CN105399867A - Preparation method for heparin sodium - Google Patents
Preparation method for heparin sodium Download PDFInfo
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- CN105399867A CN105399867A CN201510886754.6A CN201510886754A CN105399867A CN 105399867 A CN105399867 A CN 105399867A CN 201510886754 A CN201510886754 A CN 201510886754A CN 105399867 A CN105399867 A CN 105399867A
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- heparin sodium
- heparin
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Abstract
The invention discloses a preparation method for heparin sodium. After crude heparin is enzymatically degraded, protein removal treatment is conducted, a heparin sodium solution is obtained through resin exchange after oxidative decoloration is completed, the oxidative decoloration solution is precipitated, dehydrated and dried, and then fine heparin sodium is obtained. The raw material source is wide, universality is high, the activity yield is high, the technological quality is stable, production cost is low, the preparation method of heparin sodium is high in operability under the normal temperature condition, and then low-investment, low-cost, high-recovery, high-operability, stable-technological-quality and large-batch preparation of heparin sodium is conveniently achieved.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of preparation method of heparin sodium.
Background technology
Heparin, be common with sodium salt and sodium salt in hematology testing, there is unique using value, heparin chelating ability is low, little on the impact of moisture movement, few to blood ingredient interference, do not affect erythrocyte volume, do not cause haemolysis, so in the multiple inspection being sample with whole blood or blood plasma, recommendation heparin is as antithrombotics.Be applicable to fragility test, blood gas analysis, pcv experiment, lectin from hemolymph and emergency treatment biochemical measurement.In the detection to ph value, vim and vigour, ionogen and calcium ion, heparin is unique operable antithrombotics, so recommendation heparin sodium is as antithrombotics.
The preparation of heparin sodium, to material and envrionment temperature control overflow higher, hardware facility drop into larger; This method adopts strong acidic condition operation (PH1.5) at Deproteinization process procedure, to heparin activity loss, (heparin solution is very unstable under low pH value environment comparatively greatly, very easily to degrade inactivation), affect final activity yield and tire, operability is not strong; This method adopts ultrafiltration and concentration isolation technique, and ultrafiltration apparatus is expensive, and hardware drops into larger; The ionic equilibrium exchanged form that this method adopts, be the mode that constantly gets involved with sodium at high concentration ion to realize the conversion of heparin sodium to heparin sodium, adequacy and the continous-stable of Dynamic ion balance conversion cannot be ensured.Prepare heparin sodium abroad and all adopt ion exchange method, this method needs to select refined heparin sodium to be raw material, raw materials cost is high, and PH can be caused in ion exchange process sharply to decline, have a strong impact on the yield of product and tire, and exchange process needs to carry out under low temperature puies forward condition, cause heparin sodium to produce hardware facility and drop into comparatively large, production cost is higher.
Summary of the invention
The object of the present invention is to provide a kind of heparin sodium preparation method, to solve the problem.
Technical scheme of the present invention is: heparin sodium preparation method, and it is prepared with the following step:
1, crude product heparin enzymolysis: crude heparin sodium is dissolved, tune pH value is 7.5-9.0, be warming up to 50-55 DEG C, add heparin enzymolysis pancreatin, insulation 4-5 hour, adjust pH value 9.0-11, after being warming up to 85 DEG C ~ 90 DEG C, cooling, filters and removes throw out, filtrate alcohol settling, collecting precipitation thing (A);
2, Deproteinization: step 1 gained throw out (A) water dissolution, stirs, and adjusts PH to 9-11, add protein flocculating settling agent, stir, leave standstill, filter and remove precipitation, collect filtrate (D), filtrate (D) adjusts PH to 11-13 to be worth, stir, leave standstill, filter and remove precipitation, collect filtrate (E), filtrate (E) adjusts pH value to neutral, with alcohol settling, and collecting precipitation thing (B);
3, oxidative decoloration: by step 2 gained throw out (B) water dissolution, with hydrogen peroxide oxidation 24-48 hour, after adjustment PH to 10.5-11.5, filter and remove throw out, collect filtrate (F), filtrate (F) adjusts pH value to 6.5-7.5, with alcohol settling, and collecting precipitation thing (C);
4, resins exchange: by step 3 gained throw out (C) water dissolution, add LiCl, stirring and dissolving, carry out ion-exchange by resin column, collected post effluent liquid, and obtained heparin sodium aqua;
5, precipitation, dehydration, drying: by step 4 gained heparin sodium aqua, filter, with alcohol settling, collecting precipitation thing (G), throw out (G) dewaters, then vacuum-drying, and pulverizing, obtains refined heparin sodium.
