CN105037584A - Method for separating heparitin from heparin by-product waste protein - Google Patents
Method for separating heparitin from heparin by-product waste protein Download PDFInfo
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Abstract
The invention relates to a method for separating heparitin from heparin by-product waste protein. The objective of the invention is to provide the method for separating heparitin from heparin by-product waste protein so as to realize reutilization of the waste and conservation of cost; and the method has the characteristics of simple process and low cost. According to a technical scheme in the invention, the method is characterized by comprising the following steps: 1) preparing a solution from the heparin by-product waste protein; 2) adding an adsorbent into the solution for adsorption; 3) washing and eluting the adsorbent, collecting eluate and carrying out precipitation so as to obtain a solid precipitate 1; 4) preparing a solution from the solid precipitate 1 and successively carrying out pH value adjustment, oxidation, pH value adjustment, reduction, pH value adjustment, salinity adjustment and precipitation so as to obtain a solid precipitate 2; and 5) drying the solid precipitate 2 so as to obtain heparitin.
Description
Technical field
The present invention relates to a kind of preparation method of heparitin, particularly relate to a kind of method being separated heparitin from heparin byproduct.
Background technology
Heparitin is that refer to natural, synthesis with the semisynthetic material having similar effect with heparin, heparitin can combine with hundreds of protein, affect the space conformation of protein, life-span and biological activity, heparitin can also combine with the multiple proteins in extracellular matrix, controls their distribution, transfer and information transmission.Visible heparitin is a kind of important informational molecule, to the behavior of host cell as the propagation of cell, differentiation, combination play regulating and controlling effect, and the identification participated in foreign cell and control process.Heparitin product can be used as production Sulodexide, the raw material of the medicines such as arklemin, and this type of medicine is because of the safe and effective extensive approval being subject to patient.
To produce through ion-exchange and fractionation precipitation the process of heparin from pig intestinal mucosa and create a large amount of useless albumen, this is because resin also adhesion protein in resin absorption process, in by product albumen except a large amount of albumen, all the other main components are Suleparoid (heparitin), dermatan sulfate and a small amount of remaining heparin.But due to active substance separation difficulty wherein, therefore generally these by products are abandoned or store after producing heparin, cause the wasting of resources.
Summary of the invention
The object of the invention is to overcome the deficiency in above-mentioned background technology, a kind of method being separated heparitin from heparin byproduct is provided, carries out utilization of waste material, cost-saving, and also it is simple to have technique, lower-cost feature.
The technical solution adopted in the present invention is:
From heparin byproduct gives up albumen, be separated a method for heparitin, it is characterized in that comprising the steps:
1) albumen that given up by heparin byproduct makes solution;
2) in solution, add sorbent material to adsorb;
3) sorbent material is washed and wash-out, collect elutriant and precipitate, being precipitated solid one;
4) precipitated solid one is made solution, afterwards successively through overregulating pH, oxidation, adjustment pH, reduction, adjustment pH, regulating salt concn and precipitation, be precipitated solid two;
5) precipitated solid two is dried, obtain heparitin.
As preferably, step 1) in, the heparin byproduct albumen that gives up adds water and makes solution, and the give up volume ratio of albumen and water of heparin byproduct is 1:1 ~ 5.
As preferably, step 2) in, sorbent material is nonionic adsorption resin.
As preferably, described nonionic adsorption resin is strongly basic anion polymeric adsorbent, and amount of resin is 6 ~ 10g/L, and adsorption temp is 50 ~ 65 DEG C, and adsorption time is 4 ~ 8h, and absorption pH7 ~ 9, salt concn is 0.5 ~ 1mol/L.
As preferably, step 3) in, first use the sodium chloride solution agitator treating sorbent material of lower concentration, then adopt the sodium chloride solution of high density to stir wash-out sorbent material, collect elutriant also by precipitation agent precipitation, be precipitated solid one.
As preferably, the concentration of the sodium chloride solution of described lower concentration is 0.1 ~ 0.8mol/L, and washing time is 20 ~ 40min; The concentration of the sodium chloride solution of described high density is 3 ~ 5mol/L, and elution time is 1 ~ 5h; The volume of described precipitation agent is 1 ~ 2 times of elutriant, sedimentation time 12 ~ 24h.
