CN102225973A - Production method for refined heparin sodium - Google Patents
Production method for refined heparin sodium Download PDFInfo
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- CN102225973A CN102225973A CN 201110169065 CN201110169065A CN102225973A CN 102225973 A CN102225973 A CN 102225973A CN 201110169065 CN201110169065 CN 201110169065 CN 201110169065 A CN201110169065 A CN 201110169065A CN 102225973 A CN102225973 A CN 102225973A
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Abstract
The invention belongs to the field of medicines, and particularly discloses a production method for refined heparin sodium. The production method is characterized by comprising the steps of preparation of pig pancreas proteolytic enzyme powder, preparation of a crude heparin sodium solution, removal of protein by the method of enzymatic hydrolysis, removal of protein by acid regulation, oxidation treatment, ultrafiltration, deposition and drying in vacuum. Refined heparin sodium is obtained after these steps. The beneficial effects of the invention are as follows: pig pancreas proteolytic enzyme has a high activity, and a small amount of pig pancreas proteolytic enzyme is used, enabling a low cost; the activity of heparin suffers little loss, and the product of the invention has a high recovery rate, good purity, color and quality, and high potency.
Description
(1) technical field
The invention belongs to field of medicaments, particularly a kind of production method of heparin sodium elaboration.
(2) background technology
The heparin sodium elaboration is subjected to the attention of countries in the world as natural anticoagulative substance, also is one of main outlet medicine of China.Along with going deep into of research, it is found that heparin sodium not only has anti-freezing, antithrombotic to form and the effect of regulating blood fat, also have anti-inflammatory, antianaphylaxis, function such as antiviral, anticancer.Heparin is the same with most of mucopolysaccharides, many in animal body forms with heparin sodium one protein complex exist, in the leaching process since heparin sodium dissociate and not exclusively make the protein of residual some amount in the crude heparin sodium, need further refining just can be used for clinical.The treating process of heparin sodium mainly is that crude heparin sodium is carried out removal of impurities and decolouring.At present, refining secondary oxidation method production technique that adopt potassium permanganate and hydrogen peroxide two-step oxidation method or hydrogen peroxide gradation to add of China's heparin sodium more.But all there is deficiency in above-mentioned two kinds of technologies; The shortcoming of first kind of technology is that product activity loss is big, the more difficult filtering of Manganse Dioxide of shade deviation, generation, and Manganse Dioxide has the part adsorption to heparin sodium, and product recovery rate is reduced; Second kind of technology products obtained therefrom color and luster is better, and application at present is more extensive, but is difficult to obtain the heparin sodium elaboration that height is tired, and reason may be that secondary oxidation damages the heparin sodium structure.
(3) summary of the invention
The present invention is in order to remedy the deficiencies in the prior art, and the production method of a kind of product recovery rate height, heparin sodium elaboration that purity is good is provided.
The present invention is achieved through the following technical solutions:
A kind of production method of heparin sodium elaboration is characterized in that: comprise the steps:
(1) preparation of pig Trypsin lytic enzyme powder;
(2) preparation of crude heparin sodium solution;
(3) enzymolysis process removes albumen;
(4) acid adjustment removes albumen;
(5) oxide treatment;
(6) ultrafiltration;
(7) precipitation, vacuum-drying promptly get the heparin sodium elaboration.
