CN104672328A - Production method of human antithrombin III - Google Patents
Production method of human antithrombin III Download PDFInfo
- Publication number
- CN104672328A CN104672328A CN201510078211.1A CN201510078211A CN104672328A CN 104672328 A CN104672328 A CN 104672328A CN 201510078211 A CN201510078211 A CN 201510078211A CN 104672328 A CN104672328 A CN 104672328A
- Authority
- CN
- China
- Prior art keywords
- plasma
- antithrombin iii
- goods
- sodium
- gel
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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
Landscapes
- 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)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a production method of human antithrombin III. The production method comprises the following steps: removing cryoprecipitate from fresh frozen human plasma to obtain a plasma supernatant, and carrying out adsorption treatment on the plasma supernatant by using a DEAE Sephadex-A50 gel; precipitating impurity protein of the plasma supernatant, and deeply filtering plasma; carrying out Capto Heparin affinity chromatography; carrying out S/D virus inactivation and Capto Q ion exchange chromatography, and removing an S/D reagent; and filtering by virtue of a nanometer film to remove viruses, preparing, freeze-drying and dry-heating. The process is capable of preparing the antithrombin III by virtue of affinity chromatography with only one step. According to the production method disclosed by the invention, the recovery rate can reach above 40 percent, and the specific activity can reach 8-10 IU/mg.
Description
Technical field
The present invention relates to biological products and blood products production technical field, relate generally to Human Antithrombin Ⅲ's separation purification method in blood products production.
Background technology
Human Antithrombin Ⅲ is α
2sphaeroprotein, molecular weight, between 58000 ~ 65000Da, is anticoagulant substances important in human body, can play blood coagulation resisting function under the condition not having heparin.Antithrombin Ⅲ is 100 ~ 300 μ g/ml at normal adults Plasma, in human body antithrombin Ⅲ level lower than normal level 70% time, thrombotic risk can be made to raise.Human Antithrombin Ⅲ's product is applicable to treatment anticoagulation enzymoprivic patient, as inborn genetic and acquired antithrombin shortage, dispersivity blood vessel internal hemorrhage (DIC), septicemia, wound, tumour, thromboembolism, complicated pregnancy etc.
Within 1975, Thaler uses heparin affinity chromatography and polyoxyethylene glycol (PEG) precipitation to carry out plasma purification, add ammonium sulfate precipitation removing PEG again; But via the activity also containing foreign protein Antithrombin Ⅲ in the antithrombin Ⅲ of the method purifying.Major part and heparin-bounding nonspecific proteins in Mckay T 500 removing blood plasma in 1981, then be separated antithrombin Ⅲ with heparin affinity chromatography, but specific activity can only reach 4IU/mg.Above-mentioned two kinds of techniques are all starting raw material with blood plasma, by adding the foreign protein in protein precipitant removal blood plasma, complex operation step, foreign protein removal effect is bad, cause that product purity is low, specific activity is low, and affect the extraction of other albumen in blood plasma, be unfavorable for the comprehensive utilization of blood plasma.
Within 1989, Hoffman is precipitated as raw material with Cohn FIV-1, and precipitation is redissolved the supernatant liquor after clarifying through twice heparin affinity chromatography, antithrombin Ⅲ specific activity reaches 6.4IU/mg.Existing patent (the CN201210544111X disclosing and authorized at home, CN2013100304464, CN2013106047238) in production technique, it is no matter the operational path that is raw material with F IV, or take blood plasma as the operational path of raw material, more than twice or twice heparin affinity chromatography is all adopted to prepare antithrombin Ⅲ, to reach the high purity of end article antithrombin Ⅲ and high specific activity standard.Owing to all not carrying out clarification pre-treatment to initial feed liquid in above-mentioned technique, twice affinity chromatography therefore must be used to carry out purifying, and guarantee product has higher purity and specific activity.But the gel price general charged of affinity chromatography is expensive, and production cost is high, and while increasing by a step affinity chromatography, also improve the risk that affinity ligand comes off, bring hidden danger to quality product.
Summary of the invention
The object of the invention is to the deficiency for existing in current Human Antithrombin Ⅲ's production technique, a kind of processing method only needing a step affinity chromatography can obtain high specific activity antithrombin Ⅲ is provided.
The technical solution adopted in the present invention is as follows:
(1) Fresh Frozen human plasma centrifugal segregation cryoprecipitate, DEAE Sephadex-A50 gel adsorption; (2) plasma supernatant precipitated impurities albumen, Depth Filtration; (3) filtered solution Capto Heparin affinity chromatography; (4) S/D reagent inactivation of virus, Capto Q ion exchange chromatography removes S/D reagent; (5) nano-film filtration removes virus, preparation, freeze-drying, xeothermic.
