CN101735340A - Method for preparing heparin sodium by combining enzymolysis and salt decomposition - Google Patents
Method for preparing heparin sodium by combining enzymolysis and salt decomposition Download PDFInfo
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- CN101735340A CN101735340A CN201010028118A CN201010028118A CN101735340A CN 101735340 A CN101735340 A CN 101735340A CN 201010028118 A CN201010028118 A CN 201010028118A CN 201010028118 A CN201010028118 A CN 201010028118A CN 101735340 A CN101735340 A CN 101735340A
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Abstract
The invention relates to a method for preparing heparin sodium by combining enzymolysis and salt decomposition, comprises the following steps: 1) dissociation: according to the weight ratio of 4-5:1 between water and chatterling mucosa, adding the water into the chatterling mucosa, and adding salt to adjust the solution salinity to be 5; adding alkali to adjust the pH value to be 9, and stirring and heating to 55 DEG C; carrying out heat preservation for 30min, and then adding alkali protease 2709 into a reaction tank according to the weight ratio of 2000-3000:1 between the chatterling mucosa and the alkali protease 2709; and carrying out heat preservation for 2h, heating to 85 DEG C and filtering; and 2) absorbing, eluting, precipitating, filtering and drying filtrate obtained in the step 1), and finally obtaining the heparin sodium. By adopting the technique combining enzymolysis and salt decomposition, the method leads heparin to be thoroughly dissociated from the chatterling mucosa, thus completing full extraction. On the basis of the prior art, the method increases about one third of unit production value and controls the increased cost not more than 1% of the increased production value.
Description
Technical field
The invention belongs to biochemical industry field, specifically, relate to a kind of salt and separate method with enzymolysis combined preparation heparin sodium.
Background technology
Heparin sodium is the sodium-salt form product of heparin.Heparin extensively is stored in animal body fluid and the tissue.General in animal body heparin and protein binding form the protein-polysaccharide mixture and exist.The used on the market heparin overwhelming majority extracts from mucous membrane of small intestine of pig, ox and lung.
The structural formula of heparin be it is generally acknowledged as follows:
Mostly our finding heparin is the heparitin compound that is made of a plurality of tetroses unit, and its molecular weight of high reactivity heparin that extracts from pig intestinal mucosa and lung is generally between 3000-30000.
The purposes of heparin is very extensive, is mainly used in anticoagulant, antithrombotic, anti-cell aging, anti metastasis, antiviral, these several broad aspect of antienzyme.
Owing to be biotechnological formulation, it is efficient, multiple-effect, low attached effect enjoy people to favor.From the single medicine that begins to develop most, develop into present a plurality of medicines, the material medicine heparin sodium is 2000 yuan/hundred million activity units from just beginning to produce also, develop into 40000 yuan/hundred million present activity units.
Dissociate and handed over since skill extracts heparin sodium in the pig intestinal mucosa since heparin seminar of biology department of the eighties Sichuan University promotes salt, people generally need the intestinal mucosa of 3500 above pigs could produce 100,000,000 activity units.
The nineties people adopt enzymolysis to produce heparin from the skill of handing over generally needs 2500 pairs of mucous membrane of small intestine to produce 100,000,000 activity units.
Chinese patent 01108278.X also discloses a kind of technology of Zymolysis process of producing heparin sodium, and this explained hereafter heparin sodium generally needs 1800～2000 pig intestinal mucosas to produce 100,000,000 activity units.
Summary of the invention
Technical problem to be solved by this invention provides a kind of salt and separates method with enzymolysis combined preparation heparin sodium, this method can solve the problem that simple property salt is separated and simple property enzymolysis can not make heparin sodium thoroughly discharge from intestinal mucosa cells, adopt salt to separate and thoroughly make heparin from intestinal mucosa cells, dissociate out, thereby extract fully with the enzymolysis process integration.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of salt is separated the method with enzymolysis combined preparation heparin sodium, comprises the steps:
1) disassociation: according to water: the weight ratio of pig intestinal mucosa is to pig intestinal mucosa in to add entry at 4～5: 1, and adding salt, to make its solution salinity be 5 degree, add alkali and regulate pH value to 9, stirring is warming up to 55 ℃, be incubated after 30 minutes, according to pig intestinal mucosa: the weight ratio of Sumizyme MP 2709 is to retort in to add Sumizyme MP 2709 at 2000～3000: 1, is incubated after 2 hours, be warming up to 85 ℃, filter at once;
2) filtrate with the step 1) gained makes heparin sodium after absorption, wash-out, sedimentation and filtration, oven dry.
