CN1844380A - Process for preparing high-purity thrombase - Google Patents
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- CN1844380A CN1844380A CN 200610078799 CN200610078799A CN1844380A CN 1844380 A CN1844380 A CN 1844380A CN 200610078799 CN200610078799 CN 200610078799 CN 200610078799 A CN200610078799 A CN 200610078799A CN 1844380 A CN1844380 A CN 1844380A
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- zymoplasm
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- 108090000190 Thrombin Proteins 0.000 title claims abstract description 68
- 238000004519 manufacturing process Methods 0.000 title abstract description 4
- 238000000034 method Methods 0.000 claims abstract description 28
- 238000004108 freeze drying Methods 0.000 claims abstract description 9
- 238000010612 desalination reaction Methods 0.000 claims abstract description 7
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 7
- 238000004255 ion exchange chromatography Methods 0.000 claims abstract description 6
- 238000005119 centrifugation Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 51
- 229960004072 thrombin Drugs 0.000 claims description 34
- 239000007788 liquid Substances 0.000 claims description 20
- 239000000872 buffer Substances 0.000 claims description 19
- 239000003153 chemical reaction reagent Substances 0.000 claims description 19
- 210000004369 blood Anatomy 0.000 claims description 14
- 239000008280 blood Substances 0.000 claims description 12
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 10
- 229910019142 PO4 Inorganic materials 0.000 claims description 8
- 239000010452 phosphate Substances 0.000 claims description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 238000004587 chromatography analysis Methods 0.000 claims description 6
- 239000008363 phosphate buffer Substances 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- 241001494479 Pecora Species 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 3
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 2
- 238000004440 column chromatography Methods 0.000 claims description 2
- 238000013016 damping Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 2
- 239000012745 toughening agent Substances 0.000 claims description 2
- 150000002500 ions Chemical class 0.000 claims 1
- 238000010828 elution Methods 0.000 abstract description 7
- 238000002360 preparation method Methods 0.000 abstract description 4
- 238000000605 extraction Methods 0.000 abstract description 2
- 238000002523 gelfiltration Methods 0.000 abstract 2
- 238000012512 characterization method Methods 0.000 abstract 1
- 239000012501 chromatography medium Substances 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 8
- 229910000397 disodium phosphate Inorganic materials 0.000 description 7
- AURFVNDXGLQSNN-UHFFFAOYSA-K trisodium 2-hydroxypropane-1,2,3-tricarboxylic acid phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O AURFVNDXGLQSNN-UHFFFAOYSA-K 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 3
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000005227 gel permeation chromatography Methods 0.000 description 3
- 238000000703 high-speed centrifugation Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 2
- 229940045641 monobasic sodium phosphate Drugs 0.000 description 2
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 2
- 235000019799 monosodium phosphate Nutrition 0.000 description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 235000011008 sodium phosphates Nutrition 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 229910001423 beryllium ion Inorganic materials 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
This invention provides a method for preparation of high-purity thrombase from low-purity thrombase. The high-purity thrombase product is obtained through the following steps: absorbtion of the low-purity thrombase solution by the chromatography media, elution, collection of the thrombase component, gel filtration, desalination and concentration by ultrafiltration, and finally freeze-drying. This method is carried out at normal temperature and pressure, with characterization of environmental-protection process, short production circle, high extraction efficencey of thrombase with yield rate upto 80% and simple operation. With a few steps of centrifugation, ion exchange chromatography, gel filtration, desalination and concentration by ultrafiltration and freeze-drying, the invention can be eaily scaled up for industrial production.
Description
Technical field
The present invention relates to technical field of bioengineering, is the method for feedstock production high-purity thrombase with animal blood low-purity zymoplasm especially.
Technical background
Zymoplasm is a kind of serine protease with high degree of specificity in the blood plasma zymoplasm system, and its restriction enzyme site on Fibrinogen is: Gly-Gly-Val-Arg-↓-Gly-Pro-Arg-↓-.Therefore it has unique effect in the following process of the resulting fusion rotein product of genetically engineered is handled.For reaching this purposes, must use highly purified zymoplasm.The prior art for preparation method generally comprises three basic steps, i.e. extraction, purifying and freeze-drying (or preparation).China patent CN200410074753.3, CN92112188.1, CN93110426.2 and CN96103650.8 have proposed to extract the method for zymoplasm respectively from animal bloods such as pig blood, ox blood, sheep blood, the zymoplasm of these method preparations all is the low-purity zymoplasm, be also referred to as the low activity zymoplasm, its zymoplasm is generally less than 400IU/mg albumen than living, and the low-purity zymoplasm can't satisfy the needs of genetic engineering technique aspect.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, the method for preparing high-purity thrombase that a kind of technical process is simple, with short production cycle and yield is high is provided.