Step (1) Fresh Frozen human plasma centrifugal segregation cryoprecipitate described in the present invention, DEAE Sephadex-A50 gel adsorption; Concrete step is as follows:
After Fresh Frozen human plasma is melted mixing, remove the cryoprecipitate in blood plasma through continuous flow centrifuge, obtain centrifugal after plasma supernatant;
Plasma supernatant adopts the filter element filtering of 1 ~ 10 μm, regulates blood plasma pH to 6.80 ~ 7.20; Add the DEAE Sephadex-A50 gel whip attachment that balanced 30 ~ 60 minutes, adsorption temp≤15 DEG C; Adopt 0.2 ~ 1 μm of micropore filter element to filter, filtering gel obtains the plasma supernatant after adsorption treatment; The dry glue amount of DEAE Sephadex-A50 1.0 ~ 2.0g/kg blood plasma; 0.05 ~ 0.1mol/L sodium chloride solution melts swollen >=8 hour; Balance 3 ~ 5 times; Regenerate with the process of 1.0 ~ 2.0mol/L sodium chloride solution, 20% ethanol is preserved; Regeneration times is no more than 10 times; Described balance liquid: 0.015 ~ 0.025mol/L Sodium Citrate, 0.14 ~ 0.20mol/L sodium-chlor, pH 6.8 ~ 7.2, temperature 2 ~ 8 DEG C;
Add the washings of 1 ~ 2 times of gel volume, agitator treating 1 ~ 3 time, after adopting 0.2 ~ 1 μm of micropore filter element to filter washings, merge with the plasma supernatant after filtering gel, obtain the plasma supernatant after processing; Washings: 0.015 ~ 0.025mol/L Sodium Citrate, 0.14 ~ 0.20mol/L sodium-chlor, pH 6.80 ~ 7.20, temperature 2 ~ 8 DEG C.
Step (2) plasma supernatant precipitated impurities albumen described in the present invention, Depth Filtration; Concrete steps are as follows:
Plasma supernatant precipitated impurities albumen
First plasma supernatant after process is warming up to 8 ~ 10 DEG C, and adding total NaCl concentration in NaCl to plasma supernatant is 60 ~ 100mmol/L, stirs, and adjusting plasma supernatant pH with the hydrochloric acid of 0.1 ~ 0.3mol/L or sodium hydroxide is 6.80 ~ 7.00; Be cooled to 4 ~ 6 DEG C, add that to add total NaCl concentration in NaCl to plasma supernatant be 120 ~ 200mmol/L, stir, adjusting plasma supernatant pH with the hydrochloric acid of 0.1 ~ 0.3mol/L or sodium hydroxide is 7.40 ~ 7.50; Be cooled to 0 ~ 2 DEG C, after stirring 10 ~ 30min, leave standstill 30 ~ 60min;
Depth Filtration
The filtering material adopted is medicinal rank Mierocrystalline cellulose filter plate, can be that a kind of individual layer filter plate of specification or the filter plate overlapped in series of several specification use; Filter plate model selects PEK1, PEKS, PDH4, PDE2, PDE1 as PALL, 30SP, the 60SP of 3M, 90SP, 30LA, 60 LA, 90 LA, and the filter plate of other models of similar effect, or its combination;
Use the solution containing 0.1% ~ 10%EDTA to clean filtering material, consumption is 100 ~ 300L/m
2; Use the liquor sodii citratis of 0.1% ~ 10% to clean filtering material again, and wash away residual EDTA, consumption is 100 ~ 300L/m
2; The solution in equipment is drained, for plasmapheresis after having rinsed.
Plasma supernatant after filtration, while carrying out column chromatography, uses the filter core of 0.2 μm to carry out online membrane filtration.
Wherein, step (3) filtered solution carries out Capto Heparin affinity chromatography and is specially:
Filtered solution after Depth Filtration, is fixed an affinity chromatography by the chromatography column of the heparin affinity gel being filled with Capto Heparin, and the heparin be combined in gel on agarose pedestal will catch the antithrombin Ⅲ in blood plasma; By other plasma component washes clean that balance liquid will not adsorb in chromatography column, with elution by the antithrombin Ⅲ adsorbed; It is 20 ~ 30 column volumes that each applied sample amount controls; Described balance liquid: phosphoric acid disodium hydrogen 10 ~ 30mmol/L, sodium-chlor 0.3 ~ 0.6mol/L, pH6.50 ~ 7.50; Described elutriant: phosphoric acid disodium hydrogen 10 ~ 30mmol/L, sodium-chlor 1.5 ~ 2.5mol/L, pH6.50 ~ 7.50; Balance, loading and wash-out linear flow speed 180cm/h ~ 300cm/h; Column regeneration: process with 0.1mol/L sodium hydroxide, elutriant, water for injection respectively; Post is preserved: 20% ethanolic soln is preserved.
Step (4) S/D reagent inactivation of virus described in the present invention, Capto Q ion exchange chromatography removes S/D reagent; Concrete steps are as follows:
Collect the goods obtained from wash-out Capto Heparin heparin chromatography post, carry out ultrafiltration, desalination and concentrate, ultra-filtration membrane aperture 10KDa, described ultrafiltration diluent: phosphoric acid disodium hydrogen 10 ~ 30mmol/L, pH6.50 ~ 7.50; After ultrafiltration, goods conductivity value is not higher than 5mS/cm, goods protein concn≤20.0mg/ml; Antithrombin Ⅲ solution precision after ultrafiltration is not less than 1 μm of filter element filtering, carry out S/D method viral inactivation treatment, method is: Polysorbate 80 content should be 1.0 ± 0.3% (g/ml), tributyl phosphate content should be 0.3 ± 0.1% (g/ml), goods protein concn≤20.0mg/ml, inactivation temperature 24 ± 1 DEG C, maintains 6 hours;
The Capto Q anion-exchange chromatography post of goods after S/D deactivation by having balanced, goods are attracted on chromatography column, and the S/D reagent stream added passes chromatography column, rinse chromatography column with balance liquid, the S/D reagent of wash residual, then elute with the goods of elutriant by absorption; Balance liquid: phosphoric acid disodium hydrogen 10 ~ 30mmol/L, pH6.50 ~ 7.50; Elutriant: phosphoric acid disodium hydrogen 10 ~ 30mmol/L, sodium-chlor 0.4 ~ 1.0mol/L, pH6.50 ~ 7.50; Balance, loading and wash-out linear flow speed 180cm/h ~ 300cm/h; Column regeneration: process with 0.3mol/L sodium hydroxide, elutriant, water for injection respectively; Post is preserved: 20% ethanolic soln or 0.01mol/L NaOH preserve.
