CN105330738A - Method for removing purified ulinastatin through viral inactivation and medicinal composition for improving ulinastatin stability - Google Patents
Method for removing purified ulinastatin through viral inactivation and medicinal composition for improving ulinastatin stability Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
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Abstract
The invention discloses a method for removing purified ulinastatin through viral inactivation and a medicinal composition for improving ulinastatin stability. Particularly, fresh urine of a healthy adult male is adopted as a raw material, the high-purity ulinastatin is prepared through the combination of chitin adsorption, ammonium hydroxide elution, ammonium sulfate salting out, adsorption column chromatography, affinity chromatography, low-temperature ethanol precipitation and a low-PH method and tangential flow ultra-filtration and other modern protein high-end biochemical separation techniques, the total yield can be increased to 70% or above, the total titer is not smaller than 5,000 iu/mg, the column efficiency is increased by about 50%, and the virus removal rate is not smaller than 4 log.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of chilled alcohol precipitation, in conjunction with cross-flow ultrafiltration, low PH method prepares that yield is high, the preparation method of the ulinastatin of titer plateaus, low virus and improve the pharmaceutical composition of ulinastatin stability.
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, and post effect improves about 50%.We organize related personnel to carry out scientific research in the recent period, and finally adopt chilled alcohol precipitation, low PH method carrys out purifying ulinastatin in conjunction with cross-flow ultrafiltration, virus removal ratio is not less than 4log, stable processing technique, quality controllable.
Summary of the invention
The invention provides a kind of inactivation of virus remove the method for purifying ulinastatin and improve the pharmaceutical composition of ulinastatin stability, it is the shortcoming that the traditional method for extracting ulinastatin yields such as kaolin absorption, the absorption of acid resistance mixed mode are too low in order to capture, not high instability of tiring, viral index are not clear, employing chilled alcohol precipitation, low PH method carry out purifying ulinastatin in conjunction with cross-flow ultrafiltration, thus improve the yield of ulinastatin, ensure that the stable and drug safety of tiring.
For solving the problems of the technologies described above, the present invention is achieved by the following technical solutions:
A kind of inactivation of virus is removed the method for purifying ulinastatin and is improved the pharmaceutical composition of ulinastatin stability, it is characterized in that: select and adopt chilled alcohol precipitation, low PH method to carry out purifying ulinastatin in conjunction with cross-flow ultrafiltration, total recovery can be made to bring up to more than 70%, total titer is not less than 5000iu/mg, post effect improves about 50%, and virus removal ratio is not less than 4log.
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 less than-20 DEG C, the alcohol settling of more than 95% 12 hours in the ratio of 1:6 in ultrafiltrated, centrifugal, solid is again with dehydrated alcohol dehydration twice, and centrifugal, abandoning supernatant, leaves and takes throw out.
5) inactivation of virus is removed
By above-mentioned throw out anhydrous alcohol solution, regulate pH3 ~ 6 with dilute hydrochloric acid, cultivate 15 ~ 30 days under 18 ~ 25 DEG C of conditions, cross-flow ultrafiltration, centrifugal.
6) 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%, and virus removal ratio is not less than 4log.。
Compared with prior art, advantage of the present invention and positively effect are:
Select inactivation of virus removal method purifying ulinastatin, can multiple foreign protein in more effective removal ulinastatin and virus, 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, ulinastatin total recovery is made to reach more than 70%, total titer is not less than 5000iu/mg, post effect improves about 50%, and virus removal ratio is not less than 4log, has saved cost, improve drug safety, create 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 of 1TpH6.0, 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 125mL/min;
2.7) with the elutriant B solution wash-out of 37L, 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 1.85L.
3) affinity chromatography (for 0.25L cylinder)
3.1) balance affinity column with the elutriant C of 3.5L, ultrafiltrated is flowed through equilibrated resin column, and flow rate control is at 125mL/min;
3.2) after end of the sample, with the elutriant C wash-out of 12L, flow velocity about 125mL/min, collects elutriant;
3.3) the affine resin in post is after elutriant C wash-out, and wash with the elutriant D of 8.0L and remove the superincumbent impurity of absorption, flow rate control, at 125mL/min, seals stand-by;
3.4) elutriant is through ultra-filtration membrane ultrafiltration to 190mL, adds disodium phosphate soln 850mL in ultrafiltrated, continues ultrafiltration to 190mL, and then adds 850mL disodium phosphate soln, then through ultra-filtration membrane ultrafiltration to 190mL.
