CN101274956A - Method for separating and purifying alpha 1-antitrypsin from human blood plasma component FIV precipitation - Google Patents
Method for separating and purifying alpha 1-antitrypsin from human blood plasma component FIV precipitation Download PDFInfo
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Abstract
The invention discloses a method for separating and purifying alpha1-antitryptase from the FIV component precipitation of human plasma, which is characterized in that the method comprises the following steps of (A) pretreatment of plasma precipitation; (B) anion gel chromatography; (C) gel-filtration chromatography; (D) ultrafiltration desalting concentration; (E) Baculovirus inactivation; (F) lyophilization and subpackaging; (G) dry heat sterilization. The method takes the FIV component precipitation which is waste material produced from human plasma during the production process of human serum albumin as raw material and adopts chromatography technique to separate and purify a protease inhibitor, namely, alpha 1- antitryptase (alpha 1-AT) and builds the production technique of alpha 1-AT concentrate. Products prepared by the method has good pureness, high yield and simple and easy operation as well as few occupied equipment, little labor intensity, low energy consumption and low production cost due to taking the waste material generated by albumin as raw material.
Description
Technical field
The present invention relates to a kind of preparation method of alpha1-antitrypsin, especially the method for separation and purification alpha1-antitrypsin from human plasma component precipitation.
Background technology
α 1-AT mainly is by a kind of glycoprotein of liver cell synthetic, transformation period 6d in the molecular weight 54KD, body.α 1-AT is a kind of serpin the abundantest in the human plasma, can suppress multiple Serine inscribe peptase, as suppressing the activity of elastoser, trypsinase, plasmin, collagenase, zymoplasm and the Hageman factor etc.α 1-AT is a topmost antitrypsin in the blood, accounts for more than 90% of serum trypsin inhibition activity.α 1-AT has stronger vascular permeability, concentration height in lung tissue, and also more single-minded to elastase activity, its major physiological function is to suppress the activity of lung's elastoser, protection lung is not damaged by the enzymolysis of elastoser.
The directly related disease of α 1-AT shortage mainly is lung and hepatic diseases in the human plasma.As obstructive emphysema, newborn infant or adult respiratory distress syndrome, lung's cystic fibrosis, children's liver cirrhosis and neonatal hepatitis etc., being in a bad way causes infant to be died young.
Treatment for α 1-AT deficiency disease mainly is to carry out replacement therapy and gene therapy.But because gene therapy is still at the experimental stage at present, have certain limitation, so α 1-AT substitutes or this type of disease of supplement therapy is a main method.China does not still have the production of this product at present, and the market requirement mainly relies on imported product to satisfy.
All carried out the production research of α 1-AT both at home and abroad.Application two-step precipitation processes such as Gadek prepare the α 1-AT concentrate formulation of clinical application, and α 1-AT only accounts for 5% of concentrate formulation Tot Prot.Glaser etc. have set up unique α 1-AT preparation technology, adopt dithiothreitol (DTT) to handle in conjunction with ammonium sulfate precipitation DEAE-Mierocrystalline cellulose chromatography, the α 1-AT concentrate formulation of the purity 70% of purifying out from the discarded component FIV-1 of Cohn ' s cold ethanol technology.Zhu Wei etc.
[9]Purification α 1-AT from Cohn component FIV.With Cohn component FIV extract through precipitation, ultrafiltration, after ion-exchange and the gel-filtration, purification α 1-AT with Pasteur method inactivation of viruses, and investigates its effect.Detect α 1-AT protein-active with the single diffusion of immunity, bidirectional crossed immunoelectrophoresis, SDS-PAGE and synthetic substrate method, survey its purity with the zonal scanning method and reach more than 90%.
Above-mentioned technology mainly adopts the precipitator method or precipitation in conjunction with purification by chromatography α 1-AT, and product purity is about 70%-90%, and purity is lower; The present invention adopts the two-step chromatography purifying, and equipment and instrument is few, and operation is simple, the product purity height, and α 1-AT electrophoresis purity reaches 98%.
Summary of the invention
The objective of the invention is in order to remedy the deficiencies in the prior art, provide a kind of new from human plasma component FIV precipitation the method for separation and purification alpha1-antitrypsin, to reduce production costs and to improve purity.
