CN103923230A - Heparin sodium refinement method - Google Patents
Heparin sodium refinement method Download PDFInfo
- Publication number
- CN103923230A CN103923230A CN201310009732.2A CN201310009732A CN103923230A CN 103923230 A CN103923230 A CN 103923230A CN 201310009732 A CN201310009732 A CN 201310009732A CN 103923230 A CN103923230 A CN 103923230A
- Authority
- CN
- China
- Prior art keywords
- heparin sodium
- filtrate
- purification
- described step
- enzymolysis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The present invention relates to a heparin sodium refinement method, and belongs to the field of biochemical industries. The technical problem solved by the present invention is to provide a combined heparin sodium refinement method through absorption impurity removing and addition of an enzyme activator to carry out enzymolysis. With the method, the problem that the single absorption and the single enzymolysis are difficult to completely remove impurities can be solved, and the enzyme activator is used so as to substantially shorten the enzymolysis time and improve the production efficiency.
Description
Technical field
The invention belongs to biochemical industry field, specifically, relate to a kind of process for purification of heparin sodium.
Background technology
Heparin sodium is the sodium-salt form product of heparin, is the natural anticoagulative substance of a kind of acid mucopolysaccharides that contains sulfate.Can be widely used in the various cardiovascular and cerebrovascular diseases of control, in clinical application, extensively be favored.
Heparin sodium is extensively present in mammiferous intestinal mucosa, lung, liver, and particularly in pig intestinal mucosa, content is the highest.The heparin sodium that China produces at present mainly extracts taking pig intestinal mucosa as raw material, because heparin sodium exists mainly with the form of heparin sodium-protein complex in animal body, therefore make the residual a certain amount of protein of meeting in crude product, has affected the quality of product.
At present, refining main one or more production technique that adopt enzymolysis, salt solution, absorption of China's heparin sodium.Application number be 201210161795.5 and application number be 201110067346.X patent adopts the method for resin absorption to refine heparin sodium, application number is that 201110169065.5 patent adopts enzymolysis process to refine, the patent No. is 201210095896.7 employing salt solutions, the mode that enzymolysis is combined with Adsorption Phase is refined, be difficult to obtain owing to adopting single absorption or enzymolysis process to refine the high heparin sodium of tiring, and enzyme digestion reaction is because speed of response is slower, enzyme dosage is large, production cycle is long, and adopt and repeatedly dissolve reppd method when purifying, alcohol consumption is large, and product yield is low, greatly affect production efficiency.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of adsorption-edulcoration and the method for adding zymoexciter and carry out refining heparin sodium of combining of enzymolysis, the method can solve the problem that simple absorption and enzymolysis are difficult to thorough removal of impurities, and by the use of zymoexciter, greatly shorten enzymolysis time, improved production efficiency.
Of the present inventionly solve the problems of the technologies described above adopted technical scheme and be: a kind of process for purification of heparin sodium, comprises the steps:
(1) pre-treatment: crude heparin sodium is pulverized, sieved;
(2) dissolve: according to crude heparin sodium: the weight ratio of water is that dissolve 1:5 ~ 20;
(3) enzymolysis: add pancreatin and zymoexciter in the made heparin sodium aqua of step 2, enzymolysis 2 ~ 6 hours at pH6 ~ 9,45 ~ 60 DEG C;
(4) be warming up to 70 ~ 90 DEG C, cross leaching filtrate;
(5) filtrate of step 4 gained is made to heparin sodium after oxidation, absorption, filtration, ultrafiltration and concentration, precipitation, dehydration.
Optimally, in described step (1), heparin sodium crude gained after pulverizing and sieving is that diameter is less than 30 object powder.
Optimally, in described step (3), add the 2%-8% that the amount of pancreatin is solution quality.
Optimally, in described step (3), zymoexciter used is one or both of calcium chloride, calcium acetate, the 0.05%-0.15% that institute's consumption is solution quality.
