CN105384811A - Method for chromatographically purifying ulinastatin by adsorption column and drug composition improving solubility of ulinastatin - Google Patents
Method for chromatographically purifying ulinastatin by adsorption column and drug composition improving solubility of ulinastatin Download PDFInfo
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- CN105384811A CN105384811A CN201510820286.2A CN201510820286A CN105384811A CN 105384811 A CN105384811 A CN 105384811A CN 201510820286 A CN201510820286 A CN 201510820286A CN 105384811 A CN105384811 A CN 105384811A
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- CN
- China
- Prior art keywords
- ulinastatin
- elutriant
- cellulosede
- deae
- ultrafiltrated
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8114—Kunitz type inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses a method for chromatographically purifying ulinastatin by virtue of an adsorption column and a drug composition improving the solubility of the ulinastatin. The method is specifically as follows: taking fresh urine of healthy adult male as a raw material, and preparing the high-purity ulinastatin by virtue of a modern protein top-grade biochemical separation technology such as chitin adsorption, ammonia water elution, ammonium sulfate precipitation, adsorption column chromatography and the like. The total yield can be increased to 60 percent or more, and the total titer is greater than or equal to 4000 iu/mg.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind ofly apply adsorpting column chromatography method yield be high to prepare, the method for the ulinastatin of titer plateaus and improve the pharmaceutical composition of ulinastatin solubility.
Background technology
Ulinastatin (Ulinastatin) is separation and purification a kind of acid glycoprotein out from NAM's freshly voided urine, it is a kind of proteinase inhibitor of wide spectrum, mainly synthesize in liver, discharged with urine by renal metabolism, and its low molecular weight compositions be decomposed to form also has the effect of very strong suppression lytic enzyme.Ulinastatin is made up of 143 amino acid, relative molecular mass about 37000 ~ 43000, belong to proteinase inhibitor, the effect having restraining effect to multiple enzymes such as the serine protease such as trypsinase, Chymetin and granulocyte elastase, Unidasa, sulfydryl enzyme, plasmins, also there is stable lysosome membrane, suppress the release of lysosomal enzyme, suppress myocardial depressant factor (MDF) (MDF) generation, scavenging activated oxygen and inflammation-inhibiting medium to discharge.Ulinastatin also can improve operation stimulates the lower immune function, Proteometabolism exception and the renal function that cause to reduce, and preventing from performing the operation stimulates the damage to internal organs and cell caused and the recurrent state etc. improved when suffering a shock.
First Europe in 1909 report that human urine also exists trypsin inhibitor, it is found that subsequently, when human body be infected, generate heat, tumour, gestation, shock, perform the operation, give glucocorticosteroid etc. stimulate time, in human urine, UTI activity raises.Within 1985, first develop listing by Japan, be widely used in clinical as the medicine of acute pancreatitis, acute circulatory failure in Japan.
The present inventor started the production technique research experiment to UTI from 2008, adopt the high-end bio-chemistry separation technology of modern protein to produce, make the total recovery of UTI bring up to more than 60%, total titer is not less than 4000iu/mg, stable processing technique, quality controllable.
Summary of the invention
The invention provides a kind of method of adsorpting column chromatography purifying ulinastatin and improve the pharmaceutical composition of ulinastatin solubility, that the traditional method for extracting ulinastatin yields such as kaolin absorption, the absorption of acid resistance mixed mode are too low, the shortcoming of not high instability of tiring in order to capture, adsorpting column chromatography is adopted to carry out purifying ulinastatin, thus improve ulinastatin yield, ensure that tire stable.
For solving the problems of the technologies described above, the present invention is achieved by the following technical solutions:
A kind of method of adsorpting column chromatography purifying ulinastatin and the pharmaceutical composition of raising ulinastatin solubility, it is characterized in that: select resin absorption, special elutriant desorb carrys out purifying ulinastatin, total recovery can be brought up to more than 60%, total titer is not less than 4000iu/mg.
In invention technical scheme, also there is following technical characteristic: described adsorpting column chromatography comprises the steps:
1) ulinastatin roughing
Take the clarification urine that 1TpH is less than 6.5, continuous stirring, ammoniacal liquor wash-out is used after slowly adding the absorption completely of 16.5kg chitin, again through 3.5kg ammonium sulfate precipitation, overnight precipitation, centrifugal being placed on do vacuum-drying in the vacuum drier of water-retaining agent with Vanadium Pentoxide in FLAKES, namely obtain ulinastatin crude product after drying.