In step 1, described heparin enzymolysis pancreatin selects trypsinase, containing a kind of in tryptic Cotazym, neutral trypsinase, Sumizyme MP or containing two or more combinations.
In step 2, described protein flocculating settling agent selects a kind of in sodium polyacrylate, polymerize aluminum chloride, calcium polyacrylate (CPA), polymerize aluminum chloride, calcium chloride or containing two or more combinations.
In described step 5, described throw out (G) dehydration implements dehydration with dehydrated alcohol.
In described step 1, described crude heparin sodium dissolves and is: with NaCL solution by crude heparin sodium material dissolution, crude heparin sodium concentration of polymer solution is 8%-16%.
In described step 1, the mass percent adding heparin enzymolysis enzyme in crude heparin sodium solution is the 0.1-0.3% of crude heparin sodium solution quality.
In described step 4, the mass percent concentration of the aqueous solution of throw out (C) is 8%-20%; The mass percent of the sodium-chlor added in the aqueous solution of throw out (C) is the 1.5%-8.5% of throw out (C) aqueous solution quality; Resin used is ionic exchange resin.
Described crude heparin sodium is commercial goods raw material, tire into 65-105IU/mg not etc.
The present invention take crude heparin sodium as raw material, and raw material sources are extensive, and cost is lower.In addition, the present invention, hardware facility drops into little, envrionment temperature is less demanding, operate under can realizing the normal temperature condition of technique whole process (except the link that local needs the short period of time to heat), resin method dynamic ion-exchange process is gentle, does not have loss of activity, the rate of recovery reaches more than 95%, and product is tired and reached more than 160IU/mg.Contrast with exchange method of ionic equilibriom for heparin sodium disclosed in domestic patent 200510019846.0, the present invention has following innovation: (1), invention increases the enzymolysis process of crude product heparin, crude product heparin exists mainly with the form of heparin-protein complex, by the enzymolysis to crude product heparin, heparin-protein complex can be made fully efficiently to dissociate, allow heparin active substance dissociate discharge, be convenient to further extraction, this is the basic premise guaranteeing later stage high reactivity yield.(2), at protein removal process procedure, domestic patent 200510019846.0, adopt low temperature (less than 10 DEG C) low pH value (PH1.5) conditional operation, heparin is very unstable under sour environment, its activity has greater loss, adopt low temperature environment to delay or relatively reduce heparin activity loss, heparin activity can not be retained completely; And the present invention adopts the technology adding protein flocculating settling agent under alkaline environment, can realize, while efficient removal protein, the loss of activity of heparin can not being caused, without the need to cold condition, ensureing ultimate yield.(3), in the conversion links of heparin sodium to heparin sodium, domestic patent 200510019846.0, employing ionic equilibrium exchanges, and coordinate ultrafiltration and concentration, the mode constantly got involved with sodium at high concentration ion, to realize the conversion of heparin sodium to heparin sodium, cannot ensure adequacy and the continous-stable of Dynamic ion balance conversion; And the present invention adopts exchange resin method, utilize sodium ion, sodium ion to the difference of the Preferential adsorption displacement degree of resin functional group, realize being that the sodium-sodium mediated exchanges with resin, thus realize the transition of heparin sodium to heparin sodium, and whole process the carrying out under normal temperature, PH neutrallty condition of continous-stable all the time, displacement transformation process is gentle, and transition is fully, do not adsorb residual to heparin activity body, there is no loss of activity.Resin material input and working service cost be very low (use of resin, need only anticipate normally by producer's operation instruction and use operates normally) also, is applicable in enormous quantities preparation.(4), under neutral or basic conditions substance characteristics such as highly stable (even in the basic conditions can withstand higher temperatures) unstable under acidic condition based on heparin, present invention process whole process, all the time under allowing heparin be in neutrality or alkaline condition, effectively can retain heparin activity, improve yield to greatest extent, and without the need to low temperature environment, the hardware facility into creating low temperature environment can be avoided to drop into and working service, reduce costs, workable.