Optimally, be oxidized in step (4) gained filtrate and add hydrogen peroxide in described step (5), filtrate is carried out to oxide treatment, add-on is the 0.1%-1% of filtrate quality.Filtrate is carried out to oxide treatment.
Optimally, in described step (5), be adsorbed as to oxidation after filtrate in add the Ca that accounts for filtrate massfraction 0.2%-2%
3(PO
4)
2to remove the impurity in filtrate.
Optimally, described step is filtered into employing Plate Filtration in (5), adopts the cellulose acetate filter membrane of 0.22 micron.
Optimally, ultrafiltration and concentration is that to select molecular weight cut-off be 3000 cellulose acetate filter membrane in described step (5).
Optimally, be precipitated as and will in the solution after ultrafiltration and concentration, use salt acid for adjusting pH to 5.0 ~ 6.0 in described step (5), stirring limit, limit adds ethanol, makes the ethanol number of degrees at 38 ~ 45 degree, leaves standstill 10 ~ 12 hours precipitations.
Optimally, dehydrated alcohol dehydration for dehydration in described step (5), centrifugal final vacuum is dry.
Beneficial effect of the present invention is that to utilize the method for adsorption-edulcoration and enzymolysis to combine refining heparin sodium removal of impurities more thorough, the product the obtaining height of tiring, yield is high, and by adding zymoexciter, enzymolysis speed is improved greatly, reduce the consumption of enzyme, shorten the production cycle, reduce production costs, be beneficial to suitability for industrialized production.
Embodiment
Embodiment 1
Concrete steps of the present invention are first carried out autoclaving to utensil used, reagent, consumptive material before testing.Concrete steps are as follows:
1, crude heparin sodium is pulverized, sieved with pulverizer, obtain diameter and be less than 30 object powder;
2, according to crude heparin sodium: the weight ratio of water is that 1:5 dissolves;
3, in the made heparin sodium aqua of step 2, adding massfraction is 2% pancreatin and 0.05% calcium chloride, enzymolysis 2 hours at pH6,45 DEG C;
4, be warming up to 70 DEG C, cross leaching filtrate;
5, in filtrate, adding massfraction is 0.1% hydrogen peroxide and 0.2%Ca
3(PO
4)
2select the cellulose acetate filter membrane of 0.22 micron to carry out half frame filtration, select can molecular weight cut-off 3000 cellulose acetate filter membrane carry out ultrafiltration and concentration, it is 5.0 that filtrate is adjusted to pH with hydrochloric acid, add ethanol while stirring, the number of degrees are 38 degree, precipitate 10 hours, dehydrate with dehydrated alcohol dehydration, centrifugal final vacuum is dry.
By experimentation and the each data logging of product in table 1.
Embodiment 2
Before experiment, first utensil used, reagent, consumptive material are carried out to autoclaving.Concrete steps are as follows:
1, crude heparin sodium is pulverized, sieved with pulverizer, obtain diameter and be less than 30 object powder;
2, according to crude heparin sodium: the weight ratio of water is that 1:12 dissolves;
3, in the made heparin sodium aqua of step 2, adding massfraction is 4% pancreatin and 0.1% calcium acetate, enzymolysis 4 hours at pH7.5,55 DEG C;
4, be warming up to 80 DEG C, cross leaching filtrate;
5, in filtrate, add 1% hydrogen peroxide and 2% Ca
3(PO
4)
2plate Filtration is selected the cellulose acetate filter membrane of 0.22 micron, select and can molecular weight cut-off be 3000 cellulose acetate filter membrane carries out ultrafiltration and concentration, it is 5.5 that filtrate is adjusted to pH with hydrochloric acid, add ethanol while stirring, the number of degrees are 41 degree, precipitate 11 hours, dehydrate with dehydrated alcohol dehydration, centrifugal final vacuum is dry.