2) adsorpting column chromatography operation (for 0.25L cylinder)
2.1) take ulinastatin crude product 1kg, dissolve with elutriant A, stir 30 minutes, centrifugal 20 minutes, leave and take centrifugate;
2.2) anion-exchange column elutriant B balances, and above-mentioned centrifugate is flowed through the anion-exchange column balanced, flow rate control is at about 120 ~ 130mL/min;
2.3) with elutriant B solution wash-out, flow rate control, at 120 ~ 130mL/min, obtains elutriant;
2.4) by elutriant through ultra-filtration membrane, add phosphate buffered saline buffer ultrafiltration repeatedly, ultrafiltration, to 1/20th of its volume, obtains ultrafiltrated.
3) precipitate
Add the alcohol settling 12 hours of not low 95% in ultrafiltrated in the ratio of 1:6, centrifugal, solid is again with dehydrated alcohol dehydration twice, and centrifugal, abandoning supernatant, leaves and takes throw out.
4) freeze-drying
Coil into Freeze Drying Equipment by throw out loading, namely freeze-drying obtains ulinastatin.
Through adjusting, gained ulinastatin total recovery is up to more than 60%, and measure by method shown in Chinese Pharmacopoeia, total titer is up to more than 4000iu/mg.
Compared with prior art, advantage of the present invention and positively effect are:
Select adsorpting column chromatography purifying ulinastatin, can more effective removal multiple foreign protein wherein, and make its physico-chemical property and biological activity keep stable, thus make the indices of product all meet Chinese Pharmacopoeia standard.The more important thing is, by technique update and perfect, make ulinastatin total recovery reach more than 60%, total titer is not less than 4000iu/mg, brings huge economic benefit and social benefit.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, and limit never in any form.
Embodiment 1
1) ulinastatin roughing
Take the clarification urine that 1TpH is less than 6.5, constantly stir, slowly add 16.5kg chitin, and measure with accurate pH test paper, regulate urine pH to 6.0; With ammoniacal liquor wash-out 1h after absorption completely, then through 3.5kg ammonium sulfate precipitation, overnight precipitation, centrifugal be placed on to put do vacuum-drying in the vacuum drier of water-retaining agent with Vanadium Pentoxide in FLAKES, namely obtain ulinastatin crude product after drying.
2) adsorpting column chromatography operation (for 0.25L cylinder)
2.1) take ulinastatin crude product 1kg, dissolve with the elutriant A of 8.5L, stir 30 minutes, centrifugal 20 minutes, leave and take centrifugate;
2.2) the elutriant B of anion-exchange column 2.5L balances, and above-mentioned centrifugate is flowed through the anion-exchange column balanced, flow rate control is at about 120mL/min;
2.3) with the elutriant B solution wash-out of 33L, flow rate control, at 120mL/min, obtains elutriant;
2.4) by elutriant through ultra-filtration membrane, add phosphate buffered saline buffer ultrafiltration repeatedly, ultrafiltration, to 1/20th of its volume, obtains ultrafiltrated and is about 1.65L.
3) precipitate
Add the alcohol settling 12 hours of not low 95% in ultrafiltrated in the ratio of 1:6, centrifugal, solid is again with dehydrated alcohol dehydration twice, and centrifugal, abandoning supernatant, leaves and takes throw out.
4) freeze-drying
Coil into Freeze Drying Equipment by throw out loading, namely freeze-drying obtains ulinastatin.
Through adjusting, gained ulinastatin total recovery is 66%, and measure by method shown in Chinese Pharmacopoeia, total titer is up to 4312iu/mg.
Embodiment 2
1) ulinastatin roughing
Take the clarification urine that 1TpH is less than 6.5, constantly stir, slowly add 16.5kg chitin, and measure with accurate pH test paper, regulate urine pH to 6.0; With ammoniacal liquor wash-out 1h after absorption completely, then through 3.5kg ammonium sulfate precipitation, overnight precipitation, centrifugal being placed on do vacuum-drying in the vacuum drier of water-retaining agent with Vanadium Pentoxide in FLAKES, namely obtain ulinastatin crude product after drying.
2) adsorpting column chromatography operation (for 0.25L cylinder)
2.1) take ulinastatin crude product 1kg, dissolve with the elutriant A of 8.5L, stir 30 minutes, centrifugal 20 minutes, leave and take centrifugate;
2.2) the elutriant B of anion-exchange column 3.0L balances, and above-mentioned centrifugate is flowed through the anion-exchange column balanced, flow rate control is at about 130mL/min;
2.3) with the elutriant B solution wash-out of 33L, flow rate control, at 130mL/min, obtains elutriant;
2.4) by elutriant through ultra-filtration membrane, add phosphate buffered saline buffer ultrafiltration repeatedly, ultrafiltration, to 1/20th of its volume, obtains ultrafiltrated and is about 1.65L.
3) precipitate
Add the alcohol settling 12 hours of not low 95% in ultrafiltrated in the ratio of 1:6, centrifugal, solid is again with dehydrated alcohol dehydration twice, and centrifugal, abandoning supernatant, leaves and takes throw out.