As preferably, described precipitation agent is dehydrated alcohol.
As preferably, step 4) in, after precipitated solid one adds water and makes solution, add sodium hydroxide successively to regulate pH, add hydrogen peroxide and carry out being oxidized, add hydrogenchloride and regulate pH, drip sodium bisulfite and carry out reduction and eliminate dioxygen radical ion, add sodium hydroxide and regulate pH, add saturated nacl aqueous solution and regulate salt concn, finally add precipitation agent precipitation, be precipitated solid two.
As preferably, step 4) in, precipitated solid one and water are after solution is made in 1:10 ~ 20 in mass ratio, sodium hydroxide is utilized to regulate pH to 9 ~ 12 successively, 1:200 adds the hydrogen peroxide that massfraction is 30% by volume, oxidation 6 ~ 8h, hydrogenchloride is utilized to regulate pH to 6 ~ 8, the sodium bisulfite dripping 40% massfraction carries out reduction and eliminates dioxygen radical ion, add sodium hydroxide and regulate pH to 5 ~ 7, adding saturated aqueous common salt regulates salt concn at 1 ~ 2mol/L, specific conductivity is at 45 ~ 50ms, finally add precipitation agent precipitation, specific conductivity controls at 8 ~ 12ms, be precipitated solid two.
The invention has the beneficial effects as follows: the present invention efficiently solves and stores or abandon financial resources and material resources needed for a large amount of heparin byproduct, and turns waste into wealth, and is again utilized by by product, has namely saved great amount of cost, created value again.The present invention is directly separated and obtains standard compliant heparitin raw material from by product, can be used as the starting raw material medicine of other drug such as Sulodexide, it is quite difficult that will be separated in traditional mode of production mode obtains the heparitin such as Suleparoid, the price of raw material is finally caused to raise, and present method acquisition heparitin raw material process is simple, with low cost, be applicable to suitability for industrialized production.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, but the present invention is not limited to following examples.
Embodiment 1:
Taking heparin by product gives up albumen 8L, with 16L water dissolution.Add the ratio of 6g resin according to often liter of albumen, add 48g resin and adsorb, adsorption temp is 59 DEG C, and pH is 7.67, and salt concn is 0.75mol/L, and adsorption time is 8h.Absorption terminates rear collection resin, and the sodium chloride solution adding 0.82mol/L washs, and washing is after 20min terminates, then adds 4.5mol/L sodium chloride solution and carry out wash-out, and wash-out 4h terminates rear collection elutriant, adds 90% ethanol of 300ml, precipitation 16h.After precipitation terminates, outwell upper strata ethanol, add 180ml water and dissolve.Regulate pH to 11.22 with 30% sodium hydroxide, add the hydrogen peroxide 2ml of 30%, stir, oxidization time is 6h, after oxidation terminates, utilizes 8% hydrogenchloride, regulate pH to 6.05, dropwise drip the sodium bisulfite of 40%, judge whether remaining dioxygen radical ion in addition with Merck test paper.Afterwards, then add sodium hydroxide, regulate pH to 5.23, add saturated nacl aqueous solution, adjustment specific conductivity is 45ms.90% ethanol adding 410ml precipitates, and precipitation terminal specific conductivity is 11ms, and precipitation 18h, dries and obtain heparitin product, weight is 7.7057g, and tire 39u/mg, total units 300523U, absorbancy 0.561, specific rotation 21.22 °, weight loss on drying is 3%, and protein content is 0.