In order to reach the foregoing invention purpose, the present invention has adopted following technical scheme:
A kind of from human plasma component FIV precipitation the method for separation and purification alpha1-antitrypsin, it is characterized in that may further comprise the steps:
(A) blood plasma precipitation pre-treatment: get the FIV precipitation and add lysate and be diluted to protein content 2%～5%, pH8.0～8.5, ionic strength 0.09-0.12, temperature is controlled at 2～8 ℃ of stirrings and spends the night, and the ratio according to 0.5%～15% (W/V) adds the silicate auxiliary agent and stirs 1h, solid-liquid separation at 2-8 ℃, get supernatant liquor, ratio (W/V) according to 0.5%-15% adds the silicate auxiliary agent once more at 2-8 ℃ of stirring 3h, and solid-liquid separation is got supernatant liquor sample introduction chromatography column;
(B) negatively charged ion gel chromatography: get gel, room temperature is adorned post after placing and reaching temperature equilibrium, with the buffer solution elution balance, pH6.0～6.5, equal amount is 8～12 times of column volume, and supernatant liquor is splined on the good chromatography column of balance, uses buffer solution elution, pH6.0～6.5, temperature is controlled at 2～8 ℃, washes 8～10 times of column volumes of post, in the monitoring automatically down of UV-light 280nm wavelength, record chromatography collection of illustrative plates is optionally collected to contain the component that a 1-AT is a main component;
(C) gel permeation chromatography: get gel, room temperature is adorned post after placing and reaching temperature equilibrium, with the damping fluid balance, the component of the a1-AT that collects behind the chromatography is transferred to pH 7.0-7.5, be splined on the good Sephacryl5-200 post of balance, with identical buffer solution elution, simultaneously in the monitoring automatically down of UV-light 280nm wavelength, record chromatography collection of illustrative plates, the component of optionally collecting a 1-AT;
(D) desalination and concentration by ultrafiltration;
(E) Pasteur's inactivation of virus: protein concentrate liquid is added the dilution of pyrogen-free water for injection, add protective material, in 60 ± 0.5 ℃ of insulations 8～12 hours, the virus in the deactivation plasma protein products;
(F) freeze-drying packing: a 1-AT protein liquid of Pasteur's deactivation changes the Freeze Drying Equipment vacuum-freeze-dry over to through Sterile Filtration;
(G) dry heat sterilization: the freeze-dried powder of packing is positioned in the heat room and is incubated, and inactivation of viruses changes stockyard over to and examines packing entirely.
The present invention is precipitated as raw material with the material FIV component of abandoning that produces in the human serum albumin production process, adopting a kind of proteinase inhibitor of chromatographic technique separation and purification is alpha1-antitrypsin (α 1-antitrypsin, α 1-AT), set up the production technique of α 1-AT concentrate formulation.Good product purity, the yield height of present method preparation, operation is simple, and the equipment that takies simultaneously is few, labour intensity is little, energy consumption is low, makes raw material with the waste material that albumin is produced, and production cost is low.
Description of drawings
Fig. 1 is a process flow sheet.
Embodiment
Technical scheme provided by the invention is the production technique of α 1-AT concentrate formulation, and processing step is as follows:
(A) FIV precipitation pre-treatment
Get that FIV precipitation adds the phosphoric acid of 8～18mM or the acetate buffer of 30～35mM is diluted to protein content 2%-5%, pH8.0-8.5, ionic strength 0.09-0.12,2-8 ℃ of stirring of temperature control spent the night.Ratio (W/V) according to 0.5%-15% adds the silicate auxiliary agent, 2-8 ℃ is stirred 1h, solid-liquid separation, separation temperature are controlled at 4 ℃, get supernatant liquor, ratio (W/V) according to 0.5%-15% adds the silicate auxiliary agent, 2-8 ℃ is stirred 3h, regulator solution pH5.5-6.5, solid-liquid separation, temperature is controlled at 4 ℃, gets supernatant liquor sample introduction chromatography column.The silicate auxiliary agent is diatomite, perlite, and the consumption of additive is that per 100 milliliters of lysates add 0.5 gram-15 grams.