4) freeze-drying
Put into into Freeze Drying Equipment by throw out loading dish, namely freeze-drying obtains ulinastatin.
Through adjusting, gained ulinastatin total recovery is 63%, and measure by method shown in Chinese Pharmacopoeia, total titer is up to 4123iu/mg.
Embodiment 3
Ulinastatin 1.1 hundred million unit of preparation in Example 2, takes 11g N.F,USP MANNITOL, 10g glycine, NaCl33g, Na
2hPO
412H
2o21.9375g, NaH
2pO
42H
2o9.94375g, add 1000ml water for injection and dissolve, poly (ether-sulfone) ultrafiltration membrane sterile filtration, is sub-packed in 1000 processed good cillin bottles, freeze-drying, rolls and covers.
The above is only preferred embodiment of the present invention, and be not restriction the present invention being made to other form, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the Equivalent embodiments of equivalent variations.But everyly do not depart from technical solution of the present invention content, any simple modification, equivalent variations and the remodeling done above embodiment according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.
Claims (8)
1. the method for a purifying ulinastatin, the method comprises: ulinastatin crude product is carried out purifying by adsorpting column chromatography, ultrafiltrated precipitation, throw out step of freeze drying, the total recovery of the ulinastatin after gained purifying brings up to more than 60%, and total titer is not less than 4000iu/mg.
2. the method for claim 1, is characterized in that: described ulinastatin crude product is by taking Male urine as raw material, is prepared successively by raw material through chitin absorption, ammoniacal liquor wash-out, ammonium sulfate precipitation.
3. method as claimed in claim 1 or 2, is characterized in that: described adsorpting column chromatography comprises the steps:
(1) dissolve ulinastatin crude product with elutriant A, stir, centrifugal, leave and take centrifugate, described elutriant A is the acetate buffer of 0.01-0.3mol/L;
(2) with elutriant B equilibrium adsorption post, by above-mentioned centrifugate upper prop, elutriant B is the damping fluid of 0.01-0.5mol/L sodium-acetate and 0.1-5mol/LNaCl;
(3) with elutriant B wash-out adsorption column, elutriant is collected;
(4) by elutriant through ultra-filtration membrane, add phosphate buffered saline buffer ultrafiltration repeatedly, obtain ultrafiltrated.
4. method as claimed in claim 3, it is characterized in that, the concentration of described phosphate buffered saline buffer is 0.01-0.5mol/L.
5. method as claimed in claim 3, it is characterized in that, described adsorption column is anion-exchange column.
6. method as claimed in claim 5, it is characterized in that, described anion-exchange column comprises: strong anion exchange column Q-SephadexA-25, Q-SephadexA-50, Q-SephadexC-25, Q-SephadexC-50; Weak anion exchange column DEAE-CelluloseDE-22, DEAE-CelluloseDE-23, DEAE-CelluloseDE-51, DEAE-CelluloseDE-52, DEAE-CelluloseDE-53.
7. method as claimed in claim 1 or 2, is characterized in that: described ultrafiltrated settling step comprises: add alcohol settling in ultrafiltrated.
8. a pharmaceutical composition, its ulinastatin prepared containing with good grounds claim 1-7 as activeconstituents, also containing N.F,USP MANNITOL, glycine, sodium-chlor, phosphoric acid buffer.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510820286.2A CN105384811A (en) | 2015-11-24 | 2015-11-24 | Method for chromatographically purifying ulinastatin by adsorption column and drug composition improving solubility of ulinastatin |
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| CN201510820286.2A CN105384811A (en) | 2015-11-24 | 2015-11-24 | Method for chromatographically purifying ulinastatin by adsorption column and drug composition improving solubility of ulinastatin |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101954072A (en) * | 2010-10-08 | 2011-01-26 | 广东天普生化医药股份有限公司 | Use of ulinastatin in preparation of drugs for treating rheumatoid arthritis and pharmaceutical composition thereof |
| CN103073637A (en) * | 2012-12-30 | 2013-05-01 | 青岛九龙生物医药有限公司 | Method for purifying ulinastatin by adsorption column chromatography |
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2015
- 2015-11-24 CN CN201510820286.2A patent/CN105384811A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101954072A (en) * | 2010-10-08 | 2011-01-26 | 广东天普生化医药股份有限公司 | Use of ulinastatin in preparation of drugs for treating rheumatoid arthritis and pharmaceutical composition thereof |
| CN103073637A (en) * | 2012-12-30 | 2013-05-01 | 青岛九龙生物医药有限公司 | Method for purifying ulinastatin by adsorption column chromatography |
Non-Patent Citations (1)
| Title |
|---|
| 王显悦 等: "乌司他丁对法洛四联症根治术婴幼儿的心肺保护作用", 《中国体外循环杂志》 * |
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Application publication date: 20160309 |