CN105315365A - Preparation method for human antithrombin III - Google Patents
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- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 101000757319 Homo sapiens Antithrombin-III Proteins 0.000 title abstract 2
- 102000052834 human SERPINC1 Human genes 0.000 title abstract 2
- 229960004336 human antithrombin iii Drugs 0.000 title abstract 2
- 238000001914 filtration Methods 0.000 claims abstract description 24
- 102000004411 Antithrombin III Human genes 0.000 claims abstract description 18
- 108090000935 Antithrombin III Proteins 0.000 claims abstract description 18
- 229960005348 antithrombin iii Drugs 0.000 claims abstract description 18
- 241000700605 Viruses Species 0.000 claims abstract description 14
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- 238000000034 method Methods 0.000 claims abstract description 9
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- 238000001042 affinity chromatography Methods 0.000 claims abstract description 3
- 238000001179 sorption measurement Methods 0.000 claims abstract description 3
- 239000000706 filtrate Substances 0.000 claims description 33
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- 239000001509 sodium citrate Substances 0.000 claims description 15
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 15
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- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 claims description 2
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- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 abstract description 3
- 238000012797 qualification Methods 0.000 abstract description 3
- 235000019168 vitamin K Nutrition 0.000 abstract description 3
- 239000011712 vitamin K Substances 0.000 abstract description 3
- 150000003721 vitamin K derivatives Chemical class 0.000 abstract description 3
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- 239000003114 blood coagulation factor Substances 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 abstract description 2
- 230000010494 opalescence Effects 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 239000002202 Polyethylene glycol Substances 0.000 abstract 3
- 229920001223 polyethylene glycol Polymers 0.000 abstract 3
- 238000004090 dissolution Methods 0.000 abstract 2
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 abstract 1
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- 229920005654 Sephadex Polymers 0.000 abstract 1
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 18
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- 238000002560 therapeutic procedure Methods 0.000 description 2
- 229960004072 thrombin Drugs 0.000 description 2
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 208000027219 Deficiency disease Diseases 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108010080865 Factor XII Proteins 0.000 description 1
- 102000000429 Factor XII Human genes 0.000 description 1
- 108010074860 Factor Xa Proteins 0.000 description 1
- 102000011652 Formyl peptide receptors Human genes 0.000 description 1
- 108010076288 Formyl peptide receptors Proteins 0.000 description 1
- 240000004859 Gamochaeta purpurea Species 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
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- 206010019663 Hepatic failure Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 206010029164 Nephrotic syndrome Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
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- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
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- 108010064129 Thrombogen Proteins 0.000 description 1
- 206010047249 Venous thrombosis Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 239000004019 antithrombin Substances 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 208000025261 autosomal dominant disease Diseases 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000010836 blood and blood product Substances 0.000 description 1
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- 229940125691 blood product Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
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- 238000004440 column chromatography Methods 0.000 description 1
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- 229940088598 enzyme Drugs 0.000 description 1
- KAQKFAOMNZTLHT-VVUHWYTRSA-N epoprostenol Chemical compound O1C(=CCCCC(O)=O)C[C@@H]2[C@@H](/C=C/[C@@H](O)CCCCC)[C@H](O)C[C@@H]21 KAQKFAOMNZTLHT-VVUHWYTRSA-N 0.000 description 1
- 229960001123 epoprostenol Drugs 0.000 description 1
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- 238000011068 loading method Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 210000001501 megacaryocyte Anatomy 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8121—Serpins
- C07K14/8128—Antithrombin III
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a preparation method for human antithrombin III (AT-III). The preparation method comprises the following steps: (1) precipitation and dissolution of a blood plasma fraction IV; (2) polyethylene glycol (PEG) precipitation and impure protein removing; (3) DEAE Sephadex A-50 gel adsorption; (4) S/D virus inactivation; (5) heparin affinity column chromatography; (6) ultrafiltration and concentration; (7) addition of a stabilizer and regulation; (8) nanofilm virus-removing filtration; (9) sterilizing filtration; (10) freeze-drying; (11) dry and fever virus inactivation. According to the preparation method, blood coagulation factors, which remain in the raw materials and are depended by vitamin K, are removed, so as to greatly lower the possibility of protein activation in the production process, and improve the product qualification ratio; PEG is adopted for the impure protein removing, so as to reduce the load of a chromatographic column; a liquid obtained from lyophilized powder re-dissolution is clear, transparent, and free of protein precipitation and opalescence; through adoption of a three-step virus inactivation method, the safety of human AT-III for clinical use can be greatly improved.
