CN103724428A - Method for improving column efficiency purified ulinastatin through resin regeneration - Google Patents
Method for improving column efficiency purified ulinastatin through resin regeneration Download PDFInfo
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- CN103724428A CN103724428A CN201310625498.6A CN201310625498A CN103724428A CN 103724428 A CN103724428 A CN 103724428A CN 201310625498 A CN201310625498 A CN 201310625498A CN 103724428 A CN103724428 A CN 103724428A
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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 improving column efficiency purified ulinastatin through resin regeneration. Particularly, the method comprises the following steps: using fresh urine of healthy adult men as a raw material, and enabling the raw material to be subjected to modern protein high-end biochemical separation technology such as chitin adsorption, ammonia elution, ammonium sulfate salting-out, adsorption column chromatography and affinity chromatography to prepare the high-purity ulinastatin. The total yield is improved by more than 70%, the total titer is not less than 5000 iu/mg, and the column efficiency is improved by about 50%.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a species specificity plastic resin treatment regeneration and improve post effect and prepare that yield is high, the method for the stable ulinastatin of tiring.
Background technology
Separation and purification a kind of acid glycoprotein out in ulinastatin (ULinastatin) Xi Cong NAM freshly voided urine, it is a kind of proteinase inhibitor of wide spectrum, mainly synthetic in liver, by renal metabolism, with urine, discharged, and its low molecular weight compositions being decomposed to form also has the effect of very strong inhibition lytic enzyme.Ulinastatin is comprised of 143 amino acid, the about 37000-43000 of relative molecular mass, belong to proteinase inhibitor, the plurality of enzymes such as the serine proteases such as trypsinase, Chymetin and granulocyte elastase, Unidasa, sulfydryl enzyme, plasmin are had to restraining effect, also there is stable lysosome membrane, suppress lysosomal enzyme release, suppress myocardial depressant factor (MDF) (MDF) and produce, remove the effect that oxyradical and inflammation-inhibiting medium discharge.Ulinastatin also can improve operation stimulates the abnormal and renal function of lower immune function, the Proteometabolism cause to reduce, prevent operation stimulate cause to the damage of internal organs and cell and improve recurrent state while suffering a shock etc.
First Europe in 1909 report that human urine exists trypsin inhibitor, it is found that subsequently, when human body is infected, heating, tumour, gestation, shock, when performing the operation, giving glucocorticosteroid etc. and stimulating, in human urine, UTI is active raises.Within 1985, by Japan, first develop listing, in Japan, as the medicine of acute pancreatitis, acute circulatory failure, be widely used in clinical.
The inventor started the production technique research experiment to UTI from 2008, adopt the high-end biochemical isolation technique of modern protein to produce, and the total recovery of UTI is brought up to more than 70%, total titer is not less than 5000iu/mg, post effect improves approximately 50%, and stable processing technique is quality controllable.
Summary of the invention
The invention provides a kind of method that resin regeneration improves post effect purifying ulinastatin, that the traditional method for extracting ulinastatin yields such as kaolin absorption, the absorption of acid resistance mixed mode are too low, the not high unsettled shortcoming of tiring in order to capture, adopt the regeneration of specificity plastic resin treatment to improve post and follow purifying ulinastatin, thereby improved the yield of ulinastatin, what guaranteed to tire is stable.
For solving the problems of the technologies described above, the present invention is achieved by the following technical solutions:
Resin regeneration improves a method for post effect purifying ulinastatin, it is characterized in that: select specificity plastic resin treatment method of reproduction to carry out purifying ulinastatin, can make total recovery bring up to more than 70%, total titer is not less than 5000iu/mg, and post effect improves approximately 50%.
In this technical scheme, its technical characterictic is also that described method comprises the steps:
1) ulinastatin roughing
Take the clarification urine that 1T pH is less than 6.5, constantly stir, after slowly adding the absorption completely of 16.5kg chitin, use ammoniacal liquor wash-out, pass through again 3.5kg ammonium sulfate precipitation, the precipitation, centrifugal being placed on Vanadium Pentoxide in FLAKES of spending the night done vacuum-drying in the vacuum drier of water-retaining agent, obtains ulinastatin crude product after dry.
2) adsorpting column chromatography (the 0.25L DEAE resin column of take is example)
2.1) the DEAE resin in post is recoiled by purified water, pack in bucket, with purified water submergence, stir evenly, standing, repeated multiple times, purified water submergence is spent the night.
2.2) DEAE resin is filtered dry, with a small amount of purified water washing, after being again filtered dry, packs in bucket, with the submergence of 0.5mol/L hydrochloric acid, stir, filter, by purified water, be washed till neutrality, then use 0.5mol/L NaOH solution soaking, stir, filter, then be washed till neutrality by purified water.
