CN1314447C - Method for preparing medicine with proteinase inhibiting function and its detecting method - Google Patents
Method for preparing medicine with proteinase inhibiting function and its detecting method Download PDFInfo
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- CN1314447C CN1314447C CNB2004100310574A CN200410031057A CN1314447C CN 1314447 C CN1314447 C CN 1314447C CN B2004100310574 A CNB2004100310574 A CN B2004100310574A CN 200410031057 A CN200410031057 A CN 200410031057A CN 1314447 C CN1314447 C CN 1314447C
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Abstract
The present invention relates to a preparation method for a medicine capable of inhibiting protease and a detection method of the medicine. The preparation method comprises the following steps: inactivating viruses in a water solution containing alpha 1-protease inhibitor; carrying out ion-exchange resin adsorption and elution; simultaneously, utilizing the reaction of serine proteinase and the alpha 1-protease inhibitor for producing the light absorption changes of corresponding wavelengths to quantitatively measure the activity of the alpha 1-protease inhibitor. The preparation method of the present invention can effectively realize high yield, ensure the high purity and high activity of the prepared medicines, and simultaneously has the advantages of technology simplification and cost reduction; the detection method has the advantages of simplicity, feasibility and high sensitivity.
Description
[technical field]
The invention belongs to albumen purification field, be specifically related to a kind of preparation method with medicine of Profilin enzyme effect.
[background technology]
α 1-protease inhibitor (have another name called alpha1-antitrypsin, alpha-1 Antitrypsin is called for short α 1-PI) is that molecular weight is about 53,000 daltonian glycoproteins, and every liter of blood plasma on average contains 1.5 grams.This albumen mainly is the decomposition that suppresses elastoser and other serine protease in body.When activated α 1-PI concentration significantly was lower than the concentration of serine protease in the body, as occurring among the crowd with α 1-PI genetic defect, lung tissue will be suffered the destruction of serine protease and cause chronic pathological changes, as emphysema.This emophysematous patient can use α 1-PI and make secular replacement therapy, yet supply falls short of demand for the α 1-PI product on the market, and therefore, the technology of exploitation energy suitability for industrialized production high-quality α 1-PI could satisfy the demand in market.
Had in the past multiple delivered can be used for the method that suitability for industrialized production has the medicine of Profilin enzyme effect.People such as Glaser (Anal.Biochem.1982,124:364-371) the using sulfated ammonium sedimentation method one step from the CohnShi component I V-1 of human plasma obtains purity and is about 70% α 1-PI, and then it is refining through anion exchange chromatography (DEAE-cellulose), but yield is on the low side, has only about 40%.
People such as Coan (U.S. Pat 4,379,087; Vox Sang.1985 48:333-342) uses polyethylene glycol precipitation and anion exchange chromatography purification α 1-PI from the CohnShi component I V-1 of human plasma, and yield is about 50%, and purity has only about 60%.The method is the α 1-PI product P rolastin of Bayer Corp., USA (Bayer Corporation) approval listing in 1988
Prototype, in the said preparation activated α 1-PI only account for total protein 〉=35% (seeing its product description, 14-7601-001 (Rev.Jan.2002)).
People such as Burnouf (Vox Sang.1987,52:291-297) adopt anion exchange (DEAE Sepharose CL6B FF) to separate the α 1-PI that obtains purity 80-90% with gel filtration (Sephacryl S-200) chromatography from human plasma supernatant A (being equivalent to CohnShi component I I+III), yield is 65-75%.
The method of above-mentioned these purifications α 1-PI is all failed when obtaining high yield, guarantees the high-purity and the high activity of product.And said method technology is more numerous, and cost is than higher.In the practical operation, the albumen that molecular size and isoelectric point, IP are all approaching with α 1-PI, the albumin maximum as content in the blood plasma makes the work of separating high-purity α 1-PI quite difficult; To not have active α 1-PI to separate with activated α 1-PI then is bigger challenge.After the many kinds of trials to the ion-exchange chromatography condition, the present invention integrates simple two-step chromatography becomes the high-purity that can be used as medicine and the preparation method of highly active α 1-PI.
The antibody of the common application specific of existing α 1-PI detection technique comes quantitative assay α 1-PI, comprises the radioimmunoassay diffusion method, rocket immunoelectrophoresis and ELISA method., the technology of existing application specific antibody can't make a distinction the α 1-PI of activated α 1-PI and non-activity, and existing these technical costss compare higher.