Description
Technical field
The invention belongs to field of biological pharmacy, relate to a kind of preparation method of blood product, be specifically related to a kind of preparation method of people's Antithrombin III.
Background technology
Antithrombin Ⅲ (antithrombin III; AT-III) be the single chain glycoprotein that a kind of vitamin K relies on; synthesize in liver, vascular endothelial cell and megalokaryocyte; it is the inhibitor containing the proteolytic enzyme of Serine such as zymoplasm and the factor Ⅻ α, Ⅺ α, Ⅸ α, Ⅹ α; molecular mass is 58-64KD, and iso-electric point is 4.8; About 70 hours transformation period, content 260-320mg/L in blood plasma; Containing 4 N glycosylation oligonucleotide chains and 3 disulfide linkage, by 9 α-helixstructures, 3 beta sheets and 1 reactive center ring composition; The Serines such as AT-III and zymoplasm, FXa, FXIIa, XIa, IXa, plasmin form mixture with the ratio of l:l, thus these enzymes are lost activity, heparin can accelerate this reaction and reach more than thousand times, AT-III conformational change is caused after heparin and the Lysine binding contained by AT-III, arginine residues contained by AT-III is more easily combined with the serine residue of zymoplasm, once heparin-AT-III antithrombin complex is formed, heparin just dissociates from mixture, is again combined with another molecule AT-III and is recycled.
AT-III is the anticoagulant main component of composition, account for 75% of the total anticoagulation ability of human body, it is the key substance of Trombin inhibiting in blood plasma, as the most important obstruction factor of factor Xa in blood, which control solidifying and fibrinous dissolving of blood, participate in the running balance of anticoagulant functions and fibrinolytic system and blood coagulation system in holder, and play anti-freezing regulating effect.Clinically, in blood the level of AT-III with various disease, symptom and changing, in reductions such as disseminated inravascular coagulation (DIC), liver illness, nephrotic syndromes.AT-III level reduction in blood may cause heparin therapy effect to present.Therefore, the activity grasping AT-III is as the monitoring of this type of disease, ill-condition analysis, prognosis judgement and heparin therapy or use the index during dispensing of AT-III concentrate formulation significant.
In recent years find that AT-III urgees endothelial cells secrete prostacyclin, suppresses leukocyte surface chemoattractant receptor syndecan-4, amelioration of inflammation reacts.Measure AT-III content in blood plasma and can judge the thrombotic cause of disease, can directly also can form mixture with zymoplasm and suppress multiple coagulation factor activity by Trombin inhibiting.In addition, AT-III pair of platelet aggregation also has certain restraining effect, is main intrinsic coagulation inhibition.As clinical application, it treatment congenital and day after tomorrow acquired AT-III lack in deficiency disease, there is the effect being difficult to substitute.It is a kind of autosomal dominant disease that heredity AT-III lacks, this sick morbidity about 1/5000, morbidity many 10-25 year, patient is everlasting after Post operation, wound, infect after, gestation or phlebothrombosis occurs postpartum, and repeatedly can there is thrombus.In patients blood plasma, to be about the type of normal people about 50%, AT-III structure and fuction exception more for AT-III biological activity and antigenicity, and coexpress reduces the avidity of heparin, thus obviously weaken the inactivation capacity of serine protease.Acquired AT-III shortage shows as AT-III and synthesizes reduction, and see hepatic diseases, hepatic insufficiency, be mainly seen in liver cirrhosis, hepatitis gravis, later period of hepatocarcinoma, normal relevant to Disease severity, can occur together thrombosis.
Namely the research preparing AT-III from blood of human body has been carried out in the world from the sixties in last century, along with deepening continuously to AT-III structure and Study on mechanism, preparation method also reaches its maturity, AT-III goods have been widely used in clinical in the world, and have developed recombinant human AT-III product.The domestic research to AT-III is few, there is no AT-III product at present and emerges, lack the various diseases caused, lack remedy measures targetedly for because of AT-III; In recent years fragmentary research report is had to occur, as patent CN103059129A.Conventional preparation technology, yield is lower, and owing to lacking the removal measure of the thrombin that vitamin k relies on, easily causes protein activation, cause AT-III production qualification rate not high in preparation process.