2.3) the DEAE resin of above-mentioned treated mistake is added to elutriant A and repeatedly equilibrate to after pH6.5, then soak stand-by with elutriant A.
2.4) the good resin of above-mentioned balance is packed in post, after post installs, then walk post with elutriant A.
2.5) take ulinastatin crude product 1kg, with elutriant A, dissolve, stir 30 minutes, centrifugal 20 minutes, leave and take centrifugate, 60 ℃ of stirring in water bath heating 10 hours;
2.6) above-mentioned centrifugate the is flowed through good DEAE resin column of balance, flow rate control is in 120-130mL/min left and right;
2.7) with elutriant B eluant solution, 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 the about 1.65L of ultrafiltrated.
3) affinity chromatography (take 0.25L cylinder as example)
3.1), with elutriant C balance affinity column, by the ultrafiltrated resin column that balance crosses of flowing through, flow rate control is at 120-130mL/min;
3.2), after end of the sample, with elutriant C wash-out, flow velocity 120-130mL/min left and right, collects elutriant;
3.3) the affine resin in post, after elutriant C wash-out, is removed the superincumbent impurity of absorption with elutriant D washing, and flow rate control, at 120-130mL/min, seals stand-by;
3.4) elutriant, through ultra-filtration membrane ultrafiltration to certain volume, adds a certain amount of disodium phosphate soln in ultrafiltrated, continue ultrafiltration to same volume, and then add equivalent disodium phosphate soln, then through ultra-filtration membrane ultrafiltration to same volume.
4) precipitation
In ultrafiltrated, in the ratio of 1: 6, add not low 95% ethanol precipitation 12 hours, centrifugal, solid is again with dehydrated alcohol dehydration twice, and centrifugal, abandoning supernatant, leaves and takes throw out.
5) freeze-drying
Throw out is packed into and coiled into Freeze Drying Equipment, and freeze-drying obtains ulinastatin.
Through adjusting, gained ulinastatin total recovery, up to more than 70%, is measured by method shown in Chinese Pharmacopoeia, and total titer is up to more than 5000iu/mg, and post effect improves approximately 50%.
Compared with prior art, advantage of the present invention and positively effect are:
Select resin regeneration to improve post and follow purifying ulinastatin, the multiple foreign protein in can 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, ulinastatin total recovery is reached more than 70%, total titer is not less than 5000iu/mg, post effect improves approximately 50%, has saved cost, has created huge economic benefit and social benefit.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, and restriction never in any form.
Embodiment 1
1) ulinastatin roughing
Take the clarification urine that 1T pH is less than 6.5, constantly stir, after slowly adding the absorption completely of 16.5kg chitin, use ammoniacal liquor wash-out, pass through again 3.5kg ammonium sulfate precipitation, the precipitation, centrifugal being placed on Vanadium Pentoxide in FLAKES of spending the night done vacuum-drying in the vacuum drier of water-retaining agent, obtains ulinastatin crude product after dry.
2) adsorpting column chromatography (the 0.25L DEAE resin column of take is example)
2.1) the DEAE resin in post is recoiled by purified water, pack in bucket, with purified water submergence, stir evenly, standing 1 hour, incline supernatant liquor and fine particle, three times repeatedly, purified water submergence is spent the night.
2.2) DEAE resin is filtered dry, with a small amount of purified water washing, after being again filtered dry, packs in bucket, with the submergence of 0.5mol/L hydrochloric acid, stir 1 hour, filter, by purified water, be washed till neutrality, then use 0.5mol/LNaOH solution soaking, stir 1 hour, filter, then be washed till neutrality by purified water.
2.3) the DEAE resin of above-mentioned treated mistake is added to elutriant A and repeatedly equilibrate to after pH6.5, then soak stand-by with elutriant A.
2.4) the good resin of above-mentioned balance is packed in post, after post installs, then walk post with elutriant A
2.5) take ulinastatin crude product 1kg, with elutriant A, dissolve, stir 30 minutes, centrifugal 20 minutes, leave and take centrifugate, 60 ℃ of stirring in water bath heating 10 hours;
2.6) above-mentioned centrifugate the is flowed through good DEAE resin column of balance, flow rate control is in 120-130mL/min left and right;
2.7) with the elutriant B eluant solution 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 the about 1.65L of ultrafiltrated.