The method that the present invention prepares zymoplasm comprises following step:
(1), the low-purity zymoplasm is dissolved centrifugation, collection supernatant liquor with buffer reagent;
(2), with supernatant thrombin solution Shangyang ion chromatography column chromatography, chromatography column is carried out wash-out, collect the zymoplasm protein peak, thrombin solution;
(3), with gel column on the thrombin solution that obtains in the step (2), gel column is carried out wash-out, collect the zymoplasm protein peak, obtain highly purified thrombin solution;
(4), high-purity thrombase solution is carried out desalination and concentration by ultrafiltration, the high-purity thrombase concentrated solution;
(5), with the freeze-drying of high-purity thrombase concentrated solution, promptly get white powdery high-purity thrombase finished product.
The present invention prepares in the method for high-purity thrombase, and described low-purity zymoplasm derives from animal blood or human bloods such as pig blood, ox blood, sheep blood.
In the method for the present invention, the employed buffer reagent of step (1) is citric acid-phosphate buffer or phosphate buffer.The pH value of buffer reagent is 5～7.The concentration of citric acid-phosphate solution or phosphate solution is 0.01～0.025mol/L.
In the method for the present invention, step (2) is carried out wash-out to chromatography column, and employed eluent is made up of basal liquid and gradient liquid.Described basal liquid is that adjusting pH value is 5～7, and the concentration of citric acid-phosphate solution or phosphate solution is the buffer reagent of 0.01～0.025mol/L.Described gradient liquid is that to regulate pH value be 5～7, with concentration be citric acid-phosphate solution of 0.01～0.025mol/L or sodium-chlor that phosphate solution is damping fluid and 0.5～1mol/L or other inorganic salt be ion toughener composition mixed solution.
In the method for the present invention, step (3) is carried out wash-out to gel column, and employed eluent is that adjusting pH value is 5～7, and the concentration of citric acid-phosphate solution or phosphate solution is the buffer reagent of 0.01～0.025mol/L.
The present invention has following advantage:
1, high-purity thrombase purification process provided by the invention carries out at normal temperatures and pressures;
2, the yield height of zymoplasm, its yield is more than 80%;
3, simple to operate.
4, process cycle is short, and whole explained hereafter only needs about 3 days (comprising freeze-drying).
Embodiment
Embodiment 1: take by weighing pig low-purity zymoplasm 6 grams, add 0.025mol/L, pH is citric acid-Sodium phosphate dibasic buffer reagent 400ml of 5.8, fully dissolving, and high speed centrifugation is collected supernatant A 1.Getting the positively charged ion chromatography post that balance is good on the supernatant thrombin solution A1, is 5.8 with regulating pH, and concentration is that citric acid-Sodium phosphate dibasic buffer reagent of 0.025mol/L is as basal liquid; With regulating pH is 5.8, and concentration is that the citric acid-Sodium phosphate dibasic buffer reagent of 0.025mol/L and sodium chloride concentration are that the mixed solution formed of 0.5mol/L is as gradient liquid; By the gradient elution agent that above-mentioned basal liquid and gradient liquid are formed chromatography column is carried out gradient elution, collect thrombin activity peak A2, get zymoplasm elutriant 410ml.
The thrombin solution A2 that collection is got goes up Sephadex G-100 gel column, and thrombin solution A2 is carried out gel chromatography, and wash-out is also collected thrombin activity peak A3, obtains high-purity thrombase dope 200ml.
Thrombin solution A3 is carried out the ultrafiltration desalination, obtain thrombin solution less salt strong solution 50ml, be labeled as A4.The above-mentioned zymoplasm dope of freeze-drying promptly gets white powdery high-purity thrombase.
Embodiment 2: takes by weighing ox low-purity zymoplasm 6.5 grams, adds SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic buffer reagent 400ml of 0.025mol/L, and fully dissolving, high speed centrifugation is collected supernatant liquor B1.Getting the positively charged ion chromatography post that balance is good on the supernatant thrombin solution B1, is 7.0 with regulating pH, and concentration is that citric acid-Sodium phosphate dibasic buffer reagent of 0.025mol/L is as basal liquid; With regulating pH is 7.0, and concentration is that the citric acid-Sodium phosphate dibasic buffer reagent of 0.025mol/L and sodium chloride concentration are that the mixed solution formed of 1mol/L is as gradient liquid; By the gradient elution agent that above-mentioned basal liquid and gradient liquid are formed chromatography column is carried out gradient elution, collect thrombin activity peak B2, get zymoplasm elutriant 360ml.
The thrombin solution B2 that collection is got goes up Sephadex G-100 gel column, and thrombin solution B2 is carried out gel chromatography, and wash-out is also collected thrombin activity peak B3, obtains high-purity thrombase dope 220ml.