Given this, this invention exploits a kind of novel method preparing AT-III lyophilized powder, there is process stabilizing, qualification rate is high, lyophilized powder redissolution liquid clear, without the advantage separated out without opalescence, and through S/D, nano-film filtration, xeothermic three step inactivation of virus, substantially increase the security that product uses.
Summary of the invention
It is advanced that technical problem to be solved by this invention is to provide a kind of technique, easy to operate, steady quality, the preparation method of product Clinical practice safety, people's Antithrombin III capable of being industrialized.
A preparation method for people's Antithrombin III, comprises the steps:
(1) plasma component IV resolution of precipitate
In the ratio of 1:10-20, component I V precipitation dropped into and dissolve in damping fluid, temperature controls at 10-30 DEG C, stirs 2-6 hour, makes it fully dissolve, obtain unit for uniform suspension;
(2) polyoxyethylene glycol (PEG) precipitates foreigh protein removing
In the suspension described in step (1), add 40-50%(w/w) polyoxyethylene glycol, stir 0.5-1.5 hour, then to connect 0.45 μm of filter element filtering with the Supradur50P filter plate that Solution produces, collect clear filtrate, with dissolving damping fluid prewashing filter plate and filter core before filtering;
(3) DEAESephadexA-50 gel adsorption
DEAESephadexA-50 gel is added in the filtrate collected by step (2), the add-on of xerogel is 0.5-1.0g/kg, xerogel is swelling with the hot water for injection of more than 70 DEG C in advance and with the cold water for injection cooling of less than 25 DEG C, then uses equilibration buffer; Slow stirring 0.5-1.5 hour, rear standing 5-15 minute; Filter, collect filtrate; Gel is reused after zeolite regeneration;
(4) S/D inactivation of virus
Tween80 to 1.0%(wt% is added in the collected filtrate of step (3)), TNBP(tributyl phosphate) to 0.3%(wt%), be warming up to 24-26 DEG C after stirring evenly, rear insulation 6-7 hour, then uses 0.45 μm of filter core clarification filtration;
(5) heparin affinity column chromatography
The upper heparin affinity column of filtrate after S/D, pillar fully balances with level pad in advance, upper prop terminates first to use Equilibration buffer wash pillar afterwards, pillar is washed afterwards with lavation buffer solution, use elution buffer wash-out pillar again, collect elutriant, with 0.45 μm or 0.22 μm of above elutriant of filter element filtering, gained filtrate is high-purity AT-III solution;
(6) ultrafiltration and concentration
Concentrate above filtrate with the ultra-filtration membrane of 5K ~ 10K, then constant volume dialysis 4-6 doubly, tire higher than 40IU/ml to AT-III by reconcentration; After shift out ultra-filtration membrane Bao Binghou and wash film bag, collect concentrated solution;
(7) stablizer and adjustment is added
In the concentrated solution collected by step (6), add stablizer, regulate AT-III to tire to desirable value (general requirements is 30-40IU/ml), rear tune pH value is to 6.50-7.50;
(8) nanometer film is except virus filtration
Carry out except virus filtration with 0.1 μm of filter core, 20 nanometer filter cores of connecting;
(9) Sterile Filtration packing
With 0.22 μm of filter core, Sterile Filtration is carried out and packing to product;
(10) freeze-drying;
(11) xeothermic inactivation of viruses
Freeze-dried preparation step (10) obtained is incubated 30 minutes in 100 DEG C of boiling water baths, carries out xeothermic inactivation of virus.
Dissolving damping fluid described in step (1) consists of 0.01M-0.02MTRIS, 0.1M-0.15M sodium-chlor, and the pH value of described dissolving damping fluid is 7.00-9.50.
The molecular weight of the polyoxyethylene glycol described in step (2) is 2000-6000, and the add-on of described polyoxyethylene glycol is 3-6%(w/w).
Level pad 1 described in step (3) consists of 0.01M-0.02MTRIS, 0.1M-0.15M sodium-chlor, and the pH value of level pad 1 is 6.50-7.50.
