crude product: the pH of the solution in (3) is adjusted to 8.5 with 40% sodium hydroxide solution. Then adding 2709 protease accounting for 0.5 percent of the weight of the crude heparin to start enzymolysis. Controlling pH within 8.5-9.0 during enzymolysis, controlling temperature at 49 deg.C, stirring once every 20min, stirring for 10min every time, performing enzymolysis for 5h, and recording temperature and pH once every 30 min.
(5) Heating to remove foreign proteins: after the enzymolysis is finished, adjusting the pH value to 6.5 +/-0.5 by using 4mol/L hydrochloric acid, introducing steam into the interlayer, quickly heating to 85 ℃, keeping the temperature for 20min, introducing cold water into the interlayer, and cooling to 50 ℃ within about 10 min.
(6) Centrifuging: the feed liquid was centrifuged with a tubular centrifuge at about 12000 rpm. And collecting the feed liquid.
(7) Adding 1.5 times of ethanol (92-93%) into the feed liquid collected in the step (6) under the stirring of ethanol precipitation, standing and precipitating for 4 hours, vacuumizing the upper layer of ethanol solution, adding 70L of purified water (measured) into the lower layer of precipitate, and stirring and dissolving.
(8) Ion exchange purification
Adjusting the pH value of the feed liquid in the (7) to 8.5 +/-0.5 by using 40% sodium hydroxide solution, controlling the temperature of the feed liquid to be 30-45 ℃, and adsorbing the feed liquid on a resin column on an infusion pump. (resin lewatit 1074)
Controlling the speed to be 1BV/h, if the discharged liquid contains heparin sodium by using azure A detection, collecting, filtering by using a box type filter press, and adsorbing once by using resin again until the effluent liquid does not contain heparin, stopping ion exchange. The liquid surface was kept flat with the resin surface, and the exchanged mother liquor was discarded.
Thirdly, the resin adsorption is completed, each chromatographic column is washed by 2BV secondary reverse osmosis water, the water temperature is 30 +/-5 ℃, the flow rate is 2BV/h, and the washing liquid is drained. After washing, the liquid level was kept level with the resin surface.
Preparing a 5 DEG Be casing salt solution, adjusting the temperature to be 30 +/-5 ℃, adjusting the pH to be 8.5 +/-0.5 by using a 40% sodium hydroxide solution, starting to wash the chromatographic column, controlling the flow rate to be 0.8BV/h, detecting the discharged liquid by using azure A under washing, and collecting and processing the discharged liquid if the discharged liquid is purple; if blue color is developed, it is discharged. After washing, the liquid level was kept level with the resin surface.
Preparing 15 DEG Be casing salt solution, adjusting the temperature to be 30 +/-5 ℃, adjusting the pH to be 8.5 +/-0.5 by using 40% sodium hydroxide solution, starting elution, controlling the flow rate to be 0.6BV/h, detecting eluent by using azure A, collecting the eluent in a turnover barrel (the volume of the column bed is about 3-5 times that of the eluent) when the eluent is purple, filtering the eluent by using a chamber filter press to a nanofiltration machine, carrying out nanofiltration (the molecular weight cut-off value is 400-500), concentrating the eluent by 3-4 times, detecting that the permeate liquid has no heparin, adding ethanol with the same volume as that of the eluent into the concentrated solution, and precipitating the mixture for 6-8 h. The supernatant was pumped into a spent ethanol storage tank and the precipitate was dissolved with secondary reverse osmosis water to a concentration of about 10% (w/v).
(9) Oxidation by oxygen
First oxidation: the pH of the solution dissolved in (8) - (v) was adjusted to 9.5 with 40% NaOH solution, and the temperature was adjusted to 28 ℃. Adding 1% (v/v) hydrogen peroxide (original concentration is 30%, diluting at a ratio of 1: 3) into the feed liquid, adding the hydrogen peroxide in three times (adding the hydrogen peroxide in 1 time every 30min, adding one third of the hydrogen peroxide in each time), adding the hydrogen peroxide in 1 hour, intermittently stirring, and carrying out co-oxidation for 3 hours from the first addition. After oxidation, the mixture was filtered through a plate frame until clear.