By experimentation and the each data logging of product in table 1.
Embodiment 3
Before experiment, first utensil used, reagent, consumptive material are carried out to autoclaving.Concrete steps are as follows:
1, crude heparin sodium is pulverized, sieved with pulverizer, obtain diameter and be less than 30 object powder;
2, according to crude heparin sodium: the weight ratio of water is that 1:20 dissolves;
3, in the made heparin sodium aqua of step 2, adding massfraction is 8% pancreatin and 0.15% calcium chloride, enzymolysis 6 hours at pH9,60 DEG C;
4, be warming up to 90 DEG C, cross leaching filtrate;
5, in filtrate, adding massfraction is 0.5% hydrogen peroxide and 1% Ca
3(PO
4)
2, select
;the cellulose acetate filter membrane of 0.22 micron carries out Plate Filtration, select can molecular weight cut-off 3000 cellulose acetate filter membrane carry out ultrafiltration and concentration, it is 6.0 that filtrate is adjusted to pH with hydrochloric acid, add ethanol while stirring, the number of degrees are 45 degree, precipitate 12 hours, dehydrate with dehydrated alcohol dehydration, centrifugal final vacuum is dry.
By experimentation and the each data logging of product in table 1.
Comparative example 1
This comparative example only adopts absorption method to refine heparin sodium, and method is removed step 3 and 4 compared with embodiment 1, and other steps are identical.
By experimentation and the each data logging of product in table 1.
Comparative example 2
This comparative example only adopts enzyme solution and does not use zymoexciter to refine heparin sodium, and method has been removed and in step 5, added Ca compared with embodiment 1
3(PO
4)
2with in step 3, add these two processes of zymoexciter, other steps are identical.
By experimentation and the each data logging of product in table 1.
Comparative example 3
The method that this comparative example adopts is the method that absorption combines with enzymolysis, but does not add zymoexciter, and method is removed in step 3 and added this process of zymoexciter compared with embodiment 1, and other steps are identical.
By experimentation and the each data logging of product in table 1.
Comparative example 4
Before experiment, first utensil used, reagent, consumptive material are carried out to autoclaving.Concrete steps are as follows:
1, crude heparin sodium is pulverized, sieved with pulverizer, obtain diameter and be less than 30 object powder;
2, according to crude heparin sodium: the weight ratio of water is that 1:1 dissolves;
3, in the made heparin sodium aqua of step 2, add the pancreatin of pancreatin and zymoexciter 1%, 0.04% calcium chloride, enzymolysis 1 hour at pH5,40 DEG C;
4, be warming up to 60 DEG C, cross leaching filtrate;
5, in filtrate, add 0.05% hydrogen peroxide and 0.1% Ca
3(PO
4)
2select the cellulose acetate filter membrane of 0.22 micron to carry out Plate Filtration, select can molecular weight cut-off 3000 cellulose acetate filter membrane carry out ultrafiltration and concentration, it is 4.5 that filtrate is adjusted to pH with hydrochloric acid, add ethanol while stirring, the number of degrees are 35 degree, precipitate 8 hours, dehydrate with dehydrated alcohol dehydration, centrifugal final vacuum is dry.
By experimentation and the each data logging of product in table 1.
Comparative example 5
Before experiment, first utensil used, reagent, consumptive material are carried out to autoclaving.Concrete steps are as follows:
1, pulverizer for crude heparin sodium is pulverized, sieved, obtain diameter and be greater than 30 orders and be less than 50 object powder;
2, according to crude heparin sodium: the weight ratio of water is that 1:30 dissolves;
3, in the made heparin sodium aqua of step 2, add 10% pancreatin, 0.2% calcium chloride, enzymolysis 8 hours at pH10,65 DEG C;
4, be warming up to 95 DEG C, cross leaching filtrate;
5, in filtrate, add 2% hydrogen peroxide and 5% Ca
3(PO
4)
2select the cellulose acetate filter membrane of 0.22 micron to carry out Plate Filtration, select can molecular weight cut-off 3000 cellulose acetate filter membrane carry out ultrafiltration and concentration, it is 7.0 that filtrate is adjusted to pH with hydrochloric acid, add ethanol while stirring, the number of degrees are 50 degree, precipitate 15 hours, dehydrate with dehydrated alcohol dehydration, centrifugal final vacuum is dry.