The production method of this heparin sodium elaboration is characterized in that: comprise the steps:
(1) preparation of pig Trypsin lytic enzyme powder:
(1-1) preparation of aqua calcis: unslaked lime and the water mixed by 1:1-6 is stirred evenly, and it is stand-by that precipitation is got supernatant liquor;
(1-2) after being removed reticular tissue, grease, impurity, the fresh pig pancreas rubs into pasty state;
(1-3) Pancreas Sus domestica of sedimentary aqua calcis and rubbing is stuck with paste, press the mixed of 1:1-4 after, the 0.1-0.3% adding fungistat by the mixture total mass stirs again;
Be that the sodium hydroxide solution of 30-40% is transferred pH value 8-10 (1-4) with massfraction, through activation in 1-6 hour, concentrate, dry, pulverize and promptly get operable pig Trypsin lytic enzyme powder;
(2) preparation of crude heparin sodium solution:
(2-1) the preparation massfraction is 3% NaCl solution for later use;
(2-2) take by weighing heparin sodium crude and the NaCl solution for preparing, press the mixed dissolving of 1:1-9, be mixed with crude heparin sodium solution;
Be that the sodium hydroxide solution adjust pH to 8.0 of 30-40% left standstill 2 hours (2-3), remove by filter insoluble impurities, collect filtered liquid and move in the retort stand-by with massfraction;
(3) enzymolysis process removes albumen:
Be the sodium hydroxide solution adjust pH 7-9 of 30-40% (3-1) with the filtered liquid massfraction of collecting, heat temperature raising is to 35-45 ℃, and add the pig Trypsin lytic enzyme powder catalytic pyrolysis that step (1-4) prepares in the ratio of the 0.02-0.05% of solution total mass, heat temperature raising stops heating during to 45-65 ℃, is incubated 4-6 hour;
(3-2) will be incubated the enzymolysis solution that finishes and filter, and collect filtered liquid, and cooling is stand-by rapidly in the immigration plastic-steel bucket;
(4) acid adjustment removes albumen:
The Hcl solution that is 30-40% with cooled enzymolysis solution massfraction (4-1) transfers pH value to 1.0-3.0, and the sodium bisulfite of the 0.1-0.3% of adding solution total mass is made protective material in enzymolysis solution, and it is stand-by to stir;
(4-2) with the solution after the acid adjustment fast under cold condition, centrifugation removes Deproteinization, it is stand-by to obtain not having the albumen clear liquid;
(5) oxide treatment: the no albumen clear liquid that will collect moves in the oxidation tank, and the adding massfraction is 30% H
2O
2Solution, consumption are the 2-3% of solution total mass, are the sodium hydroxide solution adjust pH 7-9 of 30-40% with massfraction, and oxidization time is 9-13h, and temperature is 25-35 ℃, filter immediately after oxidation finishes, and it are stand-by to collect filtered liquid;
(6) ultrafiltration: step (5) filtered liquid is carried out ultrafiltration through the ultra-filtration membrane of 6000-10000 molecular weight, and it is stand-by to collect ultrafiltrated;
(7) precipitation, vacuum-drying: with the ultrafiltrated of collecting precipitate, vacuum-drying, promptly get the heparin sodium elaboration.
Fungistat is phenol or potassium permanganate in the step (1-3).
Cooling temperature is 4-8 ℃ in the step (3-2).
The low-temperature centrifugation separation is to carry out centrifugation at 4-8 ℃ in the step (4-2).
In enzymolysis solution, add sodium bisulfite as protective material, for preventing the heparin inactivation.
The present invention utilizes the characteristics of the single-minded protolysate of pig Trypsin lytic enzyme, and removes proteic method in conjunction with acid adjustment, this method can be when removing deproteinize little effect heparin total titer.In heparin sodium crude solution, add a spot of pig Trypsin lytic enzyme, make a small amount of protein degradation that remains in the heparin become small-molecular peptides, from heparin-protein complex, dissociate out, remove albumen and low-temperature centrifugation technology in conjunction with acid adjustment alkali and oxidation style again, the color and luster that makes the heparin sodium elaboration is good, and the good and rate of recovery of tiring and tire of quality all has in various degree and improves.
Therefore, the invention has the beneficial effects as follows: pig Trypsin hydrolytic enzyme activities height, consumption is few, cost is low; Heparin activity loss is few, and the product recovery rate height, elaboration purity is good, color and luster is good, quality is good, the height of tiring.
(4) embodiment
Embodiment 1:
The production method of heparin sodium elaboration, adopt following steps:
(1) preparation of pig Trypsin lytic enzyme powder:
(1-1) preparation of aqua calcis: 0.5 kilogram of unslaked lime mixed stirring evenly for 2.5 kilograms with water, it is stand-by that precipitation is got supernatant liquor;
(1-2) after being removed reticular tissue, grease, impurity, the fresh pig pancreas rubs into pasty state;
(1-3) 3 kilograms of sedimentary aqua calcises and the Pancreas Sus domestica that rubs are stuck with paste 6 kilograms mix after, add fungistat again and stir for 0.018 kilogram;