As the raw material of heparinoid drug, its physico-chemical property need reach certain standard, if some index exceeding standard is too many, the process of purifying can increase significant loss again, increase running cost, thus summarize the reference standard of some main physical and chemical of heparitin product, following table is the physics and chemistry result that embodiment 1 obtains.
| Project | Standard | Embodiment 1 |
| Tire | ≥35u/mg | 39u/mg |
| Absorbancy (0.4%260nm) | <0.800 | 0.561 |
| Specific rotation (4%) | ≥+18° | 21.22° |
| Weight loss on drying | <5％ | 3％ |
| Protein content | 0 | 0 |
Embodiment 2:
Taking heparin by product gives up albumen 8L, with 16L water dissolution.The ratio adding 8g resin according to often liter of albumen adds 64g resin and adsorbs, and adsorption temp is 59 DEG C, and pH is 7.79, and salt concn is 0.78mol/L, and adsorption time is 8h.Absorption terminates rear collection resin, and the sodium chloride solution adding 0.82mol/L washs, and washing is after 20min terminates, then adds 4.5mol/L sodium chloride solution and carry out wash-out, and wash-out 3h terminates rear collection elutriant, adds 90% ethanol of 300ml, precipitation 16h.After precipitation terminates, outwell upper strata ethanol, add 180ml water and dissolve.Regulate pH to 11.3 with 30% sodium hydroxide, add the hydrogen peroxide 2ml of 30%, stir, oxidization time is 6h, after oxidation terminates, utilizes 8% hydrogenchloride, regulate pH to 6.83, dropwise drip the sodium bisulfite of 40%, judge whether remaining dioxygen radical ion in addition with Merck test paper.Afterwards, then add sodium hydroxide, regulate pH to 5, add saturated nacl aqueous solution, adjustment specific conductivity is 50ms.90% ethanol adding 410ml precipitates, and precipitation terminal specific conductivity is 10ms, and precipitation 18h, dries and obtain heparitin product, weight is 10.446g, and tire 37u/mg, total units 386502U, absorbancy 0.591, specific rotation 21.31 °, weight loss on drying is 4%, and protein content is 0.
Following table is the physics and chemistry result that embodiment 2 obtains.
| Project | Standard | Embodiment 1 |
| Tire | ≥35u/mg | 37u/mg |
| Absorbancy (0.4%260nm) | <0.800 | 0.591 |
| Specific rotation (4%) | ≥+18° | 21.31° |
| Weight loss on drying | <5％ | 4％ |
| Protein content | 0 | 0 |
Embodiment 3:
Taking heparin by product gives up protein 10 L, with 25L water dissolution.The ratio adding 7g resin according to often liter of albumen adds 70g resin and adsorbs, and adsorption temp is 59 DEG C, and pH is 7.96, and salt concn is 0.78mol/L, and adsorption time is 6h.Absorption terminates rear collection resin, and the sodium chloride solution adding 0.82mol/L washs, and washing is after 20min terminates, then adds 4.5mol/L sodium chloride solution and carry out wash-out, and wash-out 4h terminates rear collection elutriant, adds 90% ethanol of 410ml, precipitation 16h.After precipitation terminates, outwell upper strata ethanol, add 360ml water and dissolve.Regulate pH to 11.36 with 30% sodium hydroxide, add the hydrogen peroxide 4ml of 30%, stir, oxidization time is 9h, after oxidation terminates, utilizes 8% hydrogenchloride, regulate pH to 7, dropwise drip the sodium bisulfite of 40%, judge whether remaining dioxygen radical ion in addition with Merck test paper.Afterwards, then add sodium hydroxide, regulate pH to 5.12, add saturated nacl aqueous solution, adjustment specific conductivity is 50ms.Add 610ml90% ethanol to precipitate, precipitation terminal specific conductivity is 12ms, and precipitation 18h, dries and obtain heparitin product, weight is 7.9051g, and tire 50.6u/mg, total units 399998U, absorbancy 0.595, specific rotation 41.16 °, weight loss on drying is 4%, and protein content is 0.