4) precipitate
Add-22 DEG C, 96% alcohol settling 12 hours in the ratio of 1:6 in ultrafiltrated, centrifugal, solid is again with dehydrated alcohol dehydration twice, and centrifugal, abandoning supernatant, leaves and takes throw out.
5) inactivation of virus is removed
By above-mentioned throw out anhydrous alcohol solution, regulate pH4.0 with dilute hydrochloric acid, cultivate 21 days under 24 DEG C of conditions, cross-flow ultrafiltration, centrifugal.
6) freeze-drying
Coil into Freeze Drying Equipment by throw out loading, namely freeze-drying obtains ulinastatin.
Through adjusting, the raising 54% of post effect, gained ulinastatin total recovery is 73%, and virus removal ratio is 5log.Measure by method shown in Chinese Pharmacopoeia, total titer is up to 5285iu/mg.
Embodiment 2
1) ulinastatin roughing
Take the clarification urine of 1TpH5.5, 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 120mL/min;
2.7) with the elutriant B solution wash-out of 35L, 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 1.75L.
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 160mL, adds disodium phosphate soln 820mL in ultrafiltrated, continues ultrafiltration to 160mL, and then adds 820mL disodium phosphate soln, then through ultra-filtration membrane ultrafiltration to 160mL.
4) precipitate
Add-23 DEG C, 97% alcohol settling 12 hours in the ratio of 1:6 in ultrafiltrated, centrifugal, solid is again with dehydrated alcohol dehydration twice, and centrifugal, abandoning supernatant, leaves and takes throw out.
5) inactivation of virus is removed
By above-mentioned throw out anhydrous alcohol solution, regulate pH4.0 with dilute hydrochloric acid, cultivate 20 days under 22 DEG C of conditions, cross-flow ultrafiltration, centrifugal.
6) freeze-drying
Coil into Freeze Drying Equipment by throw out loading, namely freeze-drying obtains ulinastatin.
Through adjusting, the raising 50% of post effect, gained ulinastatin total recovery is 71%, and virus removal ratio is 4log.Measure by method shown in Chinese Pharmacopoeia, total titer is up to 5210iu/mg.
Embodiment 3
Ulinastatin 100,000,000 unit of preparation in Example 2, takes 10.0g N.F,USP MANNITOL, NaCl5g, Na
2hPO
412H
2o43.875g, NaH
2pO
42H
2o19.8875g, 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 an inactivation of virus purifying ulinastatin, the method comprises: ulinastatin crude product is carried out purifying by chromatography, chilled alcohol precipitation, low PH method in conjunction with cross-flow ultrafiltration, 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, post effect promotes about 50%, and virus removal ratio is not less than 4log.
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 chilled alcohol precipitation in ultrafiltrated, centrifugal, solid is again with dehydrated alcohol dehydration, and centrifugal, abandoning supernatant, leaves and takes throw out;
(9) throw out is after the process of low PH method, cross-flow ultrafiltration;
(10) freeze-drying, obtains ulinastatin.
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, 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.
6. 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.
7. 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.
8. method as claimed in claim 1 or 2, it is characterized in that: in ultrafiltrated, add chilled alcohol precipitation, low PH method method carries out inactivation of virus removal in conjunction with cross-flow ultrafiltration process, specifically, select less than-20 DEG C, the ethanol of more than 95%, the double-deck poly (ether sulfone) film retaining aperture 20nm selected by ultra-filtration membrane, and low PH method treatment condition select pH3 ~ 6, cultivates 15 ~ 30 days for 18 ~ 25 DEG C.
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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| CN104497135A (en) * | 2014-12-23 | 2015-04-08 | 青岛康原药业有限公司 | Method for purifying ulinastatin by virtue of virus inactivation/removal technology and pharmaceutical composition containing ulinastatin |
| CN104513308A (en) * | 2014-12-23 | 2015-04-15 | 青岛康原药业有限公司 | Method of resin regeneration for improving column efficiency and purifying ulinastatin, and drug composition containing ulinastatin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104497135A (en) * | 2014-12-23 | 2015-04-08 | 青岛康原药业有限公司 | Method for purifying ulinastatin by virtue of virus inactivation/removal technology and pharmaceutical composition containing ulinastatin |
| CN104513308A (en) * | 2014-12-23 | 2015-04-15 | 青岛康原药业有限公司 | Method of resin regeneration for improving column efficiency and purifying ulinastatin, and drug composition containing ulinastatin |
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