Heparin affinity column filler used described in step (5) is HeparinSepharose6FF or HeparinSepharoseCL-6B; Level pad composition 2 described in step (5) is 0.01M-0.02M Trisodium Citrate, 0.1M-0.2M sodium-chlor, and the PH of level pad 2 is 6.50-7.50; Described lavation buffer solution consists of 0.01M-0.02M Trisodium Citrate, 0.25-0.5M sodium-chlor, and the PH of described lavation buffer solution is 6.50-7.50; Described elution buffer consists of 0.01M-0.02M Trisodium Citrate, 1M-2M sodium-chlor, and the PH of described elution buffer is 6.50-7.50.
Stablizer described in step (7) is glycine, and the add-on of stablizer is 0.5-5%(w/w).
The preparation method of the people's Antithrombin Ⅲ described in step (1) ~ (11), comprises S/D, nano-film filtration and xeothermic three step viruses and goes out except method.
advantage of the present invention
1, this technique eliminates the thrombin that vitamin K residual in raw material relies on, and eliminates thrombogen especially, greatly reduces the probability of protein activation in production process, improve conforming product rate;
2, adopt polyoxyethylene glycol to remove foreign protein, for the operation of follow-up column chromatography provides preferably loading condition, decrease the load of chromatography column;
3, polyoxyethylene glycol is a kind of protein protective agent, can prevent protein denaturation inactivation in process of production;
4, adopt three step virus inactivating methods, the Clinical practice security of product is improved greatly.
Embodiment
Below in conjunction with accompanying drawing and embodiment, the present invention is further illustrated; embodiment is just in order to make those skilled in the art understand the present invention better; unintelligible is restriction to right protection scope of the present invention; on the contrary; for the common scientific research personnel of this area; the change of any unsubstantiality that all the present invention of utilization carry out or adjustment, all should fall within protection scope of the present invention.
Embodiment one
1,3kg component I V precipitation put in 30kg damping fluid, damping fluid consists of 0.02MHCL-TRIS, 0.15M sodium-chlor, PH7.50-7.60, and temperature controls at 10 DEG C, stirs 6 hours, makes it fully dissolve;
2, in above suspension, add polyoxyethylene glycol to 6%, stir 0.5 hour, then to connect 0.45 μm of filter element filtering with the Supradur50P filter plate that Solution produces, collect clear filtrate, with dissolving damping fluid prewashing filter plate and filter core before filtering;
3, add DEAESephadexA-50 gel that is swelling in advance and balance in above filtrate, add 33g by dry glue, damping fluid consists of 0.02MTRIS-HCL, 0.15M sodium-chlor, PH6.50-6.50; Slow stirring 1.5 hours, latter standing 5 minutes; Then filter, collect filtrate;
4, add Tween80 to 1.0%(wt% in above filtrate), TNBP(tributyl phosphate) to 0.3%(wt%), be warming up to 24-26 DEG C after stirring evenly, rear insulation 6 hours, then use 0.45 μm of filter core clarification filtration;
5, the upper HeparinSepharose6FF chromatography column of above filtrate, pillar fully balances with level pad in advance, level pad is 0.01M Trisodium Citrate, 0.15M sodium chloride solution (PH6.50-6.60), rear lavation buffer solution (0.01M Trisodium Citrate, 0.3M sodium chloride solution, PH6.50-6.60) wash, then use elution buffer (0.01M Trisodium Citrate, 1.5M sodium chloride solution, PH6.50-6.60) wash-out, collect elutriant, with 0.45 μm of filter element filtering, collect filtrate;
6, with the ultra-filtration membrane (0.1M of 10K molecular weight cut-off
2) concentrated above filtrate, then constant volume dialyses 5 times, shifts out ultra-filtration membrane Bao Binghou and wash film bag after again concentrating, and surveys AT-III and tires as 42.8IU/ml;
7, regulate AT-III to tire to 35IU/ml, add glycine to 1%, rear tune pH value is to 6.90-7.10;
8, carry out except virus filtration with 0.1 μm of filter core, 20 nanometer filter cores of connecting
9, with 0.22 μm of filter core, Sterile Filtration is carried out and packing to product;
10, freeze-drying;
11, in 100 DEG C of boiling water baths, be incubated 30 minutes, carry out xeothermic inactivation of virus.