By experimentation and the each data logging of product in table 1.
Table 1
| Sequence number | Embodiment | Enzyme dosage | Production cycle (or enzymolysis time) | Product is tired | Product yield |
| Embodiment 1 | Absorption+enzymolysis (containing zymoexciter) | 2% | 48 hours | 180iu/mg | 92.6% |
| Embodiment 2 | Absorption+enzymolysis (containing zymoexciter) | 4% | 46 hours | 205iu/mg | 91.2% |
| Embodiment 3 | Absorption+enzymolysis (containing zymoexciter) | 8% | 45 hours | 193iu/mg | 93.1% |
| Comparative example 1 | Absorption | 0 | 36 hours | 120iu/mg | 82.5% |
| Comparative example 2 | Enzymolysis (without zymoexciter) | 2% | 56 hours | 135iu/mg | 85.2% |
| Comparative example 3 | Absorption+enzymolysis (without zymoexciter) | 2% | 53 hours | 143iu/mg | 83.3% |
| Comparative example 4 | Absorption+enzymolysis (containing zymoexciter) | 1% | 58 hours | 140iu/mg | 78.4% |
| Comparative example 5 | Absorption+enzymolysis (containing zymoexciter) | 10% | 53 hours | 146iu/mg | 76.9% |
Claims (10)
1. a process for purification for heparin sodium, comprises the steps:
(1) pre-treatment: crude heparin sodium is pulverized, sieved;
(2) dissolve: according to crude heparin sodium: the weight ratio of water is that dissolve 1:5 ~ 20;
(3) enzymolysis: add pancreatin and zymoexciter in the made heparin sodium aqua of step 2, enzymolysis 2 ~ 6 hours at pH6 ~ 9,45 ~ 60 DEG C;
(4) be warming up to 70 ~ 90 DEG C, cross leaching filtrate;
(5) filtrate of step 4 gained is made to heparin sodium after oxidation, absorption, filtration, ultrafiltration and concentration, precipitation, dehydration.
2. the process for purification of a kind of heparin sodium according to claim 1, in described step (1), heparin sodium crude gained after pulverizing and sieving is that diameter is less than 30 object powder.
3. the process for purification of a kind of heparin sodium according to claim 1, the 2%-8% that the amount that adds pancreatin in described step (3) is solution quality.
4. the process for purification of a kind of heparin sodium according to claim 1, in described step (3), zymoexciter used is one or both of calcium chloride, calcium acetate, the 0.05%-0.15% that institute's consumption is solution quality.
5. the process for purification of a kind of heparin sodium according to claim 1, in described step (5), be oxidized in step (4) gained filtrate and add hydrogen peroxide, filtrate is carried out to oxide treatment, and add-on is the 0.1%-1% of filtrate quality, and filtrate is carried out to oxide treatment.
6. the process for purification of a kind of heparin sodium according to claim 1, in described step (5), be adsorbed as to oxidation after filtrate in add the Ca that accounts for filtrate massfraction 0.2%-2%
3(PO
4)
2to remove the impurity in filtrate.
7. the process for purification of a kind of heparin sodium according to claim 1, described step is filtered into employing Plate Filtration in (5), adopts the cellulose acetate filter membrane of 0.22 micron.
8. the process for purification of a kind of heparin sodium according to claim 1, ultrafiltration and concentration is that to select molecular weight cut-off be 3000 cellulose acetate filter membrane in described step (5).