[summary of the invention]
[technical problem that will solve]
In order to realize high yield, guarantee the high-purity and the high activity of product, simplify technology simultaneously and reduce cost, the invention provides a kind of preparation method with medicine of Profilin enzyme effect; Simultaneously at existing detection technique complex operation, situation that detection sensitivity is low, the invention provides a kind of detection method of simple, medicine that detection sensitivity is high with the effect of Profilin enzyme.
[technical scheme]
In order to realize high yield, guarantee the high-purity and the high activity of product, simplify technology simultaneously and reduce cost, the invention provides a kind of preparation method with medicine of Profilin enzyme effect, this method contains and has the following steps: viral inactivation treatment contains the aqueous solution of α 1-PI, ion exchange resin absorption, eluting.
The aqueous solution that will contain α 1-PI is through viral inactivation treatment, at first by anion exchange resin absorption α 1-PI, and from the anion exchange resin the activated α 1-PI of eluting selectively; Pass through cation exchange resin then, obtain having the medicine of Profilin enzyme effect.The aqueous solution that contains α 1-PI comprises: people's blood plasma or plasma component, the microorganism that contains gene recombined alpha 1-PI or cell culture fluid and contain the genetically modified animal body fluid of α 1-PI and comprise milk and blood plasma.Normal human plasma, every liter of α 1-PI that contains the 0.83-4.0 gram, wherein quite most of some plasma component that in the commercial process of plasma protein, is enriched in.People such as Wright discover, in the milk of Transgenic Sheep, the content of α 1-PI can be up to every liter 60 grams (Biotechnology, 1991,9:830-834).At microorganism such as escherichia coli (Casolaroet al, 1987, J.Appl.Physiol., 63:2015-2023) and yeast (Hubbard et al, 1991, Proc.Natl.Acad.Sci., 86:680-684), the applying gene recombinant technique can produce a large amount of α 1-PI.The material that preferably contains α 1-PI is the CohnShi component I V of human plasma, because of it is a garbage in the conventional industrialization plasma protein purge process of application of cold temperature ethanol precipitation (CohnShi method).In order to increase the safety of α 1-PI goods, before anion exchange chromatography was handled, dissolved CohnShi component I V can be via one or more method combined removal/inactivation of viruses.Virus removal/ablation method comprises: Pasteur (Pasteur) sterilization, and dry heating method, low pH is incubated the method for putting, organic solvent/detergent (S/D) facture, sad or caprylate facture, ultrafiltration virus removal method, the nanofiltration method, or the like.
α 1-PI in the human plasma has 100 kinds of different molecular variants (variants) of surpassing, and the isoelectric point, IP of several variants that content is high is between 4.3-4.6.When the pH of solution was higher than its isoelectric point, IP, α 1-PI was electronegative, can be incorporated on the anion exchange resin.The pH of solution is than high many more of its isoelectric point, IP, and the adhesion of α 1-PI and anion exchange resin is big more.Binding site on anion in the solution and the α 1-PI competition anion exchange resin, therefore, low ionic strength helps α 1-PI and is adsorbed to the anion-exchange chromatography post.The pH of the aqueous solution that contains α 1-PI that adjusting is crossed through virus removal/inactivation treatment is to 〉=4.8, and≤12.0, and regulate its ionic strength to 〉=0.1mS/cm, and≤10.0mS/cm, make it flow through the anion-exchange chromatography post.Available anion exchange resin comprises reinforcing YIN-essence ion exchange resin and weak anion exchange resin, for example, and the Q Sepharose Fast Flow and the DEAESepharose Fast Flow of peace agate West Asia (Amersham) company.Target protein α 1-PI is attached on the anion exchange resin, and the part foreign protein is not adsorbed and is removed.Increase the salinity of chromatographic column washing liquid or regulate the pH of washing liquid, further eluting part foreign protein, the pH that continues to increase the salinity of washing liquid or regulate washing liquid just selectively elutes the α 1-PI of absorption.The anion-exchange chromatography post eluent that contains α 1-PI is refining with cation exchange chromatography subsequently.Adjusting through the pH of the anion-exchange chromatography post eluent that contains α 1-PI of dialysis treatment to 〉=4.8, and≤13.0, and regulate its ionic strength to 〉=0.1mS/cm, and≤10.0mS/cm, make it flow through the cation-exchange chromatography post.Available cation exchange resin comprises strong cation-exchanging resin and weak cation exchange resin, for example, and the SP Sepharose Fast Flow and the CM Sepharose FastFlow of peace agate West Asia company.Target protein α 1-PI flows through cation exchange resin, and remaining most foreign proteins are adsorbed and are removed.Collect cation-exchange chromatography post effluent, by ultrafiltration or dialysis treatment, aseptic filtration after filling a prescription, bottle then lyophilizing and sealing are preserved, as the medicine with the effect of Profilin enzyme of purification.