Step of the present invention (5) nano-film filtration removes virus, preparation, freeze-drying, xeothermic; Concrete steps are as follows:
Collect the goods from wash-out ion exchange column, carry out ultrafiltration desalination and concentrate, ultra-filtration membrane aperture 10KDa, goods protein content 10.0 ~ 20.0mg/ml, after ultrafiltration, goods carry out pre-filtering with the filter membrane of 0.1 μm, are then undertaken except virus filtration by the nanometer film of 20nm; 100 DEG C are undertaken, the xeothermic inactivation of viruses process of 30min by the preparation of product specification, freeze-drying, goods.
The invention has the beneficial effects as follows:
(1) with the blood plasma after DEAE Sephadex-A50 gel adsorption for raw material, do not affect the production of cryoprecipitate and Human Factor Ⅸ Complex in blood plasma production line, do not affect the production of other products such as the white and immunoglobulin (Ig) of follow-up people yet.And thrombogen in the blood plasma after DEAE Sephadex-A50 gel adsorption, VII, Ⅸ, the content of Ⅹ factor is extremely low, heparin affinity gel can be avoided the non-specific adsorption of above-mentioned thrombin, improve the purity of target protein antithrombin Ⅲ and the carrying capacity of gel.
(2) blood plasma clarifying treatment does not use the protein precipitants such as polyoxyethylene glycol (PEG), ammonium sulfate, T 500, does not introduce Adventitious impurities.By adjustment salt concn, pH value, the conditions such as temperature, again through the membrane filtration of Depth Filtration and 0.2 μm, just can remove the impurity protein that in blood plasma, major part is combined with heparin affinity chromatography gel, make blood plasma feed liquid reach required by fixed bed column chromatography close to the filtering accuracy of 0.2 μm and clarity, effectively can not only improve the linear velocity in loading process, also can the specific activity of improving product and gel carrying capacity further, the protection to gel filler can be realized further simultaneously, guarantee that the number of times of reusing of expensive Capto Heparin heparin affinity gel reaches more than 200 times.
(3) blood plasma is after removing the clarifying treatment such as cryoprecipitate, DEAE Sephadex-A50 gel adsorption, impurity protein precipitation and Depth Filtration, only need a step Capto Heparin gel heparin affinity chromatography, can realize antithrombin Ⅲ specific activity in elutriant and reach 8 ~ 10IU/mg, purity is close to 100%.
(4) Capto Q gel is for removing the S/D reagent in the middle of antithrombin Ⅲ in product.This gel carrying capacity is large, and process is convenient, simple to operate, and the single step rate of recovery reaches more than 95%, does not affect purity and the specific activity of middle product.
(5) through antithrombin Ⅲ finished product prepared by this technique, total yield can reach more than 40%, and specific activity can reach 8 ~ 10IU/mg, and purity is close to 100%.
(6) add the inactivation of virus/removal step of S/D reagent, nano-film filtration and xeothermic three kinds of different mechanisms in technological process, the fat coating that may be able to occur in fully deactivation/removal goods and non-lipid-coated virus, ensure that the security of goods.
Embodiment
In order to understand the present invention better, describe technical scheme of the present invention in detail with specific examples below, but the present invention is not limited thereto.
embodiment 1
1 Fresh Frozen human plasma centrifugal segregation cryoprecipitate, DEAE Sephadex-A50 gel adsorption;
1.1 plasma removing cryoprecipitates
Fresh frozen plasma through feeding intake, melt slurry, centrifugation cryoprecipitate, collect centrifugal after blood plasma.In process, blood plasma temperature is no more than 4 DEG C, and whizzer fluid speed is no more than 2L/min.
1.2 plasma D EAE Sephadex-A50 gel adsorption
The blood plasma removing cryoprecipitate adopts the filter element filtering of 1 μm.Regulate blood plasma pH to 7.00; Add the A-50 gel whip attachment 45min balanced, adsorption temp 4 DEG C; Filtering gel.Balance liquid: 0.01mol/L Sodium Citrate, 0.20mol/L sodium-chlor, pH 7.00,4 DEG C.
Add the washings of 1 times of gel volume, agitator treating 2 times, after being adopted by washings 1 μm of micropore filter element to filter, merge with the plasma supernatant after filtering gel, obtain the plasma supernatant after processing.Washings: 0.01mol/L Sodium Citrate, 0.20mol/L sodium-chlor, pH 7.00,4 DEG C.
2 plasma supernatant precipitated impurities albumen, Depth Filtration
2.1 plasma supernatant precipitated impurities albumen
First plasma supernatant is warming up to 10 DEG C, and adding total NaCl concentration in NaCl to plasma supernatant is 65mmol/L, stirs, and is 6.80 with the hydrochloric acid adjustment plasma supernatant pH of 0.3mol/L; Be cooled to 5 DEG C, adding total NaCl concentration in interpolation NaCl to plasma supernatant is 200mmol/L, stirs, and is 7.40 with 0.3mol/L sodium hydroxide adjustment plasma supernatant pH; Be cooled to 1 DEG C, after stirring 30min, leave standstill 60min.