3) affinity chromatography (take 0.25L cylinder as example)
3.1), with the elutriant C balance affinity column of 3.0L, by the ultrafiltrated resin column that balance crosses of flowing through, flow rate control is at 120mL/min;
3.2), after end of the sample, with the elutriant C wash-out of 10L, flow velocity 120mL/min left and right, collects elutriant;
3.3) the affine resin in post, after elutriant C wash-out, is removed the superincumbent impurity of absorption with the elutriant D washing of 5.0L, and flow rate control, at 120mL/min, seals stand-by;
3.4) elutriant, through ultra-filtration membrane ultrafiltration to 170mL, adds disodium phosphate soln 830mL in ultrafiltrated, continue ultrafiltration to 170mL, and then add 830mL disodium phosphate soln, then through ultra-filtration membrane ultrafiltration to 170mL.
4) precipitation
In ultrafiltrated, in the ratio of 1: 6, add not low 95% ethanol precipitation 12 hours, centrifugal, solid is again with dehydrated alcohol dehydration twice, and centrifugal, abandoning supernatant, leaves and takes throw out.
5) freeze-drying
Throw out is packed into and coiled into Freeze Drying Equipment, and freeze-drying obtains ulinastatin.
Through adjusting, post effect improves 51%, and gained ulinastatin total recovery is 72%, by method shown in Chinese Pharmacopoeia, measures, and total titer is up to 5295iu/mg.
Embodiment 2
1) ulinastatin roughing
Take the clarification urine that 1T pH is less than 6.5, constantly stir, after slowly adding the absorption completely of 16.5kg chitin, use ammoniacal liquor wash-out, pass through again 3.5kg ammonium sulfate precipitation, the precipitation, centrifugal being placed on Vanadium Pentoxide in FLAKES of spending the night done vacuum-drying in the vacuum drier of water-retaining agent, obtains ulinastatin crude product after dry.
2) adsorpting column chromatography (the 0.25L DEAE resin column of take is example)
2.1) the DEAE resin in post is recoiled by purified water, pack in bucket, with purified water submergence, stir evenly, standing 1 hour, incline supernatant liquor and fine particle, three times repeatedly, purified water submergence is spent the night.
2.2) DEAE resin is filtered dry, with a small amount of purified water washing, after being again filtered dry, packs in bucket, with the submergence of 0.5mol/L hydrochloric acid, stir 1 hour, filter, by purified water, be washed till neutrality, then use 0.5mol/LNaOH solution soaking, stir 1 hour, filter, then be washed till neutrality by purified water.
2.3) the DEAE resin of above-mentioned treated mistake is added to elutriant A and repeatedly equilibrate to after pH6.5, then soak stand-by with elutriant A.
2.4) the good resin of above-mentioned balance is packed in post, after post installs, then walk post with elutriant A
2.5) take ulinastatin crude product 1kg, with elutriant A, dissolve, stir 30 minutes, centrifugal 20 minutes, leave and take centrifugate, 60 ℃ of stirring in water bath heating 10 hours;
2.6) above-mentioned centrifugate the is flowed through good DEAE resin column of balance, flow rate control is in 120-130mL/min left and right;
2.7) with the elutriant B eluant solution 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 the about 1.65L of ultrafiltrated.
3) affinity chromatography (take 0.25L cylinder as example)
3.1), with the elutriant C balance affinity column of 3.0L, by the ultrafiltrated resin column that balance crosses of flowing through, flow rate control is at 130mL/min;
3.2), after end of the sample, with the elutriant C wash-out of 10L, flow velocity 130mL/min left and right, collects elutriant;
3.3) the affine resin in post, after elutriant C wash-out, is removed the superincumbent impurity of absorption with the elutriant D washing of 7.5L, and flow rate control, at 130mL/min, seals stand-by;
3.4) elutriant, through ultra-filtration membrane ultrafiltration to 170mL, adds disodium phosphate soln 830mL in ultrafiltrated, continue ultrafiltration to 170mL, and then add 830mL disodium phosphate soln, then through ultra-filtration membrane ultrafiltration to 170mL.
4) precipitation
In ultrafiltrated, in the ratio of 1: 6, add not low 95% ethanol precipitation 12 hours, centrifugal, solid is again with dehydrated alcohol dehydration twice, and centrifugal, abandoning supernatant, leaves and takes throw out.
5) freeze-drying
Throw out is packed into and coiled into Freeze Drying Equipment, and freeze-drying obtains ulinastatin.
Through adjusting, post effect improves 48%, and gained ulinastatin total recovery is 69%, by method shown in Chinese Pharmacopoeia, measures, and total titer is up to 5102iu/mg.
The above, be only preferred embodiment of the present invention, is not the present invention to be done to the restriction of other form, and any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the equivalent embodiment of equivalent variations.But every technical solution of the present invention content that do not depart from, any simple modification, equivalent variations and the remodeling above embodiment done 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 to purifying by adsorpting column chromatography, ultrafiltrated precipitation, throw out step of freeze drying, the total recovery of the ulinastatin after gained purifying is brought up to more than 70%, total titer is not less than 5000iu/mg, and post effect promotes approximately 50%.