Embodiment
Embodiment 1:
Step 1: use the NaCL solution of 1%-3% by crude heparin sodium raw material (commercial goods raw material, do not tire 65-105IU/mg not etc.) stirring and dissolving, crude heparin sodium concentration is 8%-16% (mass percent concentration), PH to 7.5-9.0 is adjusted by the NaOH solution of 5M, be warming up to 50-55 DEG C, heparin enzymolysis trypsinase is added by the 0.1-0.3% of crude heparin sodium solution quality, insulation 4-5 hour, adjust pH value to 9.0-11 by the NaOH solution of 5M, be warming up to 85 DEG C ~ 90 DEG C, be cooled to normal temperature, filter and remove throw out, filtrate is with the alcohol settling more than 4 hours of 0.8-1.5 times of volume 95%, collecting precipitation thing A,
Step 2: by throw out A pure water stirring and dissolving, concentration of ordinary dissolution is 10%-20% (mass percent), PH to 8.5-11 is adjusted by the NaOH solution of 5M, protein flocculating settling agent calcium chloride is added by the 0.1%-5% of throw out A aqueous solution quality, stir 1-2 hour, leave standstill more than 1 hour, filter and remove precipitation, collect filtrate, adjust pH value to 11-13 with NaOH (or sodium carbonate) solution of 5M, stir, leave standstill more than 30 minutes, filter and remove precipitation, collect filtrate, adjust pH value to neutral with the HCL solution of 6M, with 95% alcohol settling more than 4 hours of 0.8-1.5 times of volume, collecting precipitation thing B,
Step 3: sediment B pure water is dissolved, concentration of ordinary dissolution is 15%-30% (mass percent concentration), the hydrogen peroxide (can gradation add) that purity is 30% is added by 1% ~ 4% of sediment B aqueous solution quality, oxidation 24-48 hour, with the NaOH solution adjustment PH to 10.5-11.5 of 5M, filter and remove throw out, collect filtrate, adjust pH value to 6.5-7.5 with the HCL solution of 6M, with 95% alcohol settling more than 4 hours of 0.8-1.5 times of volume, collecting precipitation thing C;
Step 4: gained throw out C pure water is dissolved, solubleness is 8%-20% (mass percent concentration), sodium-chlor is added again by the 1.5%-8.5% of throw out C solution quality, stirring and dissolving, carries out ion-exchange by regenerating the ionic resin post made the transition, and exchanges below flow velocity 10BV/h, collected post effluent liquid, and with the pure water through post of 1-2 times amount, merge effluent liquid, obtain Calciparine/sodium salt solution;
Step 5: by gained Calciparine/sodium salt solution, filters, and with 95% alcohol settling more than 4 hours of 0.8-3 times of volume, collecting precipitation thing, with dehydrated alcohol dehydration twice, then vacuum-drying, pulverizes, obtain refined heparin sodium.
Embodiment 2
Heparin enzymolysis pancreatin in step 1 is selected containing tryptic Cotazym;
Protein flocculating settling agent in step 2 is selected polymerize aluminum chloride;
Other steps are identical with embodiment 1, obtain refined heparin sodium.
Embodiment 3
Heparin enzymolysis pancreatin in step 1 is selected neutral trypsinase;
Protein flocculating settling agent in step 2 is selected sodium polyacrylate;
Other steps are identical with embodiment 1, obtain refined heparin sodium.
Embodiment 4
Heparin enzymolysis pancreatin in step 1 is selected Sumizyme MP;
Protein flocculating settling agent in step 2 is selected sodium polyacrylate;
Other steps are identical with embodiment 1, obtain refined heparin sodium.
Embodiment 5
Heparin enzymolysis pancreatin in step 1 is selected trypsinase;
Protein flocculating settling agent in step 2 is selected calcium polyacrylate (CPA);
Other steps are identical with embodiment 1, obtain refined heparin sodium.
Show the refined heparin sodium prepared by the inventive method, all reach above-mentioned quality index.
Claims (8)
1. a preparation method for heparin sodium, its step is as follows:
(1) crude product heparin enzymolysis: crude heparin sodium is dissolved, tune pH value is 7.5-9.0, be warming up to 50-55 DEG C, add heparin enzymolysis pancreatin, insulation 4-5 hour, adjust pH value 9.0-11, after being warming up to 85 DEG C ~ 90 DEG C, cooling, filters and removes throw out, filtrate alcohol settling, collecting precipitation thing (A);
(2) Deproteinization: step (1) gained throw out (A) water dissolution, stirs, and adjusts PH to 9-11, add protein flocculating settling agent, stir, leave standstill, filter and remove precipitation, collect filtrate (D), filtrate (D) adjusts PH to 11-13 to be worth, stir, leave standstill, filter and remove precipitation, collect filtrate (E), filtrate (E) adjusts pH value to neutral, with alcohol settling, and collecting precipitation thing (B);
(3) oxidative decoloration: by step (2) gained throw out (B) water dissolution, with hydrogen peroxide oxidation 24-48 hour, after adjustment PH to 10.5-11.5, filter and remove throw out, collect filtrate (F), filtrate (F) adjusts pH value to 6.5-7.5, with ethanol, and collecting precipitation thing (C);
(4) resins exchange: by step (3) gained throw out (C) water dissolution, add LiCl, stirring and dissolving, carry out ion-exchange by resin column, collected post effluent liquid, and obtained heparin sodium aqua;
(5) precipitation, dehydration, drying: by step (4) gained heparin sodium aqua, filter, with alcohol settling, collecting precipitation thing (G), throw out (G) dewaters, then vacuum-drying, and pulverizing, obtains refined heparin sodium.