Following table is the physics and chemistry result that embodiment 3 obtains.
| Project | Standard | Embodiment 1 |
| Tire | ≥35u/mg | 50.6u/mg |
| Absorbancy (0.4%260nm) | <0.800 | 0.595 |
| Specific rotation (4%) | ≥+18° | 41.16° |
| Weight loss on drying | <5％ | 4％ |
| Protein content | 0 | 0 |
Embodiment 4:
Taking heparin by product gives up albumen 8L, with 20L water dissolution.The ratio adding 6g resin according to often liter of albumen adds 48g resin and adsorbs, and adsorption temp is 59 DEG C, and pH is 7.92, and salt concn is 0.77mol/L, and adsorption time is 6h.Absorption terminates rear collection resin, and the sodium chloride solution adding 0.82mol/L washs, and washing is after 20min terminates, then adds 4.5mol/L sodium chloride solution and carry out wash-out, and wash-out 3h terminates rear collection elutriant, adds 90% ethanol of 300ml, precipitation 16h.After precipitation terminates, outwell upper strata ethanol, add 180ml water and dissolve.Regulate pH to 11.36 with 30% sodium hydroxide, add the hydrogen peroxide 3ml of 30%, stir, oxidization time is 6h, after oxidation terminates, utilizes 8% hydrogenchloride, regulate pH to 7.13, dropwise drip the sodium bisulfite of 40%, judge whether remaining dioxygen radical ion in addition with Merck test paper.Afterwards, then add sodium hydroxide, regulate pH to 5.56, add saturated nacl aqueous solution, adjustment specific conductivity is 50ms.Add 310ml90% ethanol to precipitate, precipitation terminal specific conductivity is 11ms, and precipitation 18h, dries and obtain heparitin product, weight is 4.719g, and tire 47.9u/mg, total units 226040U, absorbancy 0.525, specific rotation 31.9 °, weight loss on drying is 3%, and protein content is 0.
Following table is the physics and chemistry result that embodiment 4 obtains.
| Project | Standard | Embodiment 1 |
| Tire | ≥35u/mg | 47.9u/mg |
| Absorbancy (0.4%260nm) | <0.800 | 0.525 |
| Specific rotation (4%) | ≥+18° | 31.9° |
| Weight loss on drying | <5％ | 3％ |
| Protein content | 0 | 0 |
Embodiment 5:
Taking heparin by product gives up albumen 8L, with 8L water dissolution.The ratio adding 10g resin according to often liter of albumen adds 80g resin and adsorbs, and adsorption temp is 65 DEG C, and pH is 9.01, and salt concn is 0.5mol/L, and adsorption time is 8h.Absorption terminates rear collection resin, and the sodium chloride solution adding 0.1mol/L washs, and washing is after 40min terminates, then adds 5mol/L sodium chloride solution and carry out wash-out, and wash-out 1h terminates rear collection elutriant, adds 90% ethanol of 300ml, precipitation 24h.After precipitation terminates, outwell upper strata ethanol, add 180ml water and dissolve.Regulate pH to 12 with 30% sodium hydroxide, add the hydrogen peroxide 2ml of 30%, stir, oxidization time is 8h, after oxidation terminates, utilizes 8% hydrogenchloride, regulate pH to 7.93, dropwise drip the sodium bisulfite of 40%, judge whether remaining dioxygen radical ion in addition with Merck test paper.Afterwards, then add sodium hydroxide, regulate pH to 6.98, add saturated nacl aqueous solution, adjustment specific conductivity is 45ms.Add 320ml90% ethanol to precipitate, precipitation terminal specific conductivity is 10.1ms, and precipitation 18h, dries and obtain heparitin product, weight is 4.612g, and tire 50.1u/mg, total units 231061U, absorbancy 0.625, specific rotation 29.1 °, weight loss on drying is 3%, and protein content is 0.
Following table is the physics and chemistry result that embodiment 5 obtains.