Be that 35% sodium hydroxide solution is transferred pH value 9 (1-4) with massfraction, through activation in 3 hours, concentrate, dry, pulverize and promptly get operable pig Trypsin lytic enzyme powder;
(2) preparation of crude heparin sodium solution:
(2-1) the preparation massfraction is 3% NaCl solution for later use;
(2-2) take by weighing heparin sodium crude double centner and the 500 kilograms of mixed dissolutions of NaCl solution that prepare, be mixed with crude heparin sodium solution;
Be that 35% sodium hydroxide solution adjust pH to 8.0 left standstill 2 hours (2-3), remove by filter insoluble impurities, collect filtered liquid and move in the retort stand-by with massfraction;
(3) enzymolysis process removes albumen:
(3-1) with the filtered liquid massfraction of collecting be 35% sodium hydroxide solution adjust pH 8, heat temperature raising to 40 ℃, and add 0.24 kilogram of catalytic pyrolysis of pig Trypsin lytic enzyme powder that step (1-4) prepares, and stop heating during heat temperature raising to 55 ℃, be incubated 5 hours;
(3-2) will be incubated the enzymolysis solution that finishes and filter, and collect filtered liquid, and cooling is stand-by rapidly in the immigration plastic-steel bucket;
(4) acid adjustment removes albumen:
Be that 35% Hcl solution is transferred pH value to 2.0 with cooled enzymolysis solution massfraction (4-1), and in enzymolysis solution, add sodium bisulfite and make protective material for 1.2 kilograms that it is stand-by to stir;
(4-2) with the solution after the acid adjustment fast under cold condition, centrifugation removes Deproteinization, it is stand-by to obtain not having the albumen clear liquid;
(5) oxide treatment: the no albumen clear liquid that will collect moves in the oxidation tank, and the adding massfraction is 30% H
2O
210 kilograms of solution are 35% sodium hydroxide solution adjust pH 8 with massfraction, and oxidization time is 11h, and temperature is 30 ℃, filter immediately after oxidation finishes, and it are stand-by to collect filtered liquid;
(6) ultrafiltration: step (5) filtered liquid is carried out ultrafiltration through the ultra-filtration membrane of 8000 molecular weight, and it is stand-by to collect ultrafiltrated;
(7) precipitation, vacuum-drying: with the ultrafiltrated of collecting precipitate, vacuum-drying, promptly get the heparin sodium elaboration.
Embodiment 2:
The production method of heparin sodium elaboration, adopt following steps:
(1) preparation of pig Trypsin lytic enzyme powder:
(1-1) preparation of aqua calcis: 0.5 kilogram of unslaked lime mixed stirring evenly for 3 kilograms with water, it is stand-by that precipitation is got supernatant liquor;
(1-2) after being removed reticular tissue, grease, impurity, the fresh pig pancreas rubs into pasty state;
(1-3) 2 kilograms of sedimentary aqua calcises and the Pancreas Sus domestica that rubs are stuck with paste 6 kilograms mix after, add fungistat again and stir for 0.018 kilogram;
Be that 30% sodium hydroxide solution is transferred pH value 8 (1-4) with massfraction, through activation in 1 hour, concentrate, dry, pulverize and promptly get operable pig Trypsin lytic enzyme powder;
(2) preparation of crude heparin sodium solution:
(2-1) the preparation massfraction is 3% NaCl solution for later use;
(2-2) take by weighing heparin sodium crude double centner and the 550 kilograms of mixed dissolutions of NaCl solution that prepare, be mixed with crude heparin sodium solution;
Be that 30% sodium hydroxide solution adjust pH to 8.0 left standstill 2 hours (2-3), remove by filter insoluble impurities, collect filtered liquid and move in the retort stand-by with massfraction;
(3) enzymolysis process removes albumen:
(3-1) with the filtered liquid massfraction of collecting be 30% sodium hydroxide solution adjust pH 7, heat temperature raising to 35 ℃, and add 0.26 kilogram of catalytic pyrolysis of pig Trypsin lytic enzyme powder that step (1-4) prepares, and stop heating during heat temperature raising to 45 ℃, be incubated 4 hours;
(3-2) will be incubated the enzymolysis solution that finishes and filter, and collect filtered liquid, and cooling is stand-by rapidly in the immigration plastic-steel bucket;
(4) acid adjustment removes albumen:
Be that 30% Hcl solution is transferred pH value to 3.0 with cooled enzymolysis solution massfraction (4-1), and in enzymolysis solution, add sodium bisulfite and make protective material for 1.3 kilograms that it is stand-by to stir;
(4-2) with the solution after the acid adjustment fast under cold condition, centrifugation removes Deproteinization, it is stand-by to obtain not having the albumen clear liquid;
(5) oxide treatment: the no albumen clear liquid that will collect moves in the oxidation tank, and the adding massfraction is 30% H
2O
211 kilograms of solution are 30% sodium hydroxide solution adjust pH 7 with massfraction, and oxidization time is 9h, and temperature is 25 ℃, filter immediately after oxidation finishes, and it are stand-by to collect filtered liquid;
(6) ultrafiltration: step (5) filtered liquid is carried out ultrafiltration through the ultra-filtration membrane of 6000 molecular weight, and it is stand-by to collect ultrafiltrated;
(7) precipitation, vacuum-drying: with the ultrafiltrated of collecting precipitate, vacuum-drying, promptly get the heparin sodium elaboration.