2.2 blood plasma Depth Filtrations
Select the PEK1 filter plate of PALL.Use the aqueous solution containing 1%EDTA to clean filtering material, consumption is 100L/m
2.Again with 1% liquor sodii citratis to carry out cleaning consumption to filtering material be 100L/m
2, drain the solution in equipment after having rinsed, carry out plasmapheresis.
Blood plasma after filtration, while carrying out column chromatography, uses the duplicature filter core of 0.2 μm of the EKV model of PALL to carry out online membrane filtration.
3 filtered solution Capto Heparin affinity chromatographys
Filtered solution after Depth Filtration, by the Capto Heparin heparin affinity chromatography post balanced.By other plasma component washes clean that balance liquid will not adsorb in chromatography column, with elution by the antithrombin Ⅲ adsorbed.It is 25 column volumes that blood plasma applied sample amount controls.Balance liquid: phosphoric acid disodium hydrogen 20mmol/L, sodium-chlor 0.5mol/L, pH7.00; Elutriant: phosphoric acid disodium hydrogen 20mmol/L, sodium-chlor 2.0mol/L, pH7.00; Balance, loading and wash-out linear flow speed 240cm/h.Column regeneration: process with 0.1mol/L sodium hydroxide, elutriant, water for injection respectively.Post is preserved: 20% ethanolic soln.
4S/D reagent inactivation of virus, Capto Q ion exchange chromatography removes S/D reagent
4.1 ultrafiltration desalinations
Collect the goods obtained from wash-out chromatography column, carry out ultrafiltration (ultra-filtration membrane aperture 10KDa) desalination and concentrate.Ultrafiltration diluent: phosphoric acid disodium hydrogen 10mmol/L pH7.00.After ultrafiltration, goods conductivity value is not higher than 4mS/cm, goods protein concn≤20.0mg/ml.
4.2 S/D method inactivation of virus
S/D method viral inactivation treatment is carried out after antithrombin Ⅲ solution precision after ultrafiltration is not less than 1 μm of filter element filtering.Method is: Polysorbate 80 content should be 1.0% (g/ml), and tributyl phosphate content should be 0.3% (g/ml), goods protein concn 20.0mg/ml, inactivation temperature 24 DEG C, maintains 6 hours.
4.3Capto Q ion exchange chromatography
The Capto Q anion-exchange chromatography post of goods after S/D deactivation by having balanced; Goods are attracted on chromatography column, and the S/D reagent added and a small amount of foreign protein stream pass chromatography column.Chromatography column is rinsed, the S/D reagent of wash residual and foreign protein with balance liquid.Elute with the goods of elutriant by absorption again.Balance liquid: phosphoric acid disodium hydrogen 20mmol/L, pH7.00; Elutriant: phosphoric acid disodium hydrogen 20mmol/L, sodium-chlor 0.8mol/L, pH7.00; Balance, loading and wash-out linear flow speed 240cm/h.Column regeneration: process with 0.3mol/L sodium hydroxide, elutriant, water for injection respectively.Post is preserved: 20% ethanolic soln.
5 inactivation of viruses, preparation, freeze-drying, xeothermic
5.1 ultrafiltration, desalination
Collect the goods from wash-out ion exchange column, carry out ultrafiltration (ultra-filtration membrane aperture 10KDa) desalination and concentrate, control goods protein content 10.0mg/ml.
5.2 virus removals (nano-film filtration)
The goods egg filter membrane of 0.1 μm carries out pre-filtering, is then undertaken except virus filtration by the nanometer film of 20nm.Nanometer film before use, after all should carry out integrity test.
5.3 work in-process preparations, degerming, packing, freeze-drying, xeothermic deactivation, is undertaken 100 DEG C by the preparation of product specification, freeze-drying, goods, the xeothermic inactivation of viruses process of 30min.
In production technique, the antithrombin Ⅲ rate of recovery in each stage is in table 1.
The antithrombin Ⅲ rate of recovery in each stage in table 1 production technique
In production technique, the antithrombin Ⅲ specific activity in each stage is in table 2.
The antithrombin Ⅲ specific activity in each stage in table 2 production technique
embodiment 2
Blood plasma Depth Filtration
Filtering material selects 30LA, 60LA combination of 3M.Use the aqueous solution containing 5%EDTA to clean filtering material, consumption is 200L/m
2.Again with 5% liquor sodii citratis to carry out cleaning consumption to filtering material be 200L/m
2.Wherein first use 50L/m
2liquor sodii citratis rinse filter plate, then use 100L/m
2liquor sodii citratis previous solu is gone out, and circulation flushing 30min, finally uses 50L/m
2liquor sodii citratis previous solu is gone out.Drain the solution in equipment after having rinsed, carry out plasmapheresis.
Blood plasma after filtration, while carrying out column chromatography, uses the duplicature filter core of the PDA model 0.2 μm of 3M to carry out online membrane filtration.
embodiment 3
1 Fresh Frozen human plasma centrifugal segregation cryoprecipitate, DEAE Sephadex-A50 gel adsorption;
1.1 plasma removing cryoprecipitates
Fresh frozen plasma through feeding intake, melt slurry, centrifugation cryoprecipitate, collect centrifugal after blood plasma.In process, blood plasma temperature is no more than 4 DEG C, and whizzer fluid speed is no more than 1L/min.