Particularly, described being adsorbed as by the per kilogram pig intestinal mucosa adds the absorption 6～8 hours 55 ℃ time of 98 type strongly basic anion exchange resins that 20g purchases (RMHS) Luo Meng Haas branch office in Shanghai.
The aqueous solution wash-out that described wash-out is spent with salinity 10～14 for the resin that will adsorb heparin sodium.
Described elution time is each 1 hour, altogether secondary.
Described sedimentation and filtration is the ethanol that adds weight concentration 95～100% to elutriant, and the regulator solution ethanol concn is 40～45 degree by the alcohol instrumentation, and mixing precipitates 24 hours and promptly filters, and press dry.The ethanol of weight concentration 95～100% can be industrial spirit herein.
Described oven dry is that the heparin sodium behind the sedimentation and filtration is got final product 65～75 ℃ of oven dry.
Described alkali is NaOH.
Described water: the weight ratio of pig intestinal mucosa is 4～5: 1.
Processing method of the present invention be the comparison supersalt dissociate friendship, enzymolysis from hand over, salt is separated the complexing precipitation, bacterium is dissociated complex-precipitation technology, and salt separate enzymolysis from crosslinked close technology after, select to establish therefrom that the most desirable, tool operability, salt that the output value is the highest are separated, enzymolysis is united from the skill of handing over.Reach every month and produce maximum-norm through test and produce heparin sodium, stablize and reach 1300 pairs of small intestines and produce 100,000,000 activity unit's heparin sodiums with the intestinal mucosa of 800,000 pigs.Reaching and stablize this yield, is a complicated system engineering.The present invention is through long-term production technical study and experiment, perfect whole process system, this wherein a part be invention, more parts are new discoveries of whole technology ideal parameters.Adopt above-mentioned processing parameter, can well reach and stablize this yield.
To sum up, the invention has the beneficial effects as follows:
1, on the prior art processes basis, improved about 1/3rd unit output value, and the cost that increases only for increase output value part 1% less than, this 1/3rd output value almost is that (as: 2000 pairs of intestinal mucosa produce 100,000,000 heparin sodiums to pure profit entirely, every pair of output value only is 20 yuan, and 1300 pairs of products 100,000,000, then every pair of small intestine output value is 33.3 yuan, every pair increases by 13.3 yuan of the output values, and only being every pair, consumes less than 0.2 yuan supplementary product onsumption) more, and product purity height, nonactive small molecules heparin also are recovered and have utilized (this heparitin is mainly used in antithrombotic).
2, adopt 85 degree high-temperature inactivation enzymes, solved the problem that poisons of ion exchange resin, ensured normally carrying out of the big production of industrial cycle.
Embodiment
Embodiment 1:
Concrete steps of the present invention are as follows: test is preceding earlier to used utensil, reagent, and consumptive material carries out autoclaving.Concrete steps are as follows:
The objective of the invention is to solve that simple property salt is separated and simple property enzymolysis can not make heparin sodium thoroughly discharge promptly to separate with the enzymolysis process integration with salt thoroughly to make heparin dissociate out from intestinal mucosa cells from intestinal mucosa cells, thus the problem of extracting fully.And more objectivity manufacturing parameters of having found the industrialness scale production.
The present invention realizes by following proposal:
1) disassociation: according to water: the weight ratio of pig intestinal mucosa is to pig intestinal mucosa in to add entry at 4～5: 1, and adding salt, to make its liquid salinity be 5 degree, add NaOH and regulate pH value to 9, stirring is warming up to 55 ℃, be incubated after 30 minutes, add 0.5g Sumizyme MP 2709 to retort, be incubated after 2 hours by the per kilogram pig intestinal mucosa, be warming up to 85 ℃, filter at once;
2) filtrate of step 1) gained is added the absorption 6～8 hours 55 ℃ time of 20g 98 type strongly basic anion exchange resins by the per kilogram pig intestinal mucosa;
3) with the above-mentioned aqueous solution wash-out that has adsorbed the resin of heparin sodium with salinity 10～14 degree, elution time is each 1 hour, altogether secondary;
4) add industrial spirit to elutriant and make that the solution ethanol concn is 40～45 degree (ethanol content 60%～70%) by the alcohol instrumentation, mixing precipitates 24 hours and promptly filters, and press dry;
5) heparin sodium behind the sedimentation and filtration is the heparin sodium crude of commercially available 90 above activity unit/milligrams 65～75 ℃ of oven dry.