The said method of same design can carry out following change: the preparation method with medicine of Profilin enzyme effect, it is characterized in that: the aqueous solution that will contain α 1-PI is after viral inactivation treatment, at first make α 1-PI flow through cation exchange resin, adsorbed by anion exchange resin subsequently, and make activated α 1-PI from anion exchange resin, elute selectively, obtain having the medicine of Profilin enzyme effect.Above-mentioned two kinds of method effects are basic identical.
Containing chemical reagent that the viral inactivation treatment of the aqueous solution of α 1-PI has an inactivation of virus effect by adding incubates to put and finishes.Chemical reagent with inactivation of virus effect joins the aqueous solution that contains α 1-PI, incubates between 0 degree Celsius and 50 degree Celsius and puts more than one hour.Can add corresponding stabilizing agent before the viral inactivation treatment.
Chemical reagent with inactivation of virus effect is organic solvent and detergent (S/D).Wherein used detergent right and wrong are Ionized.Chemical reagent with inactivation of virus effect also comprises: sad and caprylate, or the like.Stabilizing agent commonly used in the viral inactivation treatment process is the sucrose of 2%-50% and the sodium citrate of 0.001M-0.50M.
The most frequently used organic solvent is a tributyl phosphate, and the most frequently used detergent is TritonX-100.In addition, Tween 80 (Tween 80) also is the detergent of using always.The final concentration of the tributyl phosphate that is added is between 0.01% and 10.0%, and the final concentration of the Triton X-100 of Jia Ruing is between 0.01% and 10.0% simultaneously.
Detection method with medicine of Profilin enzyme effect is application serine protease and α 1-PI reaction and the light absorption variation that causes respective wavelength, thus the activity of quantitative assay α 1-PI.Serine protease and substrate reactions cause that the light absorption of respective wavelength changes, α 1-PI is by suppressing the light absorption variation that serine protease changes this wavelength, and concrete grammar comprises the steps: 1) utilize serine protease and α 1-protease inhibitor to react; 2) measuring the light absorption that causes respective wavelength changes; 3) change the activity that records quantitative α 1-protease inhibitor according to light absorption.
The final concentration of the serine protease that is added adds the corresponding substrate that is five times in this enzyme weight simultaneously between 0.1ppm and 100ppm.But the light absorption of measuring this wavelength in the certain hour changes the just activity of quantitative assay α 1-PI.Use the active method of serine protease detection α 1-PI and be called serine protease restraint algoscopy.The serine protease restraint of each unit is defined as, and compared with the control, in one minute, changes the activity of 1 unit of light absorption of respective wavelength.
The most frequently used serine protease is trypsin or elastoser.Other available serine protease comprises: Chymotrypsin, and thrombin, plasmin and collagenase, or the like.Highly purified trypsin becomes more readily available, thereby is most commonly used to the determination of activity of α 1-PI.Use the active method of trypsin detection α 1-PI and be called the TIC algoscopy.When not having mortifier, the catabolite of trypsin per minute can increase the light absorption of respective wavelength.The TIC of each unit is defined as, and compared with the control, in one minute, reduces the activity of 1 unit of light absorption of respective wavelength.Tryptic substrate commonly used is a BAEE, and the optical wavelength commonly used that TIC is measured is 253nm.
[beneficial effect]
Preparation method provided by the invention realizes high yield effectively, guarantees the high-purity and the high activity of product, simplifies technology simultaneously, reduces cost; Detection method is simple, highly sensitive.
Use method provided by the invention and use anion exchange (DEAE Sepharose CL6BFF) and gel filtration (Sephacryl S-200) purification α 1-PI, detect with conventional ELISA method at last, the result shows that method provided by the invention realizes high yield effectively, has guaranteed the high-purity and the high activity of product.
The antibody of the common application specific of existing α 1-PI detection technique comes quantitative assay α 1-PI, comprises the radioimmunoassay diffusion method, rocket immunoelectrophoresis and ELISA method., the technology of existing application specific antibody can't make a distinction the α 1-PI of activated α 1-PI and non-activity, and existing these technical costss compare higher.Detection method provided by the invention is used the activated medicine with the effect of Profilin enzyme of reaction quantitative assay of simple serine protease and corresponding substrate, has got rid of the α 1-PI of non-activity, has improved degree of accuracy; Operating procedure is simple simultaneously, and cost is corresponding have been reduced.