1.2 plasma D EAE Sephadex A-50 adsorb
The blood plasma removing cryoprecipitate adopts the filter element filtering of 1 μm.Regulate blood plasma pH to 7.15; Add the A-50 gel whip attachment 60min balanced, adsorption temp 2 DEG C; Filtering gel.Balance liquid: 0.01mol/L Sodium Citrate, 0.20mol/L sodium-chlor, pH 7.15,2 DEG C.
Add the washings of 1 times of gel volume, agitator treating 2 times, after being adopted by washings 1 μm of micropore filter element to filter, merge with the plasma supernatant after filtering gel, obtain the plasma supernatant after processing.Washings: 0.01mol/L Sodium Citrate, 0.20mol/L sodium-chlor, pH 7.15,2 DEG C.
2 plasma supernatant precipitated impurities albumen, Depth Filtration
2.1 plasma supernatant precipitated impurities albumen
First plasma supernatant is warming up to 9 DEG C, and adding total NaCl concentration in NaCl to plasma supernatant is 100mmol/L, stirs, and is 7.00 with the hydrochloric acid adjustment plasma supernatant pH of 0.1mol/L; Be cooled to 4 DEG C, adding total NaCl concentration in interpolation NaCl to plasma supernatant is 200mmol/L, stirs, and is 7.50 with 0.1mol/L sodium hydroxide adjustment plasma supernatant pH; Be cooled to 1 DEG C, after stirring 30min, leave standstill 60min.
2.2 blood plasma Depth Filtrations
Select the PEK1 filter plate of PALL.Use the aqueous solution containing 0.1%EDTA to clean filtering material, consumption is 300L/m
2.Again with 0.1% liquor sodii citratis to carry out cleaning consumption to filtering material be 300L/m
2, drain the solution in equipment after having rinsed, carry out plasmapheresis.
Blood plasma after filtration, while carrying out column chromatography, uses the filter core of 0.2 μm to carry out online membrane filtration.Select 0.2 μm of duplicature filter core of the UECV model of PALL.
3 filtered solution Capto Heparin affinity chromatographys
Filtered solution after Depth Filtration, by the Capto Heparin heparin affinity chromatography post balanced.By other plasma component washes clean that balance liquid will not adsorb in chromatography column, with elution by the antithrombin Ⅲ adsorbed.It is 20 column volumes that blood plasma applied sample amount controls.Balance liquid: phosphoric acid disodium hydrogen 20mmol/L, sodium-chlor 0.55mol/L, pH7.15; Elutriant: phosphoric acid disodium hydrogen 20mmol/L, sodium-chlor 2.0mol/L, pH7.15; Balance, loading and wash-out linear flow speed 300cm/h.Column regeneration: process with 0.1mol/L sodium hydroxide, elutriant, water for injection respectively.Post is preserved: 20% ethanolic soln.
Detect the middle product obtained after Capto Heparin heparin affinity chromatography, the specific activity of antithrombin Ⅲ is 9.21IU/mg, and purity is 99.78%.
embodiment 4
Ion exchange chromatography removes S/D reagent
The Capto Q anion-exchange chromatography post of goods after S/D deactivation by having balanced.Chromatography column is rinsed, the S/D reagent of wash residual and foreign protein with balance liquid.Elute with the goods of elutriant by absorption again.Balance liquid: phosphoric acid disodium hydrogen 20mmol/L, pH7.15; Elutriant: phosphoric acid disodium hydrogen 20mmol/L, sodium-chlor 1.0mol/L, pH7.15; Balance, loading and wash-out linear flow speed 300cm/h.Column regeneration: process with 0.3mol/L sodium hydroxide, elutriant, water for injection respectively.Post is preserved: 20% ethanolic soln.
Carried out the antithrombin Ⅲ goods processed by Capto Q ion exchange chromatography, S/D amount of reagent and specific activity detected result are as table 3.
S/D reagent and specific activity detected result in goods before and after table 3 CaptoQ chromatography
。
Claims (8)
1. a Human Antithrombin Ⅲ's production method, step is as follows: (1) Fresh Frozen human plasma centrifugal segregation cryoprecipitate, DEAE Sephadex-A50 gel adsorption; (2) plasma supernatant precipitated impurities albumen, Depth Filtration; (3) filtered solution Capto Heparin affinity chromatography; (4) S/D reagent inactivation of virus, Capto Q ion exchange chromatography removes S/D reagent; (5) nano-film filtration removes virus, preparation, freeze-drying, xeothermic.
2. the production method of a kind of Human Antithrombin Ⅲ according to claim 1, is characterized in that, described step (1) Fresh Frozen human plasma centrifugal segregation cryoprecipitate, DEAE Sephadex-A50 gel adsorption; Concrete step is as follows:
After Fresh Frozen human plasma is melted mixing, remove the cryoprecipitate in blood plasma through continuous flow centrifuge, obtain centrifugal after plasma supernatant;
Plasma supernatant adopts the filter element filtering of 1-10 μm, regulates blood plasma pH to 6.80-7.20; Add the DEAE Sephadex-A50 gel whip attachment 30-60 minute balanced, adsorption temp≤15 DEG C; Adopt 0.2-1 μm of micropore filter element to filter, filtering gel obtains the plasma supernatant after adsorption treatment; The dry glue amount of DEAE Sephadex A50 is 1.0-2.0g/kg blood plasma; 0.05-0.1mol/L sodium chloride solution melts swollen >=8 hour; Balance 3-5 time; Regenerate with the process of 1.0 ~ 2.0mol/L sodium chloride solution, 20% ethanol is preserved; Regeneration times is no more than 10 times; Described balance liquid: 0.015-0.025mol/L Sodium Citrate, 0.14-0.20mol/L sodium-chlor, pH 6.80-7.20, temperature 2-8 DEG C;
Add the washings of 1-2 times of gel volume, agitator treating 1-3 time, after being adopted by washings 0.2-1 μm of micropore filter element to filter, merge with the plasma supernatant after filtering gel, obtain the plasma supernatant after processing; Described washings: 0.015-0.025mol/L Sodium Citrate, 0.14-0.20mol/L sodium-chlor, pH 6.80-7.20, temperature 2-8 DEG C.