9. the process for purification of a kind of heparin sodium according to claim 1, in described step (5), be precipitated as and will in the solution after ultrafiltration and concentration, use salt acid for adjusting pH to 5.0 ~ 6.0, stirring limit, limit adds ethanol, makes the ethanol number of degrees at 38 ~ 45 degree, leaves standstill 10 ~ 12 hours precipitations.
10. the process for purification of a kind of heparin sodium according to claim 1, dehydrated alcohol dehydration for dehydration in described step (5), centrifugal final vacuum is dry.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310009732.2A CN103923230A (en) | 2013-01-11 | 2013-01-11 | Heparin sodium refinement method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310009732.2A CN103923230A (en) | 2013-01-11 | 2013-01-11 | Heparin sodium refinement method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103923230A true CN103923230A (en) | 2014-07-16 |
Family
ID=51141642
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310009732.2A Pending CN103923230A (en) | 2013-01-11 | 2013-01-11 | Heparin sodium refinement method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103923230A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109776696A (en) * | 2019-01-15 | 2019-05-21 | 湖北亿诺瑞生物制药有限公司 | A kind of preparation process of high purity heparin sodium |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0261088A2 (en) * | 1986-09-19 | 1988-03-23 | Farmaceutici Damor S.p.A. | Process for the production of heparanlysopeptide sulphate, the product thus obtained and its therapeutic use |
| CN1844165A (en) * | 2006-03-22 | 2006-10-11 | 南京健友生物化学制药有限公司 | Process for preparing high purity sodium heparin by purification of crude sodium heparin |
| CN101597344A (en) * | 2009-05-07 | 2009-12-09 | 张丽萍 | A kind of extraction of heparin, separation, purification process |
| CN101649336A (en) * | 2008-08-25 | 2010-02-17 | 射洪县天贵畜产品加工厂 | Novel process for producing sodium heparin |
| CN101735340A (en) * | 2010-01-18 | 2010-06-16 | 叶青理 | Method for preparing heparin sodium by combining enzymolysis and salt decomposition |
| CN101824099A (en) * | 2010-02-12 | 2010-09-08 | 淮安麦德森化学有限公司 | Method for purifying crude product heparin sodium |
| CN101831008A (en) * | 2009-03-11 | 2010-09-15 | 四川茂森生物科技有限公司 | New production process for refining crude heparin sodium |
| CN102225973A (en) * | 2011-06-22 | 2011-10-26 | 郓城绅联生物科技有限公司 | Production method for refined heparin sodium |
| WO2012036152A1 (en) * | 2010-09-14 | 2012-03-22 | 国立大学法人 宮崎大学 | High purity heparin and production method therefor |
| CN102603926A (en) * | 2012-03-27 | 2012-07-25 | 烟台东诚生化股份有限公司 | New preparing process of high-titer heparin sodium |
| CN102617756A (en) * | 2012-03-23 | 2012-08-01 | 武汉博锐通科技有限公司 | Preparation method of heparin lithium |
| CN102746421A (en) * | 2012-05-23 | 2012-10-24 | 杭州龙扬生物科技有限公司 | Impurity removing technology of crude heparin sodium |
-
2013
- 2013-01-11 CN CN201310009732.2A patent/CN103923230A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0261088A2 (en) * | 1986-09-19 | 1988-03-23 | Farmaceutici Damor S.p.A. | Process for the production of heparanlysopeptide sulphate, the product thus obtained and its therapeutic use |
| CN1844165A (en) * | 2006-03-22 | 2006-10-11 | 南京健友生物化学制药有限公司 | Process for preparing high purity sodium heparin by purification of crude sodium heparin |
| CN101649336A (en) * | 2008-08-25 | 2010-02-17 | 射洪县天贵畜产品加工厂 | Novel process for producing sodium heparin |
| CN101831008A (en) * | 2009-03-11 | 2010-09-15 | 四川茂森生物科技有限公司 | New production process for refining crude heparin sodium |