[specific embodiment]
The present invention designs following reaction condition altogether:
Table 1, reaction condition
| Anion exchange resin | Cation exchange resin | |||||||||||
| pH | 5.0 | 6.5 | 8.0 | 9.5 | 11.0 | 12.0 | 4.8 | 5.4 | 7.0 | 9.6 | 11.0 | 12.6 |
| Ionic strength (mS/cm) | 0.5 | 2.5 | 4.5 | 6.5 | 8.5 | 10.0 | 0.5 | 1.5 | 3.0 | 6.0 | 8.0 | 10.0 |
The following examples should be to further specify of the present invention, rather than the present invention is defined as cited embodiment.
Embodiment 1
CohnShi component I V adds 37% sucrose and the 0.38M sodium citrate stabilizing agent as viral inactivation treatment after adding the purified water dissolving of 20 times of weight.Adding final concentration subsequently and be 0.3% tributyl phosphate and final concentration and be 1.0% Triton X-100 incubates under 30 ℃ and puts 4 hours.Behind inactivation of virus, behind the adjusting pH to 6.5, water is reduced to 2.5mS/cm with ionic strength, is added on the DEAE Sepharose Fast Flow chromatographic column of pH 6.5 pre-equilibrations.Use 20mM sodium dihydrogen phosphate and 100mM sodium chloride solution eluting target protein α 1-PI selectively.The concentration that strengthens sodium chloride can comprise Ceruloplasmin to the foreign protein of brute force absorption, elutes from this ion exchange column.DEAE Sepharose Fast Flow resin is reusable after the sterilization of 0.5M sodium hydroxide.
The eluent that contains α 1-PI passes through the ultrafilter membrane bag (Pall Corporation) of 10kD except that freshen.Elder generation's water is reduced to 2.5mS/cm with ionic strength, and reuse 5mM sodium citrate (pH 5.4) carries out the solution exchange.The treated solution that contains α 1-PI is added on the CMSepharose Fast Flow chromatographic column of pH 5.4 pre-equilibrations, and activated α 1-PI flows through this ion exchange resin and is able to purification.Present embodiment is purified into 45.3 milligrams, the α 1-PI of purity 95.8% from 4.18 gram component I V.The yield that active alpha 1-PI is arranged is up to 63.4%.
Present embodiment is used the activity of trypsin quantitative assay α 1-PI.When not having mortifier, the product that BAEE produced of the trypsin per minute of 33.3ppm degraded 166.7ppm can increase the light absorption 0.1-0.2 unit of 253nm.The TIC of each unit is defined as, and compared with the control, in one minute, reduces the activity of 1 unit of light absorption of 253nm.Table 2 is this routine experimental data.
Table 2, α 1-PI activity
| Total protein (mg) | α 1-PI activity (yield) | |
| Component I V | 1695.2 | 612.0U(100％) |
| The DEAE chromatography | 108.6 | 432.6U(70.7％) |
| The CM chromatography | 45.3 | 388.1U(63.4％) |
Embodiment 2
Dissolved CohnShi component I V regulates pH to 5.4 with 1M hydrochloric acid, and water is regulated ionic strength to 1.5mS/cm, is added to then on the CM Sepharose Fast Flow chromatographic column of 5mM sodium citrate (pH 5.4) pre-equilibration.Collect the effluent of this ion exchange resin, behind the adjusting pH to 6.5, regulate ionic strength, be added on the DEAE Sepharose Fast Flow chromatographic column of 20mM sodium dihydrogen phosphate (pH 6.5) pre-equilibration to 2.5mS/cm.Use 20mM sodium dihydrogen phosphate and the 100mM sodium chloride solution activated α 1-PI of eluting selectively.Present embodiment is purified into 58.9 milligrams, the α 1-PI of purity 92.5% from 5.34 gram component I V.The yield that active alpha 1-PI is arranged is up to 81.2%.Table 3 is this routine experimental data.
Table 3, α 1-PI activity
| Total protein (mg) | α 1-PI activity (yield) | |
| Component I V | 1191.2 | 448.7U(100％) |
| The CM chromatography | 255.3 | 408.8U(91.1％) |
| The DEAE chromatography | 58.9 | 364.5U(81.2％) |
Remaining is identical with embodiment 1.