3. the production method of a kind of Human Antithrombin Ⅲ according to claim 1, is characterized in that, described step (2) plasma supernatant precipitated impurities albumen, Depth Filtration; Concrete steps are as follows:
Plasma supernatant precipitated impurities albumen
First plasma supernatant after process is warming up to 8-10 DEG C, and adding total NaCl concentration in NaCl to plasma supernatant is 60-100mmol/L, stirs, and is 6.80-7.00 with the hydrochloric acid of 0.1-0.3mol/L or sodium hydroxide adjustment plasma supernatant pH; Being cooled to 4-6 DEG C, adding that to add total NaCl concentration in NaCl to plasma supernatant be 120-200mmol/L, stir, is 7.40-7.50 with the hydrochloric acid of 0.1-0.3mol/L or sodium hydroxide adjustment plasma supernatant pH; Be cooled to 0-2 DEG C, after stirring 10-30min, leave standstill 30-60min;
Depth Filtration
The filtering material adopted is medicinal rank Mierocrystalline cellulose filter plate, can be that a kind of individual layer filter plate of specification or the filter plate overlapped in series of several specification use; Use the solution containing 0.1%-10%EDTA to clean filtering material, consumption is 100-300L/m
2; Clean filtering material with the liquor sodii citratis of 0.1%-10%, and wash away residual EDTA, consumption is 100-300L/m
2; The solution in equipment is drained, for plasmapheresis after having rinsed;
Plasma supernatant after filtration, while carrying out column chromatography, uses the filter core of 0.2 μm to carry out online membrane filtration.
4. the production method of a kind of Human Antithrombin Ⅲ according to claim 1 or 2 or 3, is characterized in that, step (3) filtered solution Capto Heparin affinity chromatography is specially:
Filtered solution after Depth Filtration, is fixed an affinity chromatography by the chromatography column of the heparin affinity gel being filled with Capto Heparin, and the heparin be combined in gel on agarose pedestal will catch the antithrombin Ⅲ in blood plasma; By other plasma component washes clean that balance liquid will not adsorb in chromatography column, with elution by the antithrombin Ⅲ adsorbed; Each applied sample amount controls as 20-30 column volume; Described balance liquid: phosphoric acid disodium hydrogen 10-30mmol/L, sodium-chlor 0.3-0.6mol/L, pH6.50-7.50; Described elutriant: phosphoric acid disodium hydrogen 10-30mmol/L, sodium-chlor 1.5-2.5mol/L, pH6.50-7.50; Balance, loading and wash-out linear flow speed 180cm/h-300cm/h; Column regeneration: process with 0.1mol/L sodium hydroxide, elutriant, water for injection respectively; Post is preserved: 20% ethanolic soln is preserved.
5. the production method of a kind of Human Antithrombin Ⅲ according to claim 1 or 2 or 3, is characterized in that, described step (4) S/D reagent inactivation of virus, and Capto Q ion exchange chromatography removes S/D reagent; Concrete steps are as follows:
Collect the goods obtained from wash-out Capto Heparin affinity column, carry out ultrafiltration, desalination and concentrate, ultra-filtration membrane aperture 10KDa, ultrafiltration diluent: Sodium phosphate dibasic 10-30mmol/L, pH6.5-7.5; After ultrafiltration, goods conductivity value is not higher than 5mS/cm, goods protein concn≤20.0mg/ml; Antithrombin Ⅲ solution precision after ultrafiltration is not less than 1 μm of filter element filtering, carry out S/D method viral inactivation treatment, method is: Polysorbate 80 content should be 1.0 ± 0.3% (g/ml), tributyl phosphate content should be 0.3 ± 0.1% (g/ml), goods protein concn≤20.0mg/ml, inactivation temperature 24 ± 1 DEG C, maintains 6 hours;
The Capto Q anion-exchange chromatography post of goods after S/D deactivation by having balanced, goods are attracted on chromatography column, and the S/D reagent stream added passes chromatography column, rinse chromatography column with balance liquid, the S/D reagent of wash residual, then elute with the goods of elutriant by absorption; Described balance liquid: phosphoric acid disodium hydrogen 10-30mmol/L, pH6.50-7.50; Described elutriant: phosphoric acid disodium hydrogen 10-30mmol/L, sodium-chlor 0.5-1.5mol/L, pH6.50-7.50; Balance, loading and wash-out linear flow speed 180cm/h-300cm/h; Column regeneration: process with 0.3mol/L sodium hydroxide, elutriant, water for injection respectively; Post is preserved: 20% ethanolic soln is preserved.