Add per 2709 types of basic protein in parameter that salt is separated disassociation liquid, thereby salt is separated combine with enzymolysis and reached heparin and thoroughly dissociate, attached production experiment contrast record is as follows:
Production experiment contrast recorder
From last table can find out salt separate three jars its on average amount to and need 2122 pairs of 100,000,000 activity units that dissociate out.It on average amounts to three jars of enzymolysis and needs 1873 pairs and just can dissociate 100,000,000 activity unit's heparin.Salt is separated three jars of enzymolysis process integrations, and it is on average amounted to and needs 1166 pairs and just can dissociate 100,000,000 activity unit's heparin.
The contrast of last table is large-scale production experiment contrast record, and the reliable part of these data is contrast experiment's data of doing of laboratory head and shoulders above, because its systematic error of data that the laboratory is done can be greatly.
From top each one day salt separate, enzymolysis, salt is separated with the enzymolysis process integration as can be seen, salt separates that to separate separating process than any simple property much all thorough with the process integration of enzymolysis, the rate of dissociation is much higher, the finished product yield reflects that to have demonstrate,proved detected result correct.
Embodiment 2:
In 2000 liters retort, take out 400 pairs of pig intestinal mucosas and water to the space that accounts for 90%, with the salt 120 kg, add alkali and regulate pH value to 9, be warming up to 55 ℃, be incubated after 30 minutes, add Sumizyme MP (2709 type) 1000 grams, be incubated after 2 hours, continue to be warmed up to 85 ℃ again, filtration is cooled to about 55 ℃.
(2), in 55 ℃ of liquid, add basic resin 98 types 8000 grams, whip attachment 8 hours, the most resin of diafiltration excessively.
(3), add one times of amount of resin 15 the degree salt solution, the wash-out secondary each 1 hour, mixes above-mentioned eluting water.
(4), adding industrial spirit in last elutriant, to make ethanol concn be 45 degree, left standstill 24 hours, lower sediment filtered with woven dacron do, oven dry is purity 90 above activity units/milligram heparin sodium product in 65-75 ℃ hot water bath.
Claims (8)
1. a salt is separated the method with enzymolysis combined preparation heparin sodium, it is characterized in that, comprises the steps:
1) disassociation: according to water: the weight ratio of pig intestinal mucosa is to pig intestinal mucosa in to add entry at 4～5: 1, and adding salt, to make its solution salinity be 5 degree, add alkali and regulate pH value to 9, stirring is warming up to 55 ℃, be incubated after 30 minutes, according to pig intestinal mucosa: the weight ratio of Sumizyme MP 2709 is to retort in to add Sumizyme MP 2709 at 2000～3000: 1, is incubated after 2 hours, be warming up to 85 ℃, filter at once;
2) filtrate with the step 1) gained makes heparin sodium after absorption, wash-out, sedimentation and filtration, oven dry.
2. a kind of salt according to claim 1 is separated the method with enzymolysis combined preparation heparin sodium, it is characterized in that described being adsorbed as adds 20g Shanghai Luo Meng Haas (RMHS) branch office by the per kilogram pig intestinal mucosa and produce 98 type strongly basic anion exchange resins 55 ℃ of time absorption 6～8 hours.
3. a kind of salt according to claim 1 and 2 is separated the method with enzymolysis combined preparation heparin sodium, it is characterized in that the aqueous solution wash-out that described wash-out is spent with salinity 10～14 for the resin that will adsorb heparin sodium.
4. a kind of enzymolysis according to claim 3 and salt are separated and are united the method for preparing heparin sodium, it is characterized in that, described elution time is each 1 hour, altogether secondary.
5. a kind of enzymolysis according to claim 1 and 2 and salt are separated and are united the method for preparing heparin sodium, it is characterized in that, described sedimentation and filtration is the ethanol that adds weight concentration 95～100% to elutriant, the regulator solution ethanol concn is 40～45 degree by the alcohol instrumentation, mixing, precipitate 24 hours and promptly filter, press dry.
6. a kind of enzymolysis according to claim 1 and 2 and salt are separated and are united the method for preparing heparin sodium, it is characterized in that, described oven dry is that the heparin sodium behind the sedimentation and filtration is got final product 65～75 ℃ of oven dry.
7. a kind of salt according to claim 1 is separated the method with enzymolysis combined preparation heparin sodium, it is characterized in that, described alkali is NaOH.
8. a kind of salt according to claim 1 is separated the method with enzymolysis combined preparation heparin sodium, it is characterized in that described water: the weight ratio of pig intestinal mucosa is 4～5: 1.