And (2) second oxidation: then 0.5% (v/v) hydrogen peroxide (original concentration is 30%, diluted 1:3 before adding) is added into the feed liquid for three times (1 time is added every 30min, one third of the hydrogen peroxide is added every time), the adding is finished for 1 hour, the intermittent stirring is carried out, and the co-oxidation is carried out for 8 hours from the first adding. The temperature and pH of the feed liquid are recorded every 1 hour in the oxidation process, and the pH of the solution is controlled to be 9.5 +/-0.5 and the temperature is controlled to be 28 +/-5 ℃. After oxidation, the mixture was filtered through a plate frame until clear. Sampling and detecting (4mg/ml solution A260<0.18, A280<0.13), if not, repeating the first oxidation and the second oxidation until the solution is qualified.
(10) DS (dermatan sulfate) assay
After the oxidation is finished, the feed liquid is filtered by a titanium filter, the pH value is adjusted to 6.5 +/-0.5 by 4mol/L hydrochloric acid solution, medicinal sodium chloride is added according to the proportion of 1.5 percent, medicinal ethanol with the same volume is added into the feed liquid for precipitation after the filtration, the stirring is carried out for 30min, and the precipitation is carried out for 4 h. The precipitate was taken to determine dermatan sulfate content.
(11) Degradation of sodium nitrite
Firstly, converting the precipitate in the step (10) according to the feeding amount (namely crude heparin sodium) (deducting dermatan sulfate) to prepare 10% heparin sodium solution, slowly adjusting the pH of the liquid medicine to 2.5-3.0 by using 6mol/L hydrochloric acid under stirring, weighing 5% (w/v) sodium nitrite of the heparin sodium, dissolving the sodium nitrite into 25% concentration (w/v) by using purified water, adding the sodium nitrite into a reaction system, and keeping the reaction temperature at 20-25 ℃ for continuous reaction (1.5 hours).
② after the degradation reaction, 20 percent (w/v) sodium hydroxide solution is used for adjusting the pH value of the traditional Chinese medicine liquid to 10.0.
And thirdly, weighing sodium borohydride with the weight being 1 percent of that of the heparin sodium for reduction reaction, adding the sodium borohydride into the liquid medicine for dissolution, and continuously reacting for 12 hours under stirring.
(12) Removal of dermatan sulfate
Adjusting pH of the reduced medicinal liquid in the step (11) to 6.0 with 20.0% sodium hydroxide solution, filtering, weighing sodium chloride with volume of 1.5% (w/v) of the medicinal liquid, adding into the filtrate for dissolving, adding ethanol (95% ethanol) with volume 0.3 times of the medicinal liquid under stirring, and standing for 6-12 hr. The precipitate was collected (this material was dermatan sulfate) for other use. The upper layer liquid was poured out and precipitated by adding 1.5 times of ethanol. And collecting the precipitate.
(13) Molecular weight fractionation
And (3) according to the yield of the final product of 35%, dissolving the precipitate in the step (12) by using purified water, weighing sodium chloride with the volume of 1.5% (w/v) of the liquid medicine according to the concentration of 5% (w/v), and adding the sodium chloride into the liquid medicine for dissolving. Adjusting pH to 6.0-7.0 with 6mol/L hydrochloric acid solution, adding 0.8-1.2 times volume of ethanol (anhydrous ethanol) under stirring, and standing for 6-12 hr. Sampling to detect the molecular weight. And if the molecular weight detection is not qualified, repeating the operation until the molecular weight detection is qualified.
(14) Drying
Dehydrating the feed liquid with qualified molecular weight obtained in the step (13) and drying to obtain the final product of the dalteparin sodium.
The quality of the dalteparin sodium prepared by the process reaches the index requirements of the United states pharmacopoeia and European pharmacopoeia, and the yield is greatly improved.
Example 2
In this embodiment, another preparation method of dalteparin sodium is provided, which includes the following steps:
(1) dissolving a crude product: dissolving crude heparin sodium at 14% (w/v), adding 2% (w/v) enteric salt, heating to 52 deg.C, and stirring to dissolve.