6. the production method of a kind of Human Antithrombin Ⅲ according to claim 1 or 2 or 3, is characterized in that, described step (5) nano-film filtration removes virus, preparation, freeze-drying, xeothermic; Concrete steps are as follows:
Collect the goods from wash-out ion exchange column, carry out ultrafiltration desalination and concentrate, ultra-filtration membrane aperture 10KDa, goods protein content 10.0-20.0mg/ml, after ultrafiltration, goods carry out pre-filtering with the filter membrane of 0.1 μm, are then undertaken except virus filtration by the nanometer film of 20nm; 100 DEG C are undertaken, the xeothermic inactivation of viruses process of 30min by the preparation of product specification, freeze-drying, goods.
7. the production method of a kind of Human Antithrombin Ⅲ according to claim 3, it is characterized in that, in blood plasma Depth Filtration, described medicinal rank Mierocrystalline cellulose filter plate model selects PEK1, PEKS, PDH4, PDE2, PDE1 as PALL, the filter plate of 30SP, the 60SP of 3M, 90SP, 30LA, 60 LA, 90 LA models, or its combination.
8. the production method of a kind of Human Antithrombin Ⅲ according to claim 1, is characterized in that, through antithrombin Ⅲ prepared by this technique, the rate of recovery can reach more than 40%, and specific activity can reach 8-10IU/mg.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510078211.1A CN104672328B (en) | 2015-02-13 | 2015-02-13 | A kind of production method of Human Antithrombin Ⅲ |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510078211.1A CN104672328B (en) | 2015-02-13 | 2015-02-13 | A kind of production method of Human Antithrombin Ⅲ |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104672328A true CN104672328A (en) | 2015-06-03 |
| CN104672328B CN104672328B (en) | 2019-04-16 |
Family
ID=53307956
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510078211.1A Active CN104672328B (en) | 2015-02-13 | 2015-02-13 | A kind of production method of Human Antithrombin Ⅲ |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104672328B (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105315365A (en) * | 2015-11-17 | 2016-02-10 | 上海洲跃生物科技有限公司 | Preparation method for human antithrombin III |
| CN106085992A (en) * | 2016-06-16 | 2016-11-09 | 武汉中原瑞德生物制品有限责任公司 | A kind of improve the method for F VII yield in Human Factor Ⅸ Complex |
| CN106474461A (en) * | 2015-08-24 | 2017-03-08 | 上海洲跃生物科技有限公司 | The method preparing freeze dried human zymoplasm |
| CN106676089A (en) * | 2017-03-01 | 2017-05-17 | 广东双林生物制药有限公司 | Method for preparing human prothrombin complex from plasma |
| CN107699555A (en) * | 2017-11-17 | 2018-02-16 | 浙江丰安生物制药有限公司 | A kind of preparation method of pig blood fibrin ferment |
| CN107699554A (en) * | 2017-11-17 | 2018-02-16 | 浙江丰安生物制药有限公司 | A kind of preparation method of pig blood fibrin ferment |
| CN109678969A (en) * | 2018-12-29 | 2019-04-26 | 北京东方百泰生物科技有限公司 | A kind of purification process of CTLA4-Ig fusion protein |
| CN113801221A (en) * | 2020-06-16 | 2021-12-17 | 上海赛伦生物技术股份有限公司 | Sea snake poison resisting serum nano-membrane filtration method |
| CN114249817A (en) * | 2020-09-22 | 2022-03-29 | 四川远大蜀阳药业有限责任公司 | Method for separating and purifying human antithrombin III |
| CN114395032A (en) * | 2022-02-24 | 2022-04-26 | 广东双林生物制药有限公司 | Method for extracting human antithrombin III from blood plasma |
| CN114573687A (en) * | 2022-04-18 | 2022-06-03 | 山东泰邦生物制品有限公司 | Storage method of human antithrombin III intermediate product |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0339919B1 (en) * | 1988-04-28 | 1996-03-13 | The Green Cross Corporation | Method for purifying antithrombin-III and antithrombin-III preparation containing said antithrombin-III |
| CN102228683A (en) * | 2011-06-21 | 2011-11-02 | 湖南紫光古汉南岳制药有限公司 | Method for preparing freeze-dried human blood coagulation factor VIII |
| CN102295696A (en) * | 2011-08-16 | 2011-12-28 | 山东泰邦生物制品有限公司 | Method for preparing coagulation factor VIII, fibrinogen and fibronectin from cryoprecitation |
| CN102604920A (en) * | 2012-04-06 | 2012-07-25 | 湖南紫光古汉南岳制药有限公司 | Preparation method of human prothrombin complex |
| 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 |
| CN103613662A (en) * | 2013-11-26 | 2014-03-05 | 中国医学科学院输血研究所 | Method for separating and purifying human antithrombin-III from human plasma |
-
2015
- 2015-02-13 CN CN201510078211.1A patent/CN104672328B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0339919B1 (en) * | 1988-04-28 | 1996-03-13 | The Green Cross Corporation | Method for purifying antithrombin-III and antithrombin-III preparation containing said antithrombin-III |
| CN102228683A (en) * | 2011-06-21 | 2011-11-02 | 湖南紫光古汉南岳制药有限公司 | Method for preparing freeze-dried human blood coagulation factor VIII |
| CN102295696A (en) * | 2011-08-16 | 2011-12-28 | 山东泰邦生物制品有限公司 | Method for preparing coagulation factor VIII, fibrinogen and fibronectin from cryoprecitation |