Thrombin solution B3 is carried out the ultrafiltration desalination, obtain thrombin solution less salt strong solution 50ml, be labeled as B4.The above-mentioned zymoplasm dope of freeze-drying promptly gets white powdery high-purity thrombase.
Embodiment 3: takes by weighing sheep low-purity zymoplasm 4.7 grams, adds SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic buffer reagent 400ml of 0.01mol/L, and fully dissolving, high speed centrifugation is collected supernatant C 1.Getting the positively charged ion chromatography post that balance is good on the supernatant thrombin solution C1, is 6.5 with regulating pH, and concentration is that citric acid-Sodium phosphate dibasic buffer reagent of 0.01mol/L is as basal liquid; With regulating pH is 6.5, and concentration is that the citric acid-Sodium phosphate dibasic buffer reagent of 0.01mol/L and sodium chloride concentration are that the mixed solution formed of 1mol/L is as gradient liquid; By the gradient elution agent that above-mentioned basal liquid and gradient liquid are formed chromatography column is carried out gradient elution, collect thrombin activity peak C2, get thrombin solution 320ml.
The thrombin solution C2 that collection is got goes up Sephadex G-100 gel column, and thrombin solution C2 is carried out gel chromatography, and wash-out is also collected thrombin activity peak C3, obtains high-purity thrombase dope 190ml.
Thrombin solution C3 is carried out the ultrafiltration desalination, obtain thrombin solution less salt strong solution 50ml, be labeled as C4.The above-mentioned zymoplasm dope of freeze-drying promptly gets white powdery high-purity thrombase.
Embodiment 4: according to the relevant thrombin titer standard detecting method of 2000 editions two ones the 1056th～1057 page of the Pharmacopoeia of the People's Republic of China, make typical curve, to above-mentioned thrombin solution Ai (A1, A2, A3, A4), Bi (B1, B2, B3, B4), Ci (C1, C2, C3, the C4) detection of tiring, detected result sees Table 1; Adopt the Bradord detection method that above-mentioned solution is carried out protein concentration and detect, detected result is as shown in table 1.According to thrombin titer detected result and corresponding proteins concentration detected result, calculate zymoplasm than living and yield (%), its result is as shown in table 2.
Table 1 experimental test data
| Classification | (IU/ml) tires | Protein concentration (mg/ml) | |
| Zymoplasm A | A1 | 3,000 | 7.5000 |
| A2 | 2,650 | 1.3344 | |
| A3 | 5,280 | 1.1102 | |
| A4 | 19,600 | 4.1670 | |
| Zymoplasm B | B1 | 3,560 | 8.902 |
| B2 | 3,700 | 1.9289 | |
| B3 | 5,520 | 1.2096 | |
| B4 | 23,100 | 5.1085 | |
| Zymoplasm C | C1 | 2,331 | 5.828 |
| C2 | 2,650 | 1.2582 | |
| C3 | 4,240 | 0.8614 | |
| C4 | 15,400 | 3.1793 |
Table 2 thrombin titer detects and analytical results
| Classification | Total titer (IU) | Than live (IU/mg) | The purifying multiple | Yield (%) | |
| Zymoplasm A | A1 | 1,200,000 | 400.0 | 1 | 100 |
| A2 | 1,086,500 | 1985.9 | 4.96 | 90.5 | |
| A3 | 1,056,000 | 4756.1 | 11.89 | 88.6 | |
| A4 | 980,000 | 4703.6 | -- | 81.7 | |
| Zymoplasm B | B1 | 1,424,000 | 356.0 | 1 | 100 |
| B2 | 1,332,000 | 1918.2 | 5.39 | 93.5 | |
| B3 | 1,214,400 | 4563.5 | 12.82 | 85.3 | |
| B4 | 1,155,000 | 4521.9 | -- | 81.1 | |
| Zymoplasm C | C1 | 932,400 | 310.8 | 1 | 100 |
| C2 | 848,000 | 2106.2 | 90.9 | ||
| C3 | 805,600 | 4922.5 | 86.4 | ||
| C4 | 770,000 | 4843.9 | -- | 82.6 | |
Claims (10)
1, a kind of method for preparing high-purity thrombase, this method comprises following step:
(1), the low-purity zymoplasm is dissolved centrifugation, collection supernatant liquor with buffer reagent;
(2), with supernatant thrombin solution Shangyang ion chromatography column chromatography, chromatography column is carried out wash-out, collect the zymoplasm protein peak, thrombin solution;
(3), with gel column on the thrombin solution that obtains in the step (2), gel column is carried out wash-out, collect the zymoplasm protein peak, obtain highly purified thrombin solution;
(4), high-purity thrombase solution is carried out desalination and concentration by ultrafiltration, the high-purity thrombase concentrated solution;
(5), with the freeze-drying of high-purity thrombase concentrated solution, promptly get white powdery high-purity thrombase finished product.