(2) Primary enzymolysis of a crude product: the pH of the solution from (1) is adjusted to 9.0 with 40% sodium hydroxide solution. Pancreatin, 0.4% of the weight of the crude heparin, was then added to start the enzymatic hydrolysis. Controlling pH within 8.5-9.0 during enzymolysis, controlling temperature at-52 deg.C, stirring once every 22min, stirring for 11min every time, rotating speed at 60-70rpm, and performing enzymolysis for 4h, and recording temperature and pH once every 30 min.
(3) Heating to remove foreign proteins: after enzymolysis, adjusting pH to 6.0 with 4mol/L hydrochloric acid, introducing steam into the interlayer, rapidly heating to 90 deg.C, maintaining for 20min, introducing cold water into the interlayer, and cooling to 50 deg.C within about 10 min.
(4) Secondary enzymolysis of the crude product: the pH of the solution in (3) is adjusted to 9.0 with 40% sodium hydroxide solution. Then adding 2709 protease accounting for 0.5 percent of the weight of the crude heparin to start enzymolysis. Controlling the pH value within the range of 8.5-9.0 in the enzymolysis process, controlling the temperature to be 52 ℃, stirring once every 18min, stirring for 9min every time, rotating speed of 60-70rpm, carrying out enzymolysis for 5h, and recording the temperature and the pH value once every 30 min.
(5) Heating to remove foreign proteins: after enzymolysis, adjusting pH to 6.0 with 4mol/L hydrochloric acid, introducing steam into the interlayer, rapidly heating to 90 deg.C, maintaining for 20min, introducing cold water into the interlayer, and cooling to 50 deg.C within about 10 min.
(6) Centrifuging: the feed liquid is centrifuged by a tubular centrifuge at a speed of about 10000 rpm. And collecting the feed liquid.
(7) Adding 1.5 times of ethanol (92-93%) into the feed liquid collected in the step (6) under the stirring of ethanol precipitation, standing and precipitating for 4 hours, vacuumizing the upper layer of ethanol solution, adding 70L of purified water (measured) into the lower layer of precipitate, and stirring and dissolving.
(8) Ion exchange purification
Regulating pH of the feed liquid in the liquid (7) to 8.0 with 40% sodium hydroxide solution, controlling the temperature of the feed liquid at 45 ℃, and adsorbing the feed liquid on a resin column on an infusion pump. (resin lewatit 1074)
Controlling the speed to be 1BV/h, if the discharged liquid contains heparin sodium by using azure A detection, collecting, filtering by using a box type filter press, and adsorbing once by using resin again until the effluent liquid does not contain heparin, stopping ion exchange. The liquid surface was kept flat with the resin surface, and the exchanged mother liquor was discarded.
Thirdly, the resin adsorption is completed, each chromatographic column is washed by 2BV secondary reverse osmosis water, the water temperature is 35 ℃, the flow rate is 2BV/h, and the washing liquid is drained. After washing, the liquid level was kept level with the resin surface.
Preparing a 5 DEG Be casing salt solution, adjusting the pH value to 8.5 +/-0.5 by using a 40% sodium hydroxide solution at the temperature of 35 ℃, starting to wash the chromatographic column, controlling the flow rate to be 0.8BV/h, detecting the discharged liquid under washing by using azure A, and collecting and processing the discharged liquid if the discharged liquid is purple; if blue color is developed, it is discharged. After washing, the liquid level was kept level with the resin surface.
Preparing 15 DEG Be casing salt solution, adjusting the temperature to 35 ℃, adjusting the pH to 8.5 +/-0.5 by using 40% sodium hydroxide solution, starting elution, controlling the flow rate to 0.6BV/h, detecting the eluent by using azure A, collecting the eluent in a turnover barrel (the volume of the column bed is about 3-5 times that of the eluent) when the eluent is purple, filtering the eluent by using a chamber filter press until the eluent is nano-filtered in a nano-filter (the molecular weight cut-off value is 400-500), concentrating the eluent by 3-4 times, detecting that the permeate has no heparin, and adding ethanol with the same volume as that of the eluent into the concentrated solution for precipitation for 6-8 h. The supernatant was pumped into a spent ethanol storage tank and the precipitate was dissolved with secondary reverse osmosis water to a concentration of about 10% (w/v).
