CN105399819A - Method for purifying ulinastatin through affinity chromatography and pharmaceutical composition for improving stability of ulinastatin - Google Patents
Method for purifying ulinastatin through affinity chromatography and pharmaceutical composition for improving stability of ulinastatin Download PDFInfo
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- CN105399819A CN105399819A CN201510806745.1A CN201510806745A CN105399819A CN 105399819 A CN105399819 A CN 105399819A CN 201510806745 A CN201510806745 A CN 201510806745A CN 105399819 A CN105399819 A CN 105399819A
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/55—Protease inhibitors
- A61K38/57—Protease inhibitors from animals; from humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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Abstract
The invention discloses a method for purifying ulinastatin through affinity chromatography and a pharmaceutical composition for improving stability of the ulinastatin. To be specific, with fresh urine of a healthy adult male as a raw material, the high-purity ulinastatin is prepared through chitin adsorption, ammonia water eluting, ammonium sulfate salting-out, adsorption column chromatography, affinity chromatography and other modern high-end biochemical separation technologies of protein, the total yield can be increased to 70% or above and the total titer is not lower than 5000 iu/mg.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of affinity chromatography of applying and prepare that yield is high, the method for the ulinastatin of titer plateaus.
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 70%, total titer is not less than 5000iu/mg, stable processing technique, quality controllable.
Summary of the invention
The invention provides a kind of method of affinitive layer purification ulinastatin and improve the pharmaceutical composition of ulinastatin stability, 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, affinity chromatography is adopted to carry out purifying ulinastatin, thus improve the yield of ulinastatin, 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 affinitive layer purification ulinastatin and the pharmaceutical composition of raising ulinastatin stability, it is characterized in that: select the affinity chromatography of special resin to carry out purifying ulinastatin, total recovery can be made to bring up to more than 70%, and total titer is not less than 5000iu/mg.
In this technical scheme, also there is following technical characteristic: described affinity 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 (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, ultrafiltration, to 1/20th of its volume, obtains ultrafiltrated and is about 1.65L.
3) affinity chromatography (for 0.25L cylinder)
3.1) balance affinity column with elutriant C, ultrafiltrated is flowed through equilibrated resin column, and flow rate control is at 120 ~ 130mL/min;
3.2) after end of the sample, with elutriant C wash-out, flow velocity about 120 ~ 130mL/min, collects elutriant;
3.3) the affine resin in post is after elutriant C wash-out, and wash with elutriant D and remove the superincumbent impurity of absorption, flow rate control, at 120 ~ 130mL/min, seals stand-by;
3.4) elutriant is through ultra-filtration membrane ultrafiltration to certain volume, adds a certain amount of disodium phosphate soln in ultrafiltrated, continues ultrafiltration to same volume, and then adds equivalent disodium phosphate soln, then through ultra-filtration membrane ultrafiltration to same volume.
4) 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.
5) 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 70%, and measure by method shown in Chinese Pharmacopoeia, total titer is up to more than 5000iu/mg.
Compared with prior art, advantage of the present invention and positively effect are:
Select affinitive layer purification ulinastatin, can multiple foreign protein in more effective removal ulinastatin, 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 70%, total titer is not less than 5000iu/mg, creates 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 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 (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 A 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, ultrafiltration, to 1/20th of its volume, obtains ultrafiltrated and is about 1.65L.
3) affinity chromatography (for 0.25L cylinder)
3.1) balance affinity column with the elutriant C of 3.0L, ultrafiltrated is flowed through equilibrated resin column, and flow rate control is at 120mL/min;
3.2) after end of the sample, with the elutriant C wash-out of 10L, flow velocity about 120mL/min, collects elutriant;
3.3) the affine resin in post is after elutriant C wash-out, and wash with the elutriant D of 5.0L and remove the superincumbent impurity of absorption, flow rate control, at 120mL/min, seals stand-by;
3.4) elutriant is through ultra-filtration membrane ultrafiltration to 170mL, adds disodium phosphate soln 830mL in ultrafiltrated, continues ultrafiltration to 170mL, and then adds 830mL disodium phosphate soln, then through ultra-filtration membrane ultrafiltration to 170mL.