| Project | Standard | Embodiment 1 |
| Tire | ≥35u/mg | 50.1u/mg |
| Absorbancy (0.4%260nm) | <0.800 | 0.625 |
| Specific rotation (4%) | ≥+18° | 29.1° |
| Weight loss on drying | <5％ | 3％ |
| Protein content | 0 | 0 |
Embodiment 6:
Taking heparin by product gives up albumen 8L, with 40L water dissolution.The ratio adding 8g resin according to often liter of albumen adds 64g resin and adsorbs, and adsorption temp is 50 DEG C, and pH is 7.02, and salt concn is 1mol/L, and adsorption time is 4h.Absorption terminates rear collection resin, and the sodium chloride solution adding 0.8mol/L washs, and washing is after 20min terminates, then adds 3mol/L sodium chloride solution and carry out wash-out, and wash-out 5h terminates rear collection elutriant, adds 90% ethanol of 600ml, precipitation 12h.After precipitation terminates, outwell upper strata ethanol, add 360ml water and dissolve.Regulate pH to 9 with 30% sodium hydroxide, add the hydrogen peroxide 4ml of 30%, stir, oxidization time is 6h, after oxidation terminates, utilizes 8% hydrogenchloride, regulate pH to 6.0, dropwise drip the sodium bisulfite of 40%, judge whether remaining dioxygen radical ion in addition with Merck test paper.Afterwards, then add sodium hydroxide, regulate pH to 5, add saturated nacl aqueous solution, adjustment specific conductivity is 50ms.Add 610ml90% ethanol to precipitate, precipitation terminal specific conductivity is 11ms, and precipitation 18h, dries and obtain heparitin product, weight is 5.212g, and tire 48.3u/mg, total units 251740U, absorbancy 0.431, specific rotation 30.7 °, weight loss on drying is 3%, and protein content is 0.
Following table is the physics and chemistry result that embodiment 6 obtains.
| Project | Standard | Embodiment 1 |
| Tire | ≥35u/mg | 48.3u/mg |
| Absorbancy (0.4%260nm) | <0.800 | 0.431 |
| Specific rotation (4%) | ≥+18° | 30.7° |
| Weight loss on drying | <5％ | 3％ |
| Protein content | 0 | 0 |
Finally, it should be noted that above what enumerate is only specific embodiments of the invention.Obviously, the invention is not restricted to above embodiment, a lot of distortion can also be had.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
Claims (9)
1. from heparin byproduct gives up albumen, be separated a method for heparitin, it is characterized in that comprising the steps:
1) albumen that given up by heparin byproduct makes solution;
2) in solution, add sorbent material to adsorb;
3) sorbent material is washed and wash-out, collect elutriant and precipitate, being precipitated solid one;
4) precipitated solid one is made solution, afterwards successively through overregulating pH, oxidation, adjustment pH, reduction, adjustment pH, regulating salt concn and precipitation, be precipitated solid two;
5) precipitated solid two is dried, obtain heparitin.
2. a kind of method being separated heparitin from heparin byproduct gives up albumen according to claim 1, is characterized in that: step 1) in, the heparin byproduct albumen that gives up adds water and makes solution, and the give up volume ratio of albumen and water of heparin byproduct is 1:1 ~ 5.
3. a kind of method being separated heparitin from heparin byproduct gives up albumen according to claim 1, is characterized in that: step 2) in, sorbent material is nonionic adsorption resin.
4. a kind of method being separated heparitin from heparin byproduct gives up albumen according to claim 3, it is characterized in that: described nonionic adsorption resin is strongly basic anion polymeric adsorbent, amount of resin is 6 ~ 10g/L, adsorption temp is 50 ~ 65 DEG C, adsorption time is 4 ~ 8h, absorption pH7 ~ 9, salt concn is 0.5 ~ 1mol/L.
5. a kind of method being separated heparitin from heparin byproduct gives up albumen according to claim 1, it is characterized in that: step 3) in, first use the sodium chloride solution agitator treating sorbent material of lower concentration, then the sodium chloride solution of high density is adopted to stir wash-out sorbent material, collect elutriant and by precipitation agent precipitation, be precipitated solid one.
6. a kind of method being separated heparitin from heparin byproduct gives up albumen according to claim 5, is characterized in that: the concentration of the sodium chloride solution of described lower concentration is 0.1 ~ 0.8mol/L, and washing time is 20 ~ 40min; The concentration of the sodium chloride solution of described high density is 3 ~ 5mol/L, and elution time is 1 ~ 5h; The volume of described precipitation agent is 1 ~ 2 times of elutriant, sedimentation time 12 ~ 24h.