(9) Oxidation by oxygen
First oxidation: the pH of the solution dissolved in (8) - (v) was adjusted to 9.0 with 40% NaOH solution, and the temperature was adjusted to 23 ℃. Adding 1% (v/v) hydrogen peroxide (original concentration is 30%, diluting at a ratio of 1: 3) into the feed liquid, adding the hydrogen peroxide in three times (adding the hydrogen peroxide in 1 time every 30min, adding one third of the hydrogen peroxide in each time), adding the hydrogen peroxide in 1 hour, intermittently stirring, and carrying out co-oxidation for 3 hours from the first addition. After oxidation, the mixture was filtered through a plate frame until clear.
And (2) second oxidation: then 0.5% (v/v) hydrogen peroxide (original concentration is 30%, diluted 1:3 before adding) is added into the feed liquid for three times (1 time is added every 30min, one third of the hydrogen peroxide is added every time), the adding is finished for 1 hour, the intermittent stirring is carried out, and the co-oxidation is carried out for 8 hours from the first adding. The temperature and pH of the feed liquid are recorded every 1 hour during the oxidation process, and the pH of the solution is controlled to be 9.0 and the temperature is controlled to be 23 ℃. After oxidation, the mixture was filtered through a plate frame until clear. Sampling and detecting (4mg/ml solution A260<0.18, A280<0.13), if not, repeating the first oxidation and the second oxidation until the solution is qualified.
(10) DS (dermatan sulfate) assay
After the oxidation is finished, the feed liquid is filtered by a titanium filter, the pH value of the feed liquid is adjusted to 6.0 by 4mol/L hydrochloric acid solution, medicinal sodium chloride is added according to the proportion of 1.5 percent, medicinal ethanol with the same volume is added into the feed liquid for precipitation after the filtration, the stirring is carried out for 30min, and the precipitation is carried out for 4 h. The precipitate was taken to determine dermatan sulfate content.
(11) Degradation of sodium nitrite
Firstly, converting the precipitate in the step (10) according to the feeding amount (namely crude heparin sodium) (deducting dermatan sulfate) to prepare 10% heparin sodium solution, slowly adjusting the pH of the liquid medicine to 3.0 by using 6mol/L hydrochloric acid under stirring, weighing 5% (w/v) sodium nitrite of the heparin sodium, dissolving the sodium nitrite into 25% concentration (w/v) by using purified water, adding the sodium nitrite into a reaction system, and keeping the reaction temperature at 20-25 ℃ for continuous reaction (1.5 hours).
② after the degradation reaction, 20 percent (w/v) sodium hydroxide solution is used for adjusting the pH value of the traditional Chinese medicine liquid to 10.0.
And thirdly, weighing sodium borohydride with the weight being 1 percent of that of the heparin sodium for reduction reaction, adding the sodium borohydride into the liquid medicine for dissolution, and continuously reacting for 12 hours under stirring.
(12) Removal of dermatan sulfate
Adjusting pH of the reduced medicinal liquid in the step (11) to 7.0 with 20.0% sodium hydroxide solution, filtering, weighing sodium chloride with volume of 1.5% (w/v) of the medicinal liquid, adding into the filtrate for dissolving, adding ethanol (95% ethanol) with volume 0.3 times of the medicinal liquid under stirring, and standing for 6-12 hr. The precipitate was collected (this material was dermatan sulfate) for other use. The upper layer liquid was poured out and precipitated by adding 1.5 times of ethanol. And collecting the precipitate.
(13) Molecular weight fractionation
And (3) according to the yield of the final product of 35%, dissolving the precipitate in the step (12) by using purified water, weighing sodium chloride with the volume of 1.5% (w/v) of the liquid medicine according to the concentration of 5% (w/v), and adding the sodium chloride into the liquid medicine for dissolving. Adjusting pH to 6.0-7.0 with 6mol/L hydrochloric acid solution, adding 0.8-1.2 times volume of ethanol (anhydrous ethanol) under stirring, and standing for 6-12 hr. Sampling to detect the molecular weight. And if the molecular weight detection is not qualified, repeating the operation until the molecular weight detection is qualified.
(14) Drying
Dehydrating the feed liquid with qualified molecular weight obtained in the step (13) and drying to obtain the final product of the dalteparin sodium.