Embodiment 3:
The production method of heparin sodium elaboration, adopt following steps:
(1) preparation of pig Trypsin lytic enzyme powder:
(1-1) preparation of aqua calcis: 0.5 kilogram of unslaked lime mixed stirring evenly for 2 kilograms with water, it is stand-by that precipitation is got supernatant liquor;
(1-2) after being removed reticular tissue, grease, impurity, the fresh pig pancreas rubs into pasty state;
(1-3) 2 kilograms of sedimentary aqua calcises and the Pancreas Sus domestica that rubs are stuck with paste 8 kilograms mix after, add fungistat again and stir for 0.02 kilogram;
Be that 40% sodium hydroxide solution is transferred pH value 10 (1-4) with massfraction, through activation in 6 hours, concentrate, dry, pulverize and promptly get operable pig Trypsin lytic enzyme powder;
(2) preparation of crude heparin sodium solution:
(2-1) the preparation massfraction is 3% NaCl solution for later use;
(2-2) take by weighing 150 kilograms of heparin sodium crudes and 600 kilograms of mixed dissolutions of the NaCl solution for preparing, be mixed with crude heparin sodium solution;
Be that 40% sodium hydroxide solution adjust pH to 8.0 left standstill 2 hours (2-3), remove by filter insoluble impurities, collect filtered liquid and move in the retort stand-by with massfraction;
(3) enzymolysis process removes albumen:
(3-1) with the filtered liquid massfraction of collecting be 40% sodium hydroxide solution adjust pH 9, heat temperature raising to 45 ℃, and add 0.3 kilogram of catalytic pyrolysis of pig Trypsin lytic enzyme powder that step (1-4) prepares, and stop heating during heat temperature raising to 65 ℃, be incubated 6 hours;
(3-2) will be incubated the enzymolysis solution that finishes and filter, and collect filtered liquid, and cooling is stand-by rapidly in the immigration plastic-steel bucket;
(4) acid adjustment removes albumen:
Be that 40% Hcl solution is transferred pH value to 1.0 with cooled enzymolysis solution massfraction (4-1), and in enzymolysis solution, add sodium bisulfite and make protective material for 1.5 kilograms that it is stand-by to stir;
(4-2) with the solution after the acid adjustment fast under cold condition, centrifugation removes Deproteinization, it is stand-by to obtain not having the albumen clear liquid;
(5) oxide treatment: the no albumen clear liquid that will collect moves in the oxidation tank, and the adding massfraction is 30% H
2O
213 kilograms of solution are 40% sodium hydroxide solution adjust pH 9 with massfraction, and oxidization time is 13h, and temperature is 35 ℃, filter immediately after oxidation finishes, and it are stand-by to collect filtered liquid;
(6) ultrafiltration: step (5) filtered liquid is carried out ultrafiltration through the ultra-filtration membrane of 10000 molecular weight, and it is stand-by to collect ultrafiltrated;
(7) precipitation, vacuum-drying: with the ultrafiltrated of collecting precipitate, vacuum-drying, promptly get the heparin sodium elaboration.
Claims (5)
1. the production method of a heparin sodium elaboration is characterized in that: comprise the steps:
(1) preparation of pig Trypsin lytic enzyme powder;
(2) preparation of crude heparin sodium solution;
(3) enzymolysis process removes albumen;
(4) acid adjustment removes albumen;
(5) oxide treatment;
(6) ultrafiltration;
(7) precipitation, vacuum-drying promptly get the heparin sodium elaboration.