Claims (5)
1, a kind of preparation has the method for the medicine of Profilin enzyme effect, it is characterized in that the aqueous solution that will contain α 1-protease inhibitor is after viral inactivation treatment, through ion exchange resin absorption, eluting, purification α 1-protease inhibitor, wherein ion exchange resin absorption, eluting are:
By anion exchange resin absorption α 1-protease inhibitor, and from the anion exchange resin the activated α 1-of eluting protease inhibitor selectively, then through a cation exchange resin;
Or filter by cation exchange resin, then by an anion exchange resin absorption, and from the anion exchange resin the activated α 1-of eluting protease inhibitor selectively.
2, preparation method according to claim 1, the aqueous solution that wherein contains α 1-protease inhibitor is: people's blood plasma or plasma component, contain gene recombined alpha 1-protease inhibitor microorganism or cell culture fluid, contain the genetically modified animal body fluid of α 1-protease inhibitor and comprise CohnShi component I V or IV-1 in milk and blood plasma and the conventional industrialization plasma protein purge process.
3, preparation method according to claim 1 is characterized in that the aqueous solution that viral inactivation treatment contains α 1-protease inhibitor is to have the chemical reagent of viral deactivation to incubate to put by apparatus to finish.
4, preparation method according to claim 3, the chemical reagent that it is characterized in that having in this method the inactivation of virus effect is sad or caprylate.
5, the preparation method of medicine according to claim 3, the chemical reagent that it is characterized in that having in this method the inactivation of virus effect is organic solvent and detergent, and wherein organic solvent is a tributyl phosphate, and detergent is Triton X-100 or Tween 80.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206272A (en) * | 2009-12-11 | 2011-10-05 | 普罗特奥姆技术公司 | Method for production of recombinant alpha1-antitrypsin |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NZ590257A (en) * | 2008-07-18 | 2012-08-31 | Grifols Therapeutics Inc | Method of preparing alpha-1 proteinase inhibitor |
| CN116033917A (en) * | 2020-05-01 | 2023-04-28 | 阿特拉斯生物技术股份有限公司 | Treatment and/or prevention of diseases or syndromes associated with viral infections |
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| US4379087A (en) * | 1982-06-17 | 1983-04-05 | Cutter Laboratories, Inc. | Method of preparing alpha-1-proteinase inhibitor |
| US4683294A (en) * | 1985-04-03 | 1987-07-28 | Smith Kline Rit, S.A. | Process for the extraction and purification of proteins from culture media producing them |
| US4939176A (en) * | 1988-12-20 | 1990-07-03 | Miles Inc. | Viral inactivation process |
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| CN1120439A (en) * | 1994-08-10 | 1996-04-17 | 美国拜尔公司 | Low temperature albumin fractionation using sodium caprylates as a partitioning agent |
| CN1125231A (en) * | 1994-08-24 | 1996-06-26 | 美国拜尔公司 | Purification of alpha-1 proteinase inhibitor using novel chromatographic separation conditions |
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Patent Citations (6)
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| US4379087A (en) * | 1982-06-17 | 1983-04-05 | Cutter Laboratories, Inc. | Method of preparing alpha-1-proteinase inhibitor |
| US4683294A (en) * | 1985-04-03 | 1987-07-28 | Smith Kline Rit, S.A. | Process for the extraction and purification of proteins from culture media producing them |
| US4939176A (en) * | 1988-12-20 | 1990-07-03 | Miles Inc. | Viral inactivation process |
| CN1053445A (en) * | 1989-12-21 | 1991-07-31 | 国际药品工业株式会社 | From people's blood plasma, separate alpha1-antitrypsin and detection system thereof |
| CN1120439A (en) * | 1994-08-10 | 1996-04-17 | 美国拜尔公司 | Low temperature albumin fractionation using sodium caprylates as a partitioning agent |
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Non-Patent Citations (1)
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| Purification of α1 Proteinase Inhibitorfrom Human Plasma Fraction IV-1 by Ion Exchange Chromatography Sharon.X.Chen et al,Vox Sanguinis,Vol.74 1998 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206272A (en) * | 2009-12-11 | 2011-10-05 | 普罗特奥姆技术公司 | Method for production of recombinant alpha1-antitrypsin |
| CN102206272B (en) * | 2009-12-11 | 2014-10-01 | 普罗特奥姆技术公司 | Method for production of recombinant alpha1-antitrypsin |
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