4) 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.
5) freeze-drying
Coil into Freeze Drying Equipment by throw out loading, namely freeze-drying obtains ulinastatin.
Through adjusting, gained ulinastatin total recovery is 73%, and measure by method shown in Chinese Pharmacopoeia, total titer is up to 5210iu/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 (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 A of anion-exchange column 3.0L balances, and above-mentioned centrifugate is flowed through the resin 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, ultrafiltration, to 1/20th of its volume, obtains ultrafiltrated and is about 1.65L.
3) affinity chromatography (for 0.25L cylinder)
3.1) balance affinity column with the elutriant C of 3.0L, ultrafiltrated is flowed through equilibrated resin column, and flow rate control is at 130mL/min;
3.2) after end of the sample, with the elutriant C wash-out of 10L, flow velocity about 130mL/min, collects elutriant;
3.3) the affine resin in post is after elutriant C wash-out, and wash with the elutriant D of 7.5L and remove the superincumbent impurity of absorption, flow rate control, at 130mL/min, seals stand-by;
3.4) elutriant is through ultra-filtration membrane ultrafiltration to 170mL, adds disodium phosphate soln 830mL in ultrafiltrated, continues ultrafiltration to 170mL, and then adds 830mL disodium phosphate soln, then through ultra-filtration membrane ultrafiltration to 170mL.
4) 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.
5) freeze-drying
Coil into Freeze Drying Equipment by throw out loading, namely freeze-drying obtains ulinastatin.
Through adjusting, gained ulinastatin total recovery is 71%, and measure by method shown in Chinese Pharmacopoeia, total titer is up to 5132iu/mg.
Embodiment 3
Ulinastatin 1.1 hundred million unit of preparation in Example 2, takes 10g N.F,USP MANNITOL, NaCl30g, 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 (9)
1. the method for a purifying ulinastatin, the method comprises: ulinastatin crude product is carried out purifying by ion exchange chromatography, ultrafiltrated precipitation, throw out step of freeze drying, the total recovery of the ulinastatin after gained purifying brings up to more than 70%, and total titer is not less than 5000iu/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) balance affinity column with elutriant C, by ultrafiltrated upper prop, elutriant C is the damping fluid of 0.01-0.5moL/L glycine and 0.1-5moL/LNaCl;
(5) after end of the sample, with elutriant C wash-out, elutriant is collected;
(6) the affine resin in post is after elutriant C wash-out, and wash with elutriant D and remove the superincumbent impurity of absorption, seal stand-by, elutriant D is the damping fluid of 0.01-0.3moL/L hydrochloric acid and 0.1-5moL/LNaCl;
(7) 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 7, it is characterized in that: described affinity column comprises: CM-SephadexA-25, CM-SephadexA-50, CM-SephadexC-25, CM-SephadexC-50, SP-Sepharose2B, SP-Sepharose4B, SP-Sepharose6B, SP-SepharoseCL-2B, SP-SepharoseCL-4B, SP-SepharoseCL-6B.
8. method as claimed in claim 1 or 2, is characterized in that: described ultrafiltrated settling step comprises: add alcohol settling in ultrafiltrated.
9. a pharmaceutical composition, its ulinastatin prepared containing with good grounds claim 1-8 as activeconstituents, also containing N.F,USP MANNITOL, sodium-chlor, phosphoric acid buffer.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN101439180A (en) * | 2008-12-31 | 2009-05-27 | 广东天普生化医药股份有限公司 | Medicament composition for improving stability of Ulinastatin |
CN103073638A (en) * | 2012-12-30 | 2013-05-01 | 青岛九龙生物医药有限公司 | Method for purifying ulinastatin via affinity chromatography |
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CN101439180A (en) * | 2008-12-31 | 2009-05-27 | 广东天普生化医药股份有限公司 | Medicament composition for improving stability of Ulinastatin |
CN103073638A (en) * | 2012-12-30 | 2013-05-01 | 青岛九龙生物医药有限公司 | Method for purifying ulinastatin via affinity chromatography |
Non-Patent Citations (1)
Title |
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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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