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Cited By (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101891842A (en) * | 2010-07-16 | 2010-11-24 | 杭州龙扬生物科技有限公司 | Process for producing heparin sodium |
| CN101914170A (en) * | 2010-08-19 | 2010-12-15 | 郓城绅联生物科技有限公司 | Preparation method for producing sodium heparin by using small sheep intestines |
| CN102229681A (en) * | 2011-06-22 | 2011-11-02 | 郓城绅联生物科技有限公司 | Preparation method for producing heparin sodium by using porcine small intestines |
| CN102464804A (en) * | 2010-11-14 | 2012-05-23 | 四川天成生化科技有限公司 | Sodium heparin biological decomposition agent |
| CN102643369A (en) * | 2011-03-21 | 2012-08-22 | 如皋市坝新肠衣有限公司 | Resin separation and purification process of heparin sodium |
| CN102875697A (en) * | 2012-10-19 | 2013-01-16 | 山东绅联生物科技有限公司 | Process for efficiently extracting liquaemin by utilizing porcine small intestines |
| CN102911291A (en) * | 2012-11-22 | 2013-02-06 | 北大荒丰缘集团有限公司 | Device suitable for automatic production of extraction industry of casing and sodium heparin |
| CN102924628A (en) * | 2012-11-22 | 2013-02-13 | 北大荒丰缘集团有限公司 | Industrial automatic production process for extracting casing heparin sodium |
| CN102993335A (en) * | 2011-09-09 | 2013-03-27 | 谭科 | Heparin sodium balance extraction method |
| CN103012622A (en) * | 2012-12-13 | 2013-04-03 | 南通天龙畜产品有限公司 | Entirely-stirring tank used for heparin sodium extraction process |
| CN103554306A (en) * | 2013-11-20 | 2014-02-05 | 南通天龙畜产品有限公司 | Process for extracting heparin sodium from greasy intestines |
| CN103694373A (en) * | 2013-11-23 | 2014-04-02 | 青岛九龙生物医药有限公司 | Method for removing DS impurity in refined heparin sodium |
| CN103804520A (en) * | 2013-11-22 | 2014-05-21 | 青岛九龙生物医药有限公司 | Method for improving test index of heparin sodium competitive product 260nm absorbancy |
| CN103848929A (en) * | 2014-03-21 | 2014-06-11 | 广元市申达实业有限公司 | Process for high-efficiently extracting sodium heparin |
| CN103923230A (en) * | 2013-01-11 | 2014-07-16 | 青岛亚博生物科技有限公司 | Heparin sodium refinement method |
| CN104098717A (en) * | 2014-07-21 | 2014-10-15 | 南通恒阳生物科技有限公司 | Method for extracting heparin sodium |
| CN104341539B (en) * | 2013-08-02 | 2016-10-26 | 武汉普赛特膜技术循环利用有限公司 | A kind of enzyme process combines the method that membrane technology one step prepares refined heparin sodium |
| CN109280098A (en) * | 2018-05-18 | 2019-01-29 | 山阳县恒桓生物科技有限公司 | A kind of preparation method of heparin sodium |
| CN109467622A (en) * | 2018-12-19 | 2019-03-15 | 正大食品（襄阳）有限公司 | A method of extracting heparin sodium from intestinal mucosa |
| CN110437348A (en) * | 2018-05-02 | 2019-11-12 | 山阳县恒瑞肉制品有限公司 | A method of preparing heparin sodium |
| CN116253810A (en) * | 2023-03-29 | 2023-06-13 | 常德云港生物科技股份有限公司 | Method for extracting heparin sodium from pig small intestine mucous membrane by utilizing electric adsorption |
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| CN1342714A (en) * | 2000-09-11 | 2002-04-03 | 董立胜 | Process for extracting heparin sodium by integrated biologic method |
| CN1876687A (en) * | 2006-06-27 | 2006-12-13 | 胡世辉 | Heparin sodium production process |
| CN101368196A (en) * | 2008-08-26 | 2009-02-18 | 如皋市坝新肠衣有限公司 | Resin enzymolysis process for heparin sodium |
| CN101544999B (en) * | 2009-04-10 | 2012-09-05 | 湖北五瑞生物工程有限公司 | Method for producing and purifying high purity and low molecular weight sodium heparin |
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2010
- 2010-01-18 CN CN2010100281187A patent/CN101735340B/en not_active Expired - Fee Related