The quality of the dalteparin sodium prepared by the process reaches the index requirements of the United states pharmacopoeia and European pharmacopoeia, and the yield is greatly improved.
Example 3
(1) Dissolving a crude product: dissolving crude heparin sodium at 16% (w/v), adding 5% (w/v) enteric salt, heating to 49 deg.C, and stirring to dissolve.
(2) Primary enzymolysis of a crude product: the pH of the solution from (1) is adjusted to 9.0 with 40% sodium hydroxide solution. Pancreatin, 0.5% of the weight of the crude heparin, was then added to start the enzymatic hydrolysis. Controlling the pH value within the range of 8.5-9.0 in the enzymolysis process, controlling the temperature to be 52 ℃, stirring once every 18min, stirring for 9min every time, rotating speed of 60-70rpm, carrying out enzymolysis for 6h, and recording the temperature and the pH value once every 30 min.
(3) Heating to remove foreign proteins: after the enzymolysis is finished, adjusting the pH value to 6.5 +/-0.5 by using 4mol/L hydrochloric acid, introducing steam into the interlayer, quickly heating to 90 ℃, preserving the temperature for 20min, introducing cold water into the interlayer, and cooling to 45 ℃ within about 10 min.
(4) Secondary enzymolysis of the crude product: the pH of the solution in (3) is adjusted to 9.0 with 40% sodium hydroxide solution. Then adding 2709 protease accounting for 0.5 percent of the weight of the crude heparin to start enzymolysis. Controlling the pH value within the range of 8.5-9.0 in the enzymolysis process, controlling the temperature to be 52 ℃, stirring once every 18min, stirring for 9min every time, rotating speed of 60-70rpm, carrying out enzymolysis for 6h, and recording the temperature and the pH value once every 30 min.
(5) Heating to remove foreign proteins: after enzymolysis, adjusting pH to 7.0 with 4mol/L hydrochloric acid, introducing steam into the interlayer, rapidly heating to 85-90 deg.C, maintaining for 20min, introducing cold water into the interlayer, and cooling to 50 deg.C within about 10 min.
(6) Centrifuging: the feed liquid was centrifuged with a tubular centrifuge at about 12000 rpm. And collecting the feed liquid.
(7) Adding 1.5 times of ethanol (92-93%) into the feed liquid collected in the step (6) under the stirring of ethanol precipitation, standing and precipitating for 4 hours, vacuumizing the upper layer of ethanol solution, adding 70L of purified water (measured) into the lower layer of precipitate, and stirring and dissolving.
(8) Ion exchange purification
Adjusting the pH value of the feed liquid in the (7) to 8.5 +/-0.5 by using 40% sodium hydroxide solution, controlling the temperature of the feed liquid to be 30-45 ℃, and adsorbing the feed liquid on a resin column on an infusion pump. (resin lewatit 1074)
Controlling the speed to be 1BV/h, if the discharged liquid contains heparin sodium by using azure A detection, collecting, filtering by using a box type filter press, and adsorbing once by using resin again until the effluent liquid does not contain heparin, stopping ion exchange. The liquid surface was kept flat with the resin surface, and the exchanged mother liquor was discarded.
Thirdly, the resin adsorption is completed, each chromatographic column is washed by 2BV secondary reverse osmosis water, the water temperature is 35 ℃, the flow rate is 2BV/h, and the washing liquid is drained. After washing, the liquid level was kept level with the resin surface.
Preparing a 5 DEG Be casing salt solution, adjusting the pH value to 8.5 +/-0.5 by using a 40% sodium hydroxide solution at the temperature of 25 ℃, starting to wash the chromatographic column, controlling the flow rate to be 0.8BV/h, detecting the discharged liquid under washing by using azure A, and collecting and processing the discharged liquid if the discharged liquid is purple; if blue color is developed, it is discharged. After washing, the liquid level was kept level with the resin surface.