| CN101597344A (en) * | 2009-05-07 | 2009-12-09 | 张丽萍 | A kind of extraction of heparin, separation, purification process |
| CN101735340A (en) * | 2010-01-18 | 2010-06-16 | 叶青理 | Method for preparing heparin sodium by combining enzymolysis and salt decomposition |
| CN101824099A (en) * | 2010-02-12 | 2010-09-08 | 淮安麦德森化学有限公司 | Method for purifying crude product heparin sodium |
| WO2012036152A1 (en) * | 2010-09-14 | 2012-03-22 | 国立大学法人 宮崎大学 | High purity heparin and production method therefor |
| CN102225973A (en) * | 2011-06-22 | 2011-10-26 | 郓城绅联生物科技有限公司 | Production method for refined heparin sodium |
| CN102617756A (en) * | 2012-03-23 | 2012-08-01 | 武汉博锐通科技有限公司 | Preparation method of heparin lithium |
| CN102603926A (en) * | 2012-03-27 | 2012-07-25 | 烟台东诚生化股份有限公司 | New preparing process of high-titer heparin sodium |
| CN102746421A (en) * | 2012-05-23 | 2012-10-24 | 杭州龙扬生物科技有限公司 | Impurity removing technology of crude heparin sodium |
Non-Patent Citations (1)
| Title |
|---|
| 李万才: "《生物分离技术（第1版）》", 31 August 2009, 中国轻工业出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109776696A (en) * | 2019-01-15 | 2019-05-21 | 湖北亿诺瑞生物制药有限公司 | A kind of preparation process of high purity heparin sodium |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101735340B (en) | Method for preparing heparin sodium by combining enzymolysis and salt decomposition | |
| CN102775523B (en) | Process for preparing high-purity low-molecular heparin sodium | |
| Boonmee et al. | Improved lactic acid production by in situ removal of lactic acid during fermentation and a proposed scheme for its recovery | |
| CN102146144B (en) | Method for extracting and refining inulin | |
| CN101544999A (en) | Method for producing and purifying high purity and low molecular weight sodium heparin | |
| CN104498564A (en) | Low molecular weight chondroitin sulfate preparation method | |
| CN110903488A (en) | Preparation method of chitosan @ metal organic framework antibacterial material | |
| CN104480228A (en) | Method for preparing glucosamine by utilizing shrimp and crab shells | |
| CN103772529A (en) | Process for preparing heparin sodium through membrane separation | |
| CN103130904A (en) | High-valued utilization method for patinopecten yessoensis offal | |
| CN103012609A (en) | Method for extracting fucosan sulphate from sea cucumber cooking waste liquor | |
| CN103848929B (en) | A kind of high efficiency extraction technique of heparin sodium | |
| CN103275151B (en) | A kind of process for purification of Matachrom | |
| CN104403026A (en) | Method for separating heparan sulfate from animal lungs | |
| CN103804522A (en) | Method for increasing purity of heparin sodium | |
| CN104341539A (en) | A one-step preparing method of high-quality heparin sodium by combination of an enzymatic method and a membrane technology | |
| CN107236054B (en) | Preparation method and application of low-molecular-weight ascophyllan | |
| CN103923230A (en) | Heparin sodium refinement method | |
| CN109438585B (en) | Purification process of type b haemophilus polysaccharide | |
| CN103804518A (en) | Preparation method for improving chondroitin sulfate content | |
| CN103804517A (en) | Preparation method for increasing chondroitin sulfate yield | |
| CN102617752A (en) | Production process of low molecular weight pectin | |
| CN112409426B (en) | Preparation method of sisomicin sulfate | |
| CN102372788A (en) | Mactra veneriformis glycosaminoglycan extraction method | |
| CN104558251B (en) | A kind of preparation method of liquaemin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140716 |
|
| RJ01 | Rejection of invention patent application after publication |