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| CN101891842A (en) * | 2010-07-16 | 2010-11-24 | 杭州龙扬生物科技有限公司 | Process for producing heparin sodium |
| CN101914170A (en) * | 2010-08-19 | 2010-12-15 | 郓城绅联生物科技有限公司 | Preparation method for producing sodium heparin by using small sheep intestines |
| CN101914170B (en) * | 2010-08-19 | 2012-07-11 | 郓城绅联生物科技有限公司 | Preparation method for producing sodium heparin by using small sheep intestines |
| CN102464804B (en) * | 2010-11-14 | 2016-05-18 | 四川天成生化科技有限公司 | Sodium heparin biological decomposition agent |
| CN102464804A (en) * | 2010-11-14 | 2012-05-23 | 四川天成生化科技有限公司 | Sodium heparin biological decomposition agent |
| CN102643369A (en) * | 2011-03-21 | 2012-08-22 | 如皋市坝新肠衣有限公司 | Resin separation and purification process of heparin sodium |
| CN102229681B (en) * | 2011-06-22 | 2013-01-09 | 山东绅联生物科技有限公司 | Preparation method for producing heparin sodium by using porcine small intestines |
| CN102229681A (en) * | 2011-06-22 | 2011-11-02 | 郓城绅联生物科技有限公司 | Preparation method for producing heparin sodium by using porcine small intestines |
| CN102993335A (en) * | 2011-09-09 | 2013-03-27 | 谭科 | Heparin sodium balance extraction method |
| CN102875697A (en) * | 2012-10-19 | 2013-01-16 | 山东绅联生物科技有限公司 | Process for efficiently extracting liquaemin by utilizing porcine small intestines |
| CN102911291A (en) * | 2012-11-22 | 2013-02-06 | 北大荒丰缘集团有限公司 | Device suitable for automatic production of extraction industry of casing and sodium heparin |
| CN102924628A (en) * | 2012-11-22 | 2013-02-13 | 北大荒丰缘集团有限公司 | Industrial automatic production process for extracting casing heparin sodium |
| CN103012622A (en) * | 2012-12-13 | 2013-04-03 | 南通天龙畜产品有限公司 | Entirely-stirring tank used for heparin sodium extraction process |
| CN103923230A (en) * | 2013-01-11 | 2014-07-16 | 青岛亚博生物科技有限公司 | Heparin sodium refinement method |
| CN104341539B (en) * | 2013-08-02 | 2016-10-26 | 武汉普赛特膜技术循环利用有限公司 | A kind of enzyme process combines the method that membrane technology one step prepares refined heparin sodium |
| CN103554306A (en) * | 2013-11-20 | 2014-02-05 | 南通天龙畜产品有限公司 | Process for extracting heparin sodium from greasy intestines |
| CN103804520A (en) * | 2013-11-22 | 2014-05-21 | 青岛九龙生物医药有限公司 | Method for improving test index of heparin sodium competitive product 260nm absorbancy |
| CN103694373A (en) * | 2013-11-23 | 2014-04-02 | 青岛九龙生物医药有限公司 | Method for removing DS impurity in refined heparin sodium |
| CN103848929A (en) * | 2014-03-21 | 2014-06-11 | 广元市申达实业有限公司 | Process for high-efficiently extracting sodium heparin |
| CN103848929B (en) * | 2014-03-21 | 2016-08-17 | 广元市申达实业有限公司 | A kind of high efficiency extraction technique of heparin sodium |
| CN104098717A (en) * | 2014-07-21 | 2014-10-15 | 南通恒阳生物科技有限公司 | Method for extracting heparin sodium |
| CN110437348A (en) * | 2018-05-02 | 2019-11-12 | 山阳县恒瑞肉制品有限公司 | A method of preparing heparin sodium |
| CN109280098A (en) * | 2018-05-18 | 2019-01-29 | 山阳县恒桓生物科技有限公司 | A kind of preparation method of heparin sodium |
| CN109467622A (en) * | 2018-12-19 | 2019-03-15 | 正大食品（襄阳）有限公司 | A method of extracting heparin sodium from intestinal mucosa |
| CN116253810A (en) * | 2023-03-29 | 2023-06-13 | 常德云港生物科技股份有限公司 | Method for extracting heparin sodium from pig small intestine mucous membrane by utilizing electric adsorption |
| CN116253810B (en) * | 2023-03-29 | 2024-06-11 | 常德云港生物科技股份有限公司 | Method for extracting heparin sodium from pig small intestine mucous membrane by utilizing electric adsorption |
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