Embodiment two,
1,3kg component I V precipitation put in 60kg damping fluid, damping fluid consists of 0.01MHCL-TRIS, 0.15M sodium-chlor, PH8.50-8.60, and temperature controls at 20 DEG C, stirs 4 hours, makes it fully dissolve;
2, in above suspension, adding polyoxyethylene glycol to 3%, stir 1.5 hours, to connect 0.45 μm of filter element filtering with the Supradur50P filter plate that Solution produces afterwards, collecting clear filtrate, with dissolving damping fluid prewashing filter plate and filter core before filtering;
3, add DEAESephadexA-50 gel that is swelling in advance and balance in above filtrate, add 33g by dry glue, damping fluid consists of 0.01MTRIS-HCL, 0.15M sodium-chlor, PH6.90-7.10; Slow stirring 1 hour, latter standing 10 minutes; Then with filtering, filtrate is collected;
4, with embodiment one;
5, the upper HeparinSepharose6FF chromatography column of above filtrate, pillar fully balances with level pad in advance, level pad is 0.02M Trisodium Citrate, 0.15M sodium chloride solution (PH6.90-7.10), rear lavation buffer solution (0.02M Trisodium Citrate, 0.5M sodium chloride solution, PH6.90-7.10) wash, then use elution buffer (0.02M Trisodium Citrate, 2M sodium chloride solution, PH6.90-7.10) wash-out, collect elutriant, with 0.45 μm of filter element filtering, collect filtrate;
6, with the ultra-filtration membrane (0.1M of 10K molecular weight cut-off
2) concentrated above filtrate, then constant volume dialyses 6 times, shifts out ultra-filtration membrane Bao Binghou and wash film bag after again concentrating, and surveys AT-III and tires as 44.3IU/ml;
7, regulate AT-III to tire to 35IU/ml, add glycine to 3%, rear tune pH value is to 6.90-7.10;
8-11, with embodiment one.
Embodiment three,
1,3kg component I V precipitation put in 45kg damping fluid, damping fluid consists of 0.02MHCL-TRIS, 0.1M sodium-chlor, PH9.40-9.50, and temperature controls at 25 DEG C, stirs 2.5 hours, makes it fully dissolve;
2, in above suspension, add polyoxyethylene glycol to 4.5%, stir 1 hour, then to connect 0.45 μm of filter element filtering with the Supradur50P filter plate that Solution produces, collect clear filtrate, with dissolving damping fluid prewashing filter plate and filter core before filtering;
3, add DEAESephadexA-50 gel that is swelling in advance and balance in above filtrate, add 36g by dry glue, damping fluid consists of 0.02MTRIS-HCL, 0.1M sodium-chlor, PH7.40-7.50; Slow stirring 0.5 hour, latter standing 15 minutes; Then filter, collect filtrate;
4, with embodiment one;
5, the upper HeparinSepharoseCL-6B chromatography column of above filtrate, pillar fully balances with level pad in advance, level pad is 0.02M Trisodium Citrate, 0.15M sodium chloride solution (PH7.40-7.50), rear lavation buffer solution (0.02M Trisodium Citrate, 0.35M sodium chloride solution, PH7.40-7.50) wash, then use elution buffer (0.02M Trisodium Citrate, 2M sodium chloride solution, PH7.40-7.50) wash-out, collect elutriant, with 0.45 μm of filter element filtering, collect filtrate;
6, with the ultra-filtration membrane (0.1M of 5K molecular weight cut-off
2) concentrated above filtrate, then constant volume dialyses 8 times, shifts out ultra-filtration membrane Bao Binghou and wash film bag after again concentrating, and surveys AT-III and tires as 40.9IU/ml;
7, regulate AT-III to tire to 35IU/ml, add glycine to 1.5%, rear tune pH value is to 6.90-7.10;
8-11, with embodiment one.
table preproduction AT-III yield and specific activity table look-up
Accompanying drawing explanation
Fig. 1 is preparation technology's schema of people's Antithrombin III.