7. a kind of method being separated heparitin from heparin byproduct gives up albumen according to claim 5 or 6, is characterized in that: described precipitation agent is dehydrated alcohol.
8. a kind of method being separated heparitin from heparin byproduct gives up albumen according to any one of claim 1 ~ 6, it is characterized in that: step 4) in, after precipitated solid one adds water and makes solution, add sodium hydroxide successively to regulate pH, add hydrogen peroxide and carry out being oxidized, add hydrogenchloride and regulate pH, drip sodium bisulfite and carry out reduction and eliminate dioxygen radical ion, add sodium hydroxide and regulate pH, add saturated nacl aqueous solution and regulate salt concn, finally add precipitation agent precipitation, be precipitated solid two.
9. a kind of method being separated heparitin from heparin byproduct gives up albumen according to claim 8, it is characterized in that: step 4) in, precipitated solid one and water are after solution is made in 1:10 ~ 20 in mass ratio, sodium hydroxide is utilized to regulate pH to 9 ~ 12 successively, 1:200 adds the hydrogen peroxide that massfraction is 30% by volume, oxidation 6 ~ 8h, hydrogenchloride is utilized to regulate pH to 6 ~ 8, the sodium bisulfite dripping 40% massfraction carries out reduction and eliminates dioxygen radical ion, add sodium hydroxide and regulate pH to 5 ~ 7, adding saturated aqueous common salt regulates salt concn at 1 ~ 2mol/L, specific conductivity is at 45 ~ 50ms, finally add precipitation agent precipitation, specific conductivity in precipitation process controls at 8 ~ 12ms, be precipitated solid two.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108530561A (en) * | 2018-07-16 | 2018-09-14 | 深圳市格利科生物科技有限公司 | A method of it is produced from heparin and extracts high-purity sulphuric acid heparan in waste |
| CN109402205A (en) * | 2018-09-04 | 2019-03-01 | 如皋市桦儒肠衣有限公司 | A kind of technique for extracting heparan or albumen from heparin byproduct |
| CN110540607A (en) * | 2019-09-24 | 2019-12-06 | 潢川县鹏升畜产品有限公司 | treatment method of heparin sodium extraction waste liquid |
| CN113817176A (en) * | 2021-10-28 | 2021-12-21 | 潢川县鹏升畜产品有限公司 | Desalination intestinal mucosa protein powder and heparinoid co-production process |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108530561A (en) * | 2018-07-16 | 2018-09-14 | 深圳市格利科生物科技有限公司 | A method of it is produced from heparin and extracts high-purity sulphuric acid heparan in waste |
| CN108530561B (en) * | 2018-07-16 | 2020-10-30 | 深圳市格利科生物科技有限公司 | Method for extracting high-purity heparan sulfate from heparin production waste |
| CN109402205A (en) * | 2018-09-04 | 2019-03-01 | 如皋市桦儒肠衣有限公司 | A kind of technique for extracting heparan or albumen from heparin byproduct |
| CN109402205B (en) * | 2018-09-04 | 2022-01-28 | 如皋市桦儒肠衣有限公司 | Process for extracting heparinoid or protein from heparin by-product |
| CN110540607A (en) * | 2019-09-24 | 2019-12-06 | 潢川县鹏升畜产品有限公司 | treatment method of heparin sodium extraction waste liquid |
| CN113817176A (en) * | 2021-10-28 | 2021-12-21 | 潢川县鹏升畜产品有限公司 | Desalination intestinal mucosa protein powder and heparinoid co-production process |
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| CN105037584B (en) | 2017-10-31 |
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Effective date of registration: 20230803 Address after: 324109 No. 19, Heda Road, Lianhuashan Industrial Park, Jiangshan Economic Development Zone, Jiangshan City, Quzhou City, Zhejiang Province Patentee after: Zhejiang Huishun Animal Products Processing Co.,Ltd. Address before: No. 371 Dongxing Road, Economic Development Zone, Tonglu County, Hangzhou City, Zhejiang Province, 311599 Patentee before: HANGZHOU HUISHUN BIOLOGICAL SCIENCE & TECHNOLOGY Co.,Ltd. |