2. heparin sodium preparation method as claimed in claim 1, is characterized in that in step (1), and described heparin enzymolysis pancreatin selects trypsinase, containing a kind of in tryptic Cotazym, neutral trypsinase, Sumizyme MP or containing two or more combinations.
3. heparin sodium preparation method as claimed in claim 1, it is characterized in that in step (2), described protein flocculating settling agent selects a kind of in sodium polyacrylate, polymerize aluminum chloride, calcium polyacrylate (CPA), polymerize aluminum chloride, calcium chloride or containing two or more combinations.
4. heparin sodium preparation method as claimed in claim 1, is characterized in that in described step (5), and described throw out (G) dehydration implements dehydration with dehydrated alcohol.
5. heparin sodium preparation method as claimed in claim 1, is characterized in that in described step (1), and described crude heparin sodium dissolves and is: with NaCL solution by crude heparin sodium material dissolution, crude heparin sodium concentration of polymer solution is 8%-16%.
6. heparin sodium preparation method as claimed in claim 5, the mass percent that it is characterized in that adding heparin enzymolysis enzyme in crude heparin sodium solution is the 0.1-0.3% of crude heparin sodium solution quality.
7. heparin sodium preparation method as claimed in claim 1, is characterized in that, in described step (4), the mass percent concentration of the aqueous solution of throw out (C) is 8%-20%; The mass percent of the sodium-chlor added in the aqueous solution of throw out (C) is the 1.5%-8.5% of throw out (C) aqueous solution quality.
8. heparin sodium preparation method as claimed in claim 1, is characterized in that, in described step (4), resin column used is ionic exchange resin.
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| CN201510886754.6A CN105399867A (en) | 2015-12-07 | 2015-12-07 | Preparation method for heparin sodium |
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| CN201510886754.6A CN105399867A (en) | 2015-12-07 | 2015-12-07 | Preparation method for heparin sodium |
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| CN105399867A true CN105399867A (en) | 2016-03-16 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101780643B1 (en) * | 2016-12-05 | 2017-09-21 | (주)우리비앤비 | Method for purifying heparin using enzymolysis |
| CN108456262A (en) * | 2018-03-13 | 2018-08-28 | 广元市海天实业有限责任公司 | A kind of preparation process of high purity heparin sodium |
| KR20190061322A (en) * | 2017-11-27 | 2019-06-05 | (주)우리비앤비 | Method for producing unfractionated heparin |
| CN110183550A (en) * | 2019-06-25 | 2019-08-30 | 广元市海天实业有限责任公司 | A kind of preparation process of refined heparin sodium |
| CN113004436A (en) * | 2021-04-30 | 2021-06-22 | 山东万邦赛诺康生化制药股份有限公司 | Preparation method of dalteparin sodium and application of method in preparation of low-molecular-weight heparin sodium |
| CN115028757A (en) * | 2022-06-29 | 2022-09-09 | 江苏麦德森制药有限公司 | Decolorizing method of heparin sodium |
-
2015
- 2015-12-07 CN CN201510886754.6A patent/CN105399867A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101780643B1 (en) * | 2016-12-05 | 2017-09-21 | (주)우리비앤비 | Method for purifying heparin using enzymolysis |
| KR20190061322A (en) * | 2017-11-27 | 2019-06-05 | (주)우리비앤비 | Method for producing unfractionated heparin |
| KR102298179B1 (en) | 2017-11-27 | 2021-09-06 | (주)우리비앤비 | Method for producing unfractionated heparin |
| CN108456262A (en) * | 2018-03-13 | 2018-08-28 | 广元市海天实业有限责任公司 | A kind of preparation process of high purity heparin sodium |
| CN110183550A (en) * | 2019-06-25 | 2019-08-30 | 广元市海天实业有限责任公司 | A kind of preparation process of refined heparin sodium |
| CN110183550B (en) * | 2019-06-25 | 2021-06-01 | 广元市海天实业有限责任公司 | Preparation process of fine heparin sodium |
| CN113004436A (en) * | 2021-04-30 | 2021-06-22 | 山东万邦赛诺康生化制药股份有限公司 | Preparation method of dalteparin sodium and application of method in preparation of low-molecular-weight heparin sodium |
| CN115028757A (en) * | 2022-06-29 | 2022-09-09 | 江苏麦德森制药有限公司 | Decolorizing method of heparin sodium |
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