Claims (8)
1. a preparation method for people's Antithrombin III, is characterized in that: comprise the steps:
(1) plasma component IV resolution of precipitate
In the ratio of 1:10-20, component I V precipitation dropped into and dissolve in damping fluid, temperature controls at 10-30 DEG C, stirs 2-6 hour, makes it fully dissolve, obtain unit for uniform suspension;
(2) polyoxyethylene glycol (PEG) precipitates foreigh protein removing
In the suspension described in step (1), add 40-50%(w/w) polyoxyethylene glycol, stir 0.5-1.5 hour, then to connect 0.45 μm of filter element filtering with the Supradur50P filter plate that Solution produces, collect clear filtrate, with dissolving damping fluid prewashing filter plate and filter core before filtering;
(3) DEAESephadexA-50 gel adsorption
DEAESephadexA-50 gel is added in the filtrate collected by step (2), the add-on of xerogel is 0.5-1.0g/kg, xerogel is swelling with the hot water for injection of more than 70 DEG C in advance and with the cold water for injection cooling of less than 25 DEG C, then uses equilibration buffer; Slow stirring 0.5-1.5 hour, rear standing 5-15 minute; Filter, collect filtrate; Gel is reused after zeolite regeneration;
(4) S/D inactivation of virus
Tween80 to 1.0%(wt% is added in the collected filtrate of step (3)), TNBP(tributyl phosphate) to 0.3%(wt%), be warming up to 24-26 DEG C after stirring evenly, rear insulation 6-7 hour, then uses 0.45 μm of filter core clarification filtration;
(5) heparin affinity column chromatography
The upper heparin affinity column of filtrate after S/D, pillar fully balances with level pad in advance, upper prop terminates first to use Equilibration buffer wash pillar afterwards, pillar is washed afterwards with lavation buffer solution, use elution buffer wash-out pillar again, collect elutriant, with 0.45 μm or 0.22 μm of above elutriant of filter element filtering, gained filtrate is high-purity AT-III solution;
(6) ultrafiltration and concentration
Concentrate above filtrate with the ultra-filtration membrane of 5K ~ 10K, then constant volume dialysis 4-6 doubly, tire higher than 40IU/ml to AT-III by reconcentration; After shift out ultra-filtration membrane Bao Binghou and wash film bag, collect concentrated solution;
(7) stablizer and adjustment is added
In the concentrated solution collected by step (6), add stablizer, regulate AT-III to tire to desirable value (general requirements is 30-40IU/ml), rear tune pH value is to 6.50-7.50;
(8) nanometer film is except virus filtration
Carry out except virus filtration with 0.1 μm of filter core, 20 nanometer filter cores of connecting;
(9) Sterile Filtration packing
With 0.22 μm of filter core, Sterile Filtration is carried out and packing to product;
(10) freeze-drying;
(11) xeothermic inactivation of viruses
Freeze-dried preparation step (10) obtained is incubated 30 minutes in 100 DEG C of boiling water baths, carries out xeothermic inactivation of virus.
2. according to the preparation method of a kind of people's Antithrombin Ⅲ according to claim 1, it is characterized in that: the dissolving damping fluid described in step (1) consists of 0.01M-0.02MTRIS, 0.1M-0.15M sodium-chlor, the pH value of described dissolving damping fluid is 7.00-9.50.
3. according to the preparation method of a kind of people's Antithrombin Ⅲ according to claim 1, it is characterized in that: the molecular weight of the polyoxyethylene glycol described in step (2) is 2000-6000, the add-on of described polyoxyethylene glycol is 3-6%(w/w).
4. according to the preparation method of a kind of people's Antithrombin Ⅲ according to claim 1, it is characterized in that: the level pad 1 described in step (3) consists of 0.01M-0.02MTRIS, 0.1M-0.15M sodium-chlor, the pH value of level pad 1 is 6.50-7.50.
5. according to the preparation method of a kind of people's Antithrombin Ⅲ according to claim 1, it is characterized in that: the heparin affinity column filler used described in step (5) is HeparinSepharose6FF or HeparinSepharoseCL-6B.
6. according to the preparation method of a kind of people's Antithrombin Ⅲ according to claim 1, it is characterized in that: the level pad composition 2 described in step (5) is 0.01M-0.02M Trisodium Citrate, 0.1M-0.2M sodium-chlor, and the PH of level pad 2 is 6.50-7.50; Described lavation buffer solution consists of 0.01M-0.02M Trisodium Citrate, 0.25-0.5M sodium-chlor, and the PH of described lavation buffer solution is 6.50-7.50; Described elution buffer consists of 0.01M-0.02M Trisodium Citrate, 1M-2M sodium-chlor, and the PH of described elution buffer is 6.50-7.50.