2. the production method of heparin sodium elaboration according to claim 1 is characterized in that: comprise the steps:
(1) preparation of pig Trypsin lytic enzyme powder:
(1-1) preparation of aqua calcis: unslaked lime and the water mixed by 1:1-6 is stirred evenly, and it is stand-by that precipitation is got supernatant liquor;
(1-2) after being removed reticular tissue, grease, impurity, the fresh pig pancreas rubs into pasty state;
(1-3) Pancreas Sus domestica of sedimentary aqua calcis and rubbing is stuck with paste, press the mixed of 1:1-4 after, the 0.1-0.3% adding fungistat by the mixture total mass stirs again;
Be that the sodium hydroxide solution of 30-40% is transferred pH value 8-10 (1-4) with massfraction, through activation in 1-6 hour, concentrate, dry, pulverize and promptly get operable pig Trypsin lytic enzyme powder;
(2) preparation of crude heparin sodium solution:
(2-1) the preparation massfraction is 3% NaCl solution for later use;
(2-2) take by weighing heparin sodium crude and the NaCl solution for preparing, press the mixed dissolving of 1:1-9, be mixed with crude heparin sodium solution;
Be that the sodium hydroxide solution adjust pH to 8.0 of 30-40% left standstill 2 hours (2-3), remove by filter insoluble impurities, collect filtered liquid and move in the retort stand-by with massfraction;
(3) enzymolysis process removes albumen:
Be the sodium hydroxide solution adjust pH 7-9 of 30-40% (3-1) with the filtered liquid massfraction of collecting, heat temperature raising is to 35-45 ℃, and add the pig Trypsin lytic enzyme powder catalytic pyrolysis that step (1-4) prepares in the ratio of the 0.02-0.05% of solution total mass, heat temperature raising stops heating during to 45-65 ℃, is incubated 4-6 hour;
(3-2) will be incubated the enzymolysis solution that finishes and filter, and collect filtered liquid, and cooling is stand-by rapidly in the immigration plastic-steel bucket;
(4) acid adjustment removes albumen:
The Hcl solution that is 30-40% with cooled enzymolysis solution massfraction (4-1) transfers pH value to 1.0-3.0, and the sodium bisulfite of the 0.1-0.3% of adding solution total mass is made protective material in enzymolysis solution, and it is stand-by to stir;
(4-2) with the solution after the acid adjustment fast under cold condition, centrifugation removes Deproteinization, it is stand-by to obtain not having the albumen clear liquid;
(5) oxide treatment: the no albumen clear liquid that will collect moves in the oxidation tank, and the adding massfraction is 30% H
2O
2Solution, consumption are the 2-3% of solution total mass, are the sodium hydroxide solution adjust pH 7-9 of 30-40% with massfraction, and oxidization time is 9-13h, and temperature is 25-35 ℃, filter immediately after oxidation finishes, and it are stand-by to collect filtered liquid;
(6) ultrafiltration: step (5) filtered liquid is carried out ultrafiltration through the ultra-filtration membrane of 6000-10000 molecular weight, and it is stand-by to collect ultrafiltrated;
(7) precipitation, vacuum-drying: with the ultrafiltrated of collecting precipitate, vacuum-drying, promptly get the heparin sodium elaboration.
3. the production method of heparin sodium elaboration according to claim 2 is characterized in that: fungistat is phenol or potassium permanganate in the step (1-3).
4. the production method of heparin sodium elaboration according to claim 2 is characterized in that: cooling temperature is 4-8 ℃ in the step (3-2).
5. the production method of heparin sodium elaboration according to claim 2 is characterized in that: the low-temperature centrifugation separation is to carry out centrifugation at 4-8 ℃ in the step (4-2).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110169065 CN102225973A (en) | 2011-06-22 | 2011-06-22 | Production method for refined heparin sodium |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110169065 CN102225973A (en) | 2011-06-22 | 2011-06-22 | Production method for refined heparin sodium |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103183746A (en) * | 2012-09-19 | 2013-07-03 | 杭州龙扬生物科技有限公司 | Process for extracting heparin sodium comprehensively by small intestines of pigs |
| 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 |
| CN108250324A (en) * | 2018-03-08 | 2018-07-06 | 广元市海鹏生物科技有限公司 | A kind of process for refining of heparin sodium |
| 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 |
| CN109485750A (en) * | 2018-12-12 | 2019-03-19 | 大英县添峰生物制品有限公司 | A kind of heparin sodium low-temperature vacuum drying technology |
| 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 |
| CN113735995A (en) * | 2020-05-29 | 2021-12-03 | 江苏唯高生物科技有限公司 | Novel process for preparing heparin sodium crude product |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2011
- 2011-06-22 CN CN 201110169065 patent/CN102225973A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103183746A (en) * | 2012-09-19 | 2013-07-03 | 杭州龙扬生物科技有限公司 | Process for extracting heparin sodium comprehensively by small intestines of pigs |
| 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 |
| CN108250324A (en) * | 2018-03-08 | 2018-07-06 | 广元市海鹏生物科技有限公司 | A kind of process for refining of heparin sodium |
| 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 |
| CN109485750A (en) * | 2018-12-12 | 2019-03-19 | 大英县添峰生物制品有限公司 | A kind of heparin sodium low-temperature vacuum drying technology |
| 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 |
| CN113735995A (en) * | 2020-05-29 | 2021-12-03 | 江苏唯高生物科技有限公司 | Novel process for preparing heparin sodium crude product |
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Application publication date: 20111026 |