2, the method for preparing high-purity thrombase according to claim 1 is characterized in that: described low-purity zymoplasm derives from pig blood, ox blood, sheep blood or human blood.
3, the method for preparing high-purity thrombase according to claim 1 is characterized in that: the employed buffer reagent of step (1) is citric acid-phosphate buffer or phosphate buffer.
4, the method for preparing high-purity thrombase according to claim 3 is characterized in that: the pH value of buffer reagent is 5～7.
5, according to claim 3 or the 4 described methods that prepare zymoplasm, it is characterized in that: the concentration of citric acid-phosphate solution or phosphate solution is 0.01～0.025mol/L.
6, the method for preparing high-purity thrombase according to claim 1 is characterized in that: the employed eluent of step (2) is made up of basal liquid and gradient liquid.
7, according to the described method for preparing high-purity thrombase of claim 6, it is characterized in that: described basal liquid is that adjusting pH value is 5～7, and the concentration of citric acid-phosphate solution or phosphate solution is the buffer reagent of 0.01～0.025mol/L.
8, according to claim 6 or the 7 described methods that prepare high-purity thrombase, it is characterized in that: described gradient liquid is that adjusting pH value is 5～7, with concentration be citric acid-phosphate solution of 0.01～0.025mol/L or sodium-chlor that phosphate solution is damping fluid and 0.5～1mol/L or other inorganic salt be the ion toughener form mixed solution.
9, the method for preparing high-purity thrombase according to claim 1 is characterized in that: the employed eluent of step (2) is citric acid-phosphate buffer or phosphate buffer.
10, the method for preparing zymoplasm according to claim 9 is characterized in that: employed eluent is that adjusting pH value is 5～7, and the concentration of citric acid-phosphate solution or phosphate solution is the buffer reagent of 0.01～0.025mol/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN 200610078799 CN1844380A (en) | 2006-05-12 | 2006-05-12 | Process for preparing high-purity thrombase |
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| CN 200610078799 CN1844380A (en) | 2006-05-12 | 2006-05-12 | Process for preparing high-purity thrombase |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002153A (en) * | 2015-07-08 | 2015-10-28 | 黄耀江 | Thrombin preparation method |
| CN105624134A (en) * | 2016-02-26 | 2016-06-01 | 福建华灿制药有限公司 | Method for preparing restrictive thrombin |
| CN107406840A (en) * | 2015-02-25 | 2017-11-28 | 奥姆里克斯生物药品有限公司 | Method for purifying and quantifying thrombin and its degrading polypeptides |
| CN108504671A (en) * | 2018-03-28 | 2018-09-07 | 宁波市医疗中心李惠利医院 | A kind of PTEN-Long protein purifications preparation method |
-
2006
- 2006-05-12 CN CN 200610078799 patent/CN1844380A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107406840A (en) * | 2015-02-25 | 2017-11-28 | 奥姆里克斯生物药品有限公司 | Method for purifying and quantifying thrombin and its degrading polypeptides |
| US11008560B2 (en) | 2015-02-25 | 2021-05-18 | Omrix Biopharmaceuticals Ltd. | Method for purifying and quantifying thrombin and its degradation polypeptides |
| CN107406840B (en) * | 2015-02-25 | 2021-05-28 | 奥姆里克斯生物药品有限公司 | Methods for purification and quantification of thrombin and its degraded polypeptides |
| US11149263B2 (en) | 2015-02-25 | 2021-10-19 | Omrix Biopharmaceuticals Ltd. | Method for purifying and quantifying thrombin and its degradation polypeptides |
| US12351843B2 (en) | 2015-02-25 | 2025-07-08 | Omrix Biopharmaceuticals Ltd. | Method for purifying and quantifying thrombin and its degradation polypeptides |
| CN105002153A (en) * | 2015-07-08 | 2015-10-28 | 黄耀江 | Thrombin preparation method |
| CN105002153B (en) * | 2015-07-08 | 2019-03-15 | 黄耀江 | A kind of preparation method of fibrin ferment |
| CN105624134A (en) * | 2016-02-26 | 2016-06-01 | 福建华灿制药有限公司 | Method for preparing restrictive thrombin |
| CN105624134B (en) * | 2016-02-26 | 2019-05-21 | 福建华灿制药有限公司 | The preparation method of restricted fibrin ferment |
| CN108504671A (en) * | 2018-03-28 | 2018-09-07 | 宁波市医疗中心李惠利医院 | A kind of PTEN-Long protein purifications preparation method |
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