| CN102604920A (en) * | 2012-04-06 | 2012-07-25 | 湖南紫光古汉南岳制药有限公司 | Preparation method of human prothrombin complex |
| 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 |
| CN103613662A (en) * | 2013-11-26 | 2014-03-05 | 中国医学科学院输血研究所 | Method for separating and purifying human antithrombin-III from human plasma |
Non-Patent Citations (1)
| Title |
|---|
| ANTONIETTA MORRICA等: "Manufacturing process of Anti-thrombin III concentrate:viral safety validation studies and effect of column re-use on viral clearance", 《BIOLOGICALS》 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106474461A (en) * | 2015-08-24 | 2017-03-08 | 上海洲跃生物科技有限公司 | The method preparing freeze dried human zymoplasm |
| CN106474461B (en) * | 2015-08-24 | 2019-12-27 | 上海洲跃生物科技有限公司 | Method for preparing freeze-dried human thrombin |
| CN105315365A (en) * | 2015-11-17 | 2016-02-10 | 上海洲跃生物科技有限公司 | Preparation method for human antithrombin III |
| CN106085992A (en) * | 2016-06-16 | 2016-11-09 | 武汉中原瑞德生物制品有限责任公司 | A kind of improve the method for F VII yield in Human Factor Ⅸ Complex |
| CN106676089B (en) * | 2017-03-01 | 2020-01-10 | 广东双林生物制药有限公司 | Method for preparing human prothrombin complex from blood plasma |
| CN106676089A (en) * | 2017-03-01 | 2017-05-17 | 广东双林生物制药有限公司 | Method for preparing human prothrombin complex from plasma |
| CN107699555A (en) * | 2017-11-17 | 2018-02-16 | 浙江丰安生物制药有限公司 | A kind of preparation method of pig blood fibrin ferment |
| CN107699554A (en) * | 2017-11-17 | 2018-02-16 | 浙江丰安生物制药有限公司 | A kind of preparation method of pig blood fibrin ferment |
| CN109678969A (en) * | 2018-12-29 | 2019-04-26 | 北京东方百泰生物科技有限公司 | A kind of purification process of CTLA4-Ig fusion protein |
| CN113801221A (en) * | 2020-06-16 | 2021-12-17 | 上海赛伦生物技术股份有限公司 | Sea snake poison resisting serum nano-membrane filtration method |
| CN114249817A (en) * | 2020-09-22 | 2022-03-29 | 四川远大蜀阳药业有限责任公司 | Method for separating and purifying human antithrombin III |
| CN114249817B (en) * | 2020-09-22 | 2023-11-14 | 四川远大蜀阳药业有限责任公司 | Method for separating and purifying human antithrombin III |
| CN114395032A (en) * | 2022-02-24 | 2022-04-26 | 广东双林生物制药有限公司 | Method for extracting human antithrombin III from blood plasma |
| CN114573687A (en) * | 2022-04-18 | 2022-06-03 | 山东泰邦生物制品有限公司 | Storage method of human antithrombin III intermediate product |
| CN114573687B (en) * | 2022-04-18 | 2024-01-26 | 山东泰邦生物制品有限公司 | Storage method of human antithrombin III intermediate product |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104672328B (en) | 2019-04-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104672328A (en) | Production method of human antithrombin III | |
| JP3094167B2 (en) | Purification method of immune serum globulin | |
| US4322275A (en) | Fractionation of protein mixtures | |
| US4351710A (en) | Fractionation of protein mixtures | |
| JP5704918B2 (en) | Method for purifying factor VIII and von Willebrand factor | |
| JP6012053B2 (en) | Method for isolating osteopontin using concentrated feed | |
| CN104109202B (en) | Method for adsorbing human prothrombin complex from plasma | |
| JP2013528183A5 (en) | ||
| CN107857811A (en) | A kind of preparation technology of human serum albumin | |
| US5112949A (en) | Method of and apparatus for separating proteins | |
| CN109456407B (en) | Preparation method of plasma human immunoglobulin | |
| CN106676089B (en) | Method for preparing human prothrombin complex from blood plasma | |
| CN109651502B (en) | Method for simultaneously separating and purifying blood coagulation factors IX, X and VII from human plasma | |
| CN115925890A (en) | Method for purifying anti-new coronavirus neutralizing antibody | |
| CN111378029B (en) | Preparation method of human coagulation factor IX | |
| CN106928344A (en) | Method for being reduced from the solution containing clotting factor and/or remove FXI and FXIa | |
| CN104450656A (en) | Method for preparing and purifying thrombin in rabbit blood | |
| CN109705208A (en) | A kind of technique of single step chromatography preparation high-purity vWF ELISA | |
| US7214319B2 (en) | Method of separating protein from animal milk | |
| CN103087184B (en) | Method for controlling prekallikrein activator in human serum albumin product | |
| CN113831405B (en) | Purification method of human serum albumin | |
| JP7445332B2 (en) | Optimized method for bevacizumab purification | |
| CN104004090A (en) | Preparing method for human immune globulin | |
| JP2004307344A (en) | Method for producing lactoferrin | |
| CN108441490B (en) | Process for preparing human prothrombin complex by flow adsorption method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder |
Address after: 271000 5666 Longquan Road, hi tech Zone, Tai'an, Shandong Patentee after: SHANDONG TAIBANG BIOLOGICAL PRODUCTS Co.,Ltd. Address before: 271000 No. 14 East Tiger Hill Road, Shandong, Tai'an Patentee before: SHANDONG TAIBANG BIOLOGICAL PRODUCTS Co.,Ltd. |
|
| CP02 | Change in the address of a patent holder |