CN105399820A - Method for purifying ulinastatin by improving column efficiency through resin regeneration and pharmaceutical composition for improving stability of ulinastatin - Google Patents
Method for purifying ulinastatin by improving column efficiency through resin regeneration and pharmaceutical composition for improving stability of ulinastatin Download PDFInfo
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- CN105399820A CN105399820A CN201510820566.3A CN201510820566A CN105399820A CN 105399820 A CN105399820 A CN 105399820A CN 201510820566 A CN201510820566 A CN 201510820566A CN 105399820 A CN105399820 A CN 105399820A
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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
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a method for purifying ulinastatin by improving column efficiency through resin regeneration and a pharmaceutical composition for improving stability of the ulinastatin. Specifically, fresh urine of a healthy adult man serves as a raw material, and is subjected to modern high-end protein biochemical separation technologies such as chitin adsorption, ammonia water elution, ammonium sulfate precipitation, adsorption column chromatography and affinity chromatography to prepare the high-purity ulinastatin, the total yield can be increased to 70% or above, the total effective value is not lower than 5,000 iu/mg, and the column efficiency is improved about 50%.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of specific resins process regeneration and improve post effect 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, post effect improves about 50%, stable processing technique, quality controllable.
Summary of the invention
The invention provides a kind of resin regeneration improve the method for post effect purifying 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, adopt specific resins process regeneration to improve post and follow 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 resin regeneration improves the method for post effect purifying ulinastatin and improves the pharmaceutical composition of ulinastatin stability, it is characterized in that: select specific resins process method of reproduction to carry out purifying ulinastatin, total recovery can be made to bring up to more than 70%, total titer is not less than 5000iu/mg, and post effect improves about 50%.
In this technical scheme, its technical characteristic is also that described method 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 0.25LDEAE resin column)
2.1) the DEAE resin purified water in post recoiled, load in bucket, stir evenly with purified water submergence, leave standstill, repeated multiple times, purified water submergence is spent the night;
2.2) DEAE resin is filtered dry, washs by a small amount of purified water, be again filtered dry in rear loading bucket, with the submergence of 0.5mol/L hydrochloric acid, stir, filter, be washed till neutrality by purified water, then use 0.5mol/LNaOH solution soaking, stir, filter, then be washed till neutrality by purified water;
2.3) above-mentioned processed DEAE resin is added after elutriant A equilibrates to pH6.5 repeatedly, then soak stand-by with elutriant A;
2.4) resin good for above-mentioned balance is loaded in post, after post installs, then walk post with elutriant A;
2.5) take ulinastatin crude product 1kg, dissolve with elutriant A, stir 30 minutes, centrifugal 20 minutes, leave and take centrifugate, heat 10 hours 60 DEG C of stirring in water bath;
2.6) above-mentioned centrifugate is flowed through the DEAE resin column balanced, flow rate control is at about 120 ~ 130mL/min;
2.7) with elutriant B solution wash-out, flow rate control, at 120 ~ 130mL/min, obtains elutriant;
2.8) 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, up to more than 70%, measures by method shown in Chinese Pharmacopoeia, and total titer is up to more than 5000iu/mg, and post effect improves about 50%.
Compared with prior art, advantage of the present invention and positively effect are:
Select resin regeneration to improve post and follow purifying 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, post effect improve about 50%, saved cost, created 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, 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 0.25LDEAE resin column)
2.1) the DEAE resin purified water in post recoiled, load in bucket, stir evenly with purified water submergence, leave standstill 1 hour, incline supernatant liquor and fine particle, and three times repeatedly, purified water submergence is spent the night;
2.2) DEAE resin is filtered dry, washs by a small amount of purified water, be again filtered dry in rear loading bucket, with the submergence of 0.5mol/L hydrochloric acid, stir 1 hour, filter, be washed till neutrality by purified water, then use 0.5mol/LNaOH solution soaking, stir 1 hour, filter, then be washed till neutrality by purified water;
2.3) above-mentioned processed DEAE resin is added after elutriant A equilibrates to pH6.5 repeatedly, then soak stand-by with elutriant A;
2.4) resin good for above-mentioned balance is loaded in post, after post installs, then walk post with elutriant A;
2.5) take ulinastatin crude product 1kg, dissolve with elutriant A, stir 30 minutes, centrifugal 20 minutes, leave and take centrifugate, heat 10 hours 60 DEG C of stirring in water bath;
2.6) above-mentioned centrifugate is flowed through the DEAE resin column balanced, flow rate control is at about 120 ~ 130mL/min;
2.7) with the elutriant B solution wash-out of 33L, flow rate control, at 120mL/min, obtains elutriant;
2.8) 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, the raising 51% of post effect, gained ulinastatin total recovery is 72%, and measure by method shown in Chinese Pharmacopoeia, total titer is up to 5295iu/mg.
Embodiment 2
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 0.25LDEAE resin column)
2.1) the DEAE resin purified water in post recoiled, load in bucket, stir evenly with purified water submergence, leave standstill 1 hour, incline supernatant liquor and fine particle, and three times repeatedly, purified water submergence is spent the night;
2.2) DEAE resin is filtered dry, washs by a small amount of purified water, be again filtered dry in rear loading bucket, with the submergence of 0.5mol/L hydrochloric acid, stir 1 hour, filter, be washed till neutrality by purified water, then use 0.5mol/LNaOH solution soaking, stir 1 hour, filter, then be washed till neutrality by purified water;
2.3) above-mentioned processed DEAE resin is added after elutriant A equilibrates to pH6.5 repeatedly, then soak stand-by with elutriant A;
2.4) resin good for above-mentioned balance is loaded in post, after post installs, then walk post with elutriant A;
2.5) take ulinastatin crude product 1kg, dissolve with elutriant A, stir 30 minutes, centrifugal 20 minutes, leave and take centrifugate, heat 10 hours 60 DEG C of stirring in water bath;
2.6) above-mentioned centrifugate is flowed through the DEAE resin column balanced, flow rate control is at about 120 ~ 130mL/min;
2.7) with the elutriant B solution wash-out of 33L, flow rate control, at 130mL/min, obtains elutriant;
2.8) 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, the raising 48% of post effect, gained ulinastatin total recovery is 69%, and measure by method shown in Chinese Pharmacopoeia, total titer is up to 5102iu/mg.
Embodiment 3
Ulinastatin 1.1 hundred million unit of preparation in Example 2, takes 10g N.F,USP MANNITOL, NaCl5.5g, Na
2hPO
412H
2o43.875g, NaH
2pO
42H
2o19.8875, 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 (10)
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 70%, total titer is not less than 5000iu/mg, and post effect promotes about 50%.
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 operation 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 the adsorption column of elutriant B balance through specificity process regeneration, 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;
(8) add alcohol settling in ultrafiltrated, centrifugal, solid is again with dehydrated alcohol dehydration, and centrifugal, abandoning supernatant, leaves and takes throw out;
(9) by throw out freeze-drying, ulinastatin is obtained.
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 3, is characterized in that, the described resin regeneration method i.e. washing of a graded, pickling, alkali cleaning process, improves the efficiency of resin column.
8. method as claimed in claim 3, 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.
9. method as claimed in claim 1 or 2, is characterized in that: described ultrafiltrated settling step comprises: add alcohol settling in ultrafiltrated.
10. 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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Application publication date: 20160316 |