Preparing 15 DEG Be casing salt solution, adjusting the temperature to 35 ℃, adjusting the pH value to 8.0 by using 40% sodium hydroxide solution, starting elution, controlling the flow rate to be 0.6BV/h, detecting the eluent by using azure A, collecting the eluent in a turnover barrel (the volume of the column bed is about 3-5 times that of the eluent) when the eluent is purple, filtering the eluent by using a box type filter press until the eluent is nanofilter for nanofiltration (the molecular weight cut-off value is 400-500), concentrating the eluent by 3-4 times, detecting that the permeate liquid has no heparin, and adding ethanol with the same volume to the elution concentrated liquid for precipitation for 6-8 h. The supernatant was pumped into a spent ethanol storage tank and the precipitate was dissolved with secondary reverse osmosis water to a concentration of about 10% (w/v).
(9) Oxidation by oxygen
First oxidation: the pH of the solution dissolved in (8) - (v) was adjusted to 10.0 with 40% NaOH solution, and the temperature was adjusted to 33 ℃. Adding 1% (v/v) hydrogen peroxide (original concentration is 30%, diluting at a ratio of 1: 3) into the feed liquid, adding the hydrogen peroxide in three times (adding the hydrogen peroxide in 1 time every 30min, adding one third of the hydrogen peroxide in each time), adding the hydrogen peroxide in 1 hour, intermittently stirring, and carrying out co-oxidation for 3 hours from the first addition. After oxidation, the mixture was filtered through a plate frame until clear.
And (2) second oxidation: then 0.5% (v/v) hydrogen peroxide (original concentration is 30%, diluted 1:3 before adding) is added into the feed liquid for three times (1 time is added every 30min, one third of the hydrogen peroxide is added every time), the adding is finished for 1 hour, the intermittent stirring is carried out, and the co-oxidation is carried out for 7 hours from the first adding. The temperature and pH of the feed liquid are recorded every 1 hour in the oxidation process, and the pH of the solution is controlled to be 9.5 +/-0.5 and the temperature is controlled to be 28 +/-5 ℃. After oxidation, the mixture was filtered through a plate frame until clear. Sampling and detecting (4mg/ml solution A260<0.18, A280<0.13), if not, repeating the first oxidation and the second oxidation until the solution is qualified.
(10) DS (dermatan sulfate) assay
After the oxidation is finished, the feed liquid is filtered by a titanium filter, the pH value is adjusted to 7.0 by 4mol/L hydrochloric acid solution, medicinal sodium chloride is added according to the proportion of 1.5 percent, medicinal ethanol with the same volume is added into the feed liquid for precipitation after the filtration, the stirring is carried out for 30min, and the precipitation is carried out for 4 h. The precipitate was taken to determine dermatan sulfate content.
(11) Degradation of sodium nitrite
Firstly, converting the precipitate in the step (10) according to the feeding amount (namely crude heparin sodium) (deducting dermatan sulfate) to prepare 10% heparin sodium solution, slowly adjusting the pH of the liquid medicine to 2.5-3.0 by using 6mol/L hydrochloric acid under stirring, weighing 5% (w/v) sodium nitrite of the heparin sodium, dissolving the sodium nitrite into 25% concentration (w/v) by using purified water, adding the sodium nitrite into a reaction system, and keeping the reaction temperature at 20-25 ℃ for continuous reaction (1.5 hours).
② after the degradation reaction, 20 percent (w/v) sodium hydroxide solution is used for adjusting the pH value of the traditional Chinese medicine liquid to 10.0.
And thirdly, weighing sodium borohydride with the weight being 1 percent of that of the heparin sodium for reduction reaction, adding the sodium borohydride into the liquid medicine for dissolution, and continuously reacting for 12 hours under stirring.
(12) Removal of dermatan sulfate
Adjusting pH of the reduced liquid medicine in the step (11) to 6.5 with 20.0% sodium hydroxide solution, filtering, weighing sodium chloride with volume of 1.5% (w/v) of the liquid medicine, adding into the filtrate for dissolving, adding ethanol (95% ethanol) with volume of 0.3 times of the liquid medicine under stirring, and standing for 6-12 hr. The precipitate was collected (this material was dermatan sulfate) for other use. The upper layer liquid was poured out and precipitated by adding 1.5 times of ethanol. And collecting the precipitate.