7. according to the preparation method of a kind of people's Antithrombin Ⅲ according to claim 1, it is characterized in that: the stablizer described in step (7) is glycine, the add-on of stablizer is 0.5-5%(w/w).
8. according to the preparation method of a kind of people's Antithrombin Ⅲ described in claim 1-7, it is characterized in that: product goes out except method through S/D, nano-film filtration and xeothermic three step viruses.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105669858A (en) * | 2016-02-23 | 2016-06-15 | 兰州生物制品研究所有限责任公司 | Method for extracting antithrombase III and multiple kinds of functional protein from plasma Cohn method component IV sediment |
| CN113584006A (en) * | 2021-08-20 | 2021-11-02 | 华兰生物工程股份有限公司 | Freeze-dried human prothrombin complex and preparation method thereof |
| CN114395032A (en) * | 2022-02-24 | 2022-04-26 | 广东双林生物制药有限公司 | A method for extracting human antithrombin Ⅲ from plasma |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5679776A (en) * | 1989-09-05 | 1997-10-21 | Centre Regional De Transfusion Sanguine De Lille | Process for preparing a concentrate of blood coagulation factor VIII-von willebrand factor complex from total plasma |
| CN1189502A (en) * | 1996-11-20 | 1998-08-05 | 株式会社绿十字 | Method for producing antithrombin-III, method for purifying it, and preparation containing it |
| CN102977207A (en) * | 2012-12-14 | 2013-03-20 | 成都蓉生药业有限责任公司 | Method for preparing antithrombin |
| CN103059129A (en) * | 2013-01-28 | 2013-04-24 | 贵州泰邦生物制品有限公司 | Method for preparing human antithrombin-III product |
| CN104672328A (en) * | 2015-02-13 | 2015-06-03 | 山东泰邦生物制品有限公司 | Production method of human antithrombin III |
-
2015
- 2015-11-17 CN CN201510786133.0A patent/CN105315365A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5679776A (en) * | 1989-09-05 | 1997-10-21 | Centre Regional De Transfusion Sanguine De Lille | Process for preparing a concentrate of blood coagulation factor VIII-von willebrand factor complex from total plasma |
| CN1189502A (en) * | 1996-11-20 | 1998-08-05 | 株式会社绿十字 | Method for producing antithrombin-III, method for purifying it, and preparation containing it |
| CN102977207A (en) * | 2012-12-14 | 2013-03-20 | 成都蓉生药业有限责任公司 | Method for preparing antithrombin |
| CN103059129A (en) * | 2013-01-28 | 2013-04-24 | 贵州泰邦生物制品有限公司 | Method for preparing human antithrombin-III product |
| CN104672328A (en) * | 2015-02-13 | 2015-06-03 | 山东泰邦生物制品有限公司 | Production method of human antithrombin III |
Non-Patent Citations (1)
| Title |
|---|
| 顾其胜: "《玻璃酸钠生产与临床应用》", 31 May 2012, 上海科学技术出版社 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105669858A (en) * | 2016-02-23 | 2016-06-15 | 兰州生物制品研究所有限责任公司 | Method for extracting antithrombase III and multiple kinds of functional protein from plasma Cohn method component IV sediment |
| CN105669858B (en) * | 2016-02-23 | 2019-07-26 | 兰州生物制品研究所有限责任公司 | A method for extracting antithrombin III and various functional proteins from plasma Cohn method fraction IV precipitation |
| CN113584006A (en) * | 2021-08-20 | 2021-11-02 | 华兰生物工程股份有限公司 | Freeze-dried human prothrombin complex and preparation method thereof |
| CN114395032A (en) * | 2022-02-24 | 2022-04-26 | 广东双林生物制药有限公司 | A method for extracting human antithrombin Ⅲ from plasma |
| CN114395032B (en) * | 2022-02-24 | 2024-11-26 | 广东双林生物制药有限公司 | A method for extracting human antithrombin III from plasma |
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