2. the method for claim 1, is characterized in that: described ulinastatin crude product is by take Male urine as raw material, and raw material is prepared through chitin absorption, ammoniacal liquor wash-out, ammonium sulfate precipitation successively.
3. method as claimed in claim 1 or 2, is characterized in that described operation comprises the steps:
(1) with elutriant A, dissolve ulinastatin crude product, stirring, centrifugal, leaves and takes centrifugate, the acetate buffer that described elutriant A is 0.01-0.3mol/L;
(2) by elutriant B balance, process the adsorption column of regeneration through specificity, by above-mentioned centrifugate upper prop, elutriant B is the damping fluid of 0.01-0.5mol/L sodium-acetate and 0.1-5mol/L NaCl;
(3) with elutriant B wash-out adsorption column, collect elutriant;
(4) with elutriant C balance affinity column, by ultrafiltrated upper prop, elutriant C is the damping fluid of 0.01-0.5mol/L glycine and 0.1-5mol/L NaCl;
(5), after end of the sample, with elutriant C wash-out, collect elutriant;
(6) the affine resin in post is after elutriant C wash-out, with elutriant D washing, removes the superincumbent impurity of absorption, seals stand-byly, and elutriant D is the damping fluid of 0.01-0.3mol/L hydrochloric acid and 0.1-5mol/L NaCl;
(7) by elutriant through ultra-filtration membrane, add phosphate buffered saline buffer ultrafiltration repeatedly, obtain ultrafiltrated.
(8) in ultrafiltrated, add ethanol precipitation, centrifugal, solid is again with dehydrated alcohol dehydration, and centrifugal, abandoning supernatant, leaves and takes throw out;
(9) by throw out freeze-drying, obtain ulinastatin.
4. method as claimed in claim 3, 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, is characterized in that, described adsorption column is anion-exchange column.
6. method as claimed in claim 5, is characterized in that, described anion-exchange column comprises: reinforcing yin essence ion exchange column Q-Sephadex A-25, Q-Sephadex A-50, Q-Sephadex C-25, Q-Sephadex C-50; Weak anionic exchange column DEAE-Cellulose DE-22, DEAE-Cellulose DE-23, DEAE-Cellulose DE-51, DEAE-Cellulose DE-52, DEAE-Cellulose DE-53.
7. method as claimed in claim 3, is characterized in that, described resin regeneration method i.e. washing, pickling, the alkali cleaning of a graded is processed, and improves the efficiency of resin column.
8. method as claimed in claim 3, it is characterized in that: described affinity column comprises: CM-Sephadex A-25, CM-Sephadex A-50, CM-Sephadex C-25, CM-Sephadex C-50, SP-Sepharose2B, SP-Sepharose4B, SP-Sepharose6B, SP-Sepharose CL-2B, SP-Sepharose CL-4B, SP-Sepharose CL-6B.
9. method as claimed in claim 1 or 2, is characterized in that: described ultrafiltrated settling step comprises: in ultrafiltrated, add ethanol precipitation.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104497134A (en) * | 2014-12-23 | 2015-04-08 | 青岛康原药业有限公司 | Method for purifying ulinastatin by virtue of affinity chromatography and pharmaceutical compositions containing ulinastatin |
| 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 |
| CN105384812A (en) * | 2015-11-24 | 2016-03-09 | 青岛康原药业有限公司 | Method for purifying ulinastatin through increasing column efficiency by resin regeneration and pharmaceutical composition for improving resolubility of ulinastatin |
| CN105399820A (en) * | 2015-11-24 | 2016-03-16 | 青岛康原药业有限公司 | Method for purifying ulinastatin by improving column efficiency through resin regeneration and pharmaceutical composition for improving stability of ulinastatin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104497134A (en) * | 2014-12-23 | 2015-04-08 | 青岛康原药业有限公司 | Method for purifying ulinastatin by virtue of affinity chromatography and pharmaceutical compositions containing ulinastatin |
| 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 |
| CN105384812A (en) * | 2015-11-24 | 2016-03-09 | 青岛康原药业有限公司 | Method for purifying ulinastatin through increasing column efficiency by resin regeneration and pharmaceutical composition for improving resolubility of ulinastatin |
| CN105399820A (en) * | 2015-11-24 | 2016-03-16 | 青岛康原药业有限公司 | Method for purifying ulinastatin by improving column efficiency through resin regeneration and pharmaceutical composition for improving stability of ulinastatin |
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Application publication date: 20140416 |