(13) Molecular weight fractionation
And (3) according to the yield of the final product of 35%, dissolving the precipitate in the step (12) by using purified water, weighing sodium chloride with the volume of 1.8% (w/v) of the liquid medicine according to the concentration of 5% (w/v), and adding the sodium chloride into the liquid medicine for dissolving. Adjusting pH to 6.0-7.0 with 6mol/L hydrochloric acid solution, adding 0.8-1.2 times volume of ethanol (anhydrous ethanol) under stirring, and standing for 6-12 hr. Sampling to detect the molecular weight. And if the molecular weight detection is not qualified, repeating the operation until the molecular weight detection is qualified.
(14) Drying
Dehydrating the feed liquid with qualified molecular weight obtained in the step (13) and drying to obtain the final product of the dalteparin sodium.
The quality of the dalteparin sodium prepared by the process reaches the index requirements of the United states pharmacopoeia and European pharmacopoeia, and the yield is greatly improved.
The invention was examined on the potency and yield of multiple batches of daparinux sodium prepared as described in methods of examples 1-3, with the results shown in table 1 below:
TABLE 1
The results of the above investigation can be summarized that the potency of the daparinux sodium prepared from different crude products can be stabilized above 110, and the yield in the preparation process can be maintained above 70%. The dalteparin sodium provided by the invention also has good clarity and stability.
Comparative example
In this example, the effects of the technical route provided by the present invention and the existing route for preparing dalteparin sodium were compared. The inventor uses the same batch of crude heparin sodium to carry out comparative experiments, wherein the route 1 is to remove the dermatan after degradation, and the route 2 is to degrade the refined crude heparin sodium after the refined crude heparin sodium is refined heparin sodium (containing the dermatan removal), wherein the routes 1(1) and 1(2) and the routes 2(1) and 2(2) are parallel experiments, and the results are shown in table 1:
TABLE 1
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A preparation method of dalteparin sodium comprises enzymolysis, resin separation, oxidation, sodium nitrite degradation and molecular weight grading treatment, and is characterized in that a crude product of heparin sodium is used as a raw material, and the degradation of sodium nitrite is carried out to treat dermatan sulfate.
2. The method for preparing dalteparin sodium of claim 1, wherein the enzymolysis process comprises sequentially carrying out enzymolysis with pancreatin and alkaline protease;
preferably, the enzymolysis process comprises the following specific steps: adjusting the pH value of the crude heparin sodium solution to 8.5-9.0, adding pancreatin, and controlling the temperature to be 49-52 ℃ to react for 4-6 h; adjusting pH to 6.5 +/-0.5 after enzymolysis, heating for a period of time, and cooling to remove impure protein; regulating the pH value to 8.5-9.0 again, adding alkaline protease, controlling the temperature to 49-52 ℃, reacting for 4-6 h, carrying out secondary enzymolysis, regulating the pH value to 6.5 +/-0.5 after the second enzymolysis is finished, heating for a period of time, and cooling to remove foreign proteins; adding ethanol solution into the material liquid obtained by centrifugation, standing and precipitating to obtain a precipitate part.
3. The method of claim 2, wherein the alkaline protease is an alkaline protease produced by Bacillus licheniformis;
preferably, the alkaline protease is 2709 protease;
further, the concentration of the crude heparin sodium solution is 14-16%, further, the concentration of the crude heparin sodium is 14%, 15% or 16%, and in the best effect embodiment, the concentration of the crude heparin sodium is 15%;
further, the pancreatin or the alkaline protease accounts for 0.4-0.6% of the weight of the crude heparin sodium; specifically 0.4%, 0.5% or 0.6%, and in the embodiment with the better effect, 0.5%;
furthermore, in the enzymolysis reaction process, a stirring step is also provided, and the stirring mode is not limited to a mechanical stirring mode or a manual stirring mode.
4. The method for preparing dalteparin sodium of claim 2, wherein the specific steps of heating for a period of time and cooling to remove impure proteins are as follows: after the enzymolysis is finished, adjusting the pH value of a reaction system to 6.5 +/-0.5, heating to 85-90 ℃, preserving heat for 18-22 min, then cooling, and reducing the temperature of the reaction system to 48-52 ℃ within 8-12 min;
preferably, the heating mode can adopt a direct heating reaction system or a heat transfer mode to heat;
further, the heating uses steam for heat transfer; the cooling process can adopt a mode of introducing cold water into the interlayer of the reaction device to realize rapid cooling;
further, the concentration of the ethanol solution is 92% -93%, and the volume of the ethanol solution is 1-3 times of that of the feed liquid part;
further, the standing and precipitating time is 3-5 h.
5. The method for preparing dalteparin sodium of claim 1, wherein the resin separation method uses strong anion resin;
preferably, the resin separation mode is as follows: adjusting the pH of the feed liquid to 8.5 +/-0.5, adding the feed liquid into resin for adsorption, washing by using a salt solution after adsorption is finished to obtain an eluent, and adding ethanol into the eluent for precipitation for a period of time to obtain a precipitate part.
6. The method for preparing dalteparin sodium of claim 1, wherein the oxidation is performed with hydrogen peroxide, and the oxidation is performed once or twice;
preferably, two oxidations are required;
further, the oxidation treatment comprises the following specific steps: adjusting the pH value of the solution to 9.5 +/-0.5, controlling the reaction temperature to 28 +/-5 ℃, adding 0.5-1.5% of hydrogen peroxide solution into the reaction liquid, oxidizing for 2-4 hours, and filtering to obtain a liquid part; repeating the operation till the two times of oxidation and co-oxidation are carried out for 7-9 hours;
furthermore, in the oxidation treatment process, the hydrogen peroxide solution is added in a manner of adding for a plurality of times, in some specific embodiments, the hydrogen peroxide solution is added for 3-4 times, and the interval time is 20-30 min;
further, after the oxidation is finished, adjusting the pH value of the feed liquid to 6.5 +/-0.5, adding salt into the reaction feed liquid until the concentration is 1-2%, filtering, adding ethanol into the obtained filtrate for precipitation, and obtaining a precipitate.
7. The method for preparing dalteparin sodium of claim 1, wherein the degradation of sodium nitrite is performed as follows: preparing the oxidized heparin sodium into a solution, adjusting the pH to 2.5-3.0, adding a sodium nitrite solution, and continuously reacting for 1-2 hours at the temperature of 20-25 ℃; after the reaction is finished, adjusting the pH value to 9.5-10.5%, adding sodium borohydride accounting for 0.8-1.2% of the weight of the heparin sodium, and reacting for 10-14 hours.
8. The method of claim 1, wherein said dermatan sulfate is treated by the following method: obtaining a solution after sodium nitrite degradation reaction, adjusting the pH value to 6.0-7.0, weighing sodium chloride, adding the sodium chloride into the filtrate, adding an ethanol solution, standing and precipitating, wherein a precipitate part is dermatan sulfate, and a liquid part is obtained;
preferably, the concentration of the sodium chloride added into the filtrate is 1-2% (w/v);
preferably, the concentration of the ethanol solution is 90-98%, and the adding amount is 0.2-0.5 time of the volume of the filtrate;
preferably, the obtained liquid part is obtained by continuously adding ethanol with the volume of 1-2 times to precipitate, and the obtained precipitate part is the low-molecular heparin sodium.
9. The method for preparing dalteparin sodium of claim 1, wherein the molecular weight fractionation is performed by: obtaining low-molecular-weight heparin sodium treated by dermatan sulfate, preparing feed liquid with the concentration of 4-6%, and adding sodium chloride until the concentration of sodium chloride in the feed liquid is 1-2%; adjusting the pH value of the solution to 6.0-7.0, adding ethanol, and standing;
preferably, the ethanol is absolute ethanol, and the volume of the added ethanol is 0.8-1.2 of the volume of the feed liquid;
preferably, the step of classifying the molecular weight further comprises a step of detecting, and if the molecular weight is not detected, the processing mode is repeated.
10. Use of the process for the preparation of dalteparin sodium of any one of claims 1-9 in the preparation of low molecular weight heparin sodium.
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| CN107011463A (en) * | 2017-05-31 | 2017-08-04 | 广元市海天实业有限责任公司 | A kind of production method of low molecular weight heparin sodium |
| CN112029015A (en) * | 2020-10-20 | 2020-12-04 | 德清亚泰肠衣有限公司 | Production and purification process of high-purity low-molecular-weight heparin sodium |
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