(B) ion exchange chromatography
Take out DEAF-Sepharose Fast Flow gel, room temperature is adorned post after placing and reaching temperature equilibrium, with the phosphate buffered saline buffer of 8～18mM, the acetate buffer of 30～35mM or the citrate buffer wash-out balance of 12～25mM, pH6.0-6.5, equal amount is 8-12 a times of column volume.Supernatant liquor is splined on the good chromatography column of balance, with the phosphate buffered saline buffer of 8～18mM, the acetate buffer of 30～35mM or the citrate buffer wash-out of 12～25mM, pH6.0-6.5, flow rate control is at 2.0～4.5 times of column volumes per hour, temperature is controlled at 2-8 ℃, washes post 8-10 times column volume.In the monitoring automatically down of UV-light 280nm wavelength, record chromatography collection of illustrative plates is optionally collected to contain the component that a 1-AT is a main component.
(C) gel permeation chromatography
Get Sephacryl S-200 gel, room temperature is adorned post after placing and reaching temperature equilibrium, with the phosphate buffered saline buffer of 8～18mM, the acetate buffer of 30～35mM or the citrate buffer balance of 12～25mM, pH7.0～7.5, equal amount is 8～12 times of column volume.The component of a 1-AT that collects behind the chromatography is transferred to pH 7.0～7.5, be splined on the good Sephacryl5-200 post of balance, with identical buffer solution elution, elution amount is 8-12 a times of column volume, flow rate control is at 1.5～3.5 times of column volumes per hour, and eluting temperature is controlled at 2～8 ℃.Simultaneously in the monitoring automatically down of UV-light 280nm wavelength, record chromatography collection of illustrative plates, the component of optionally collecting a 1-AT.
(D) desalination and concentration by ultrafiltration
Go into ultrafilter through a of chromatography purification 1-AT solution pump, with pyrogen-free water for injection ultrafiltration desalination, the ultra-filtration membrane molecular weight cut-off is 100KD.
(E) pasteurization
Protein concentrate liquid is added pyrogen-free water for injection be diluted to protein content 1.5%-2.5%; pH6.4～6.8; add the hybrid protection agent of glycine and maltose as pasteurization; glycine content is controlled at 45-55mM; maltose content is controlled at 25%～35% in 60 ± 0.5 ℃ of insulations 10 hours, the virus in the deactivation plasma protein products.
(F) freeze-drying packing
The a 1-AT protein liquid of Pasteur's deactivation is crossed 0.22 μ m millipore filtration Sterile Filtration, changes the Freeze Drying Equipment vacuum-freeze-dry over to, and water content is controlled at below 3%, and lyophilized powder is pressed 500mg albumen/bottle packing, changes product to be checked storehouse over to.
(G) dry heat sterilization
The freeze-dried powder of packing is positioned in 80 ℃ of heat rooms, is incubated 60～72 hours inactivation of viruses, changes stockyard over to and examines packing entirely.
Embodiment 1:
(1) FIV precipitation pre-treatment
Take by weighing Cohn method component precipitation FIV 3kg, add 15mM, the Sodium phosphate dibasic one sodium dihydrogen phosphate buffer 50L of pH8.0, spend the night in 2～8 ℃ of stirrings, add 3kg diatomite, stirred 1 hour at 2～8 ℃, high speed centrifugation separates, temperature is controlled at 4 ℃, adds perlite 1.5kg in the filtrate, and 2-8 ℃ is stirred 3h, transfer pH value of solution 6.2 with 1M HCl, high speed centrifugation separates, and temperature is controlled at 4 ℃, and getting supernatant liquor is chromatography upper prop liquid.
(2) DEAE-Sepharose Fast Flow anion-exchange chromatography
Take out DEAF-Sepharose Fast Flow gel, room temperature is placed to reach temperature equilibrium, gets 6L gel dress post, and with 15mM, the sodium phosphate buffer balance of pH6.0 is washed post, 8 times of column volumes of balance liquid consumption.Upper prop liquid 10L is splined on the good DEAF-Sepharose Fast Flow post (9.5x110cm) of balance, use pH6.0, the sodium phosphate buffer of 15mM is washed post, wash 8 times of column volumes of post liquid consumption, flow velocity 2.2L/h, with pH6.0, the citric acid one sodium citrate buffer solution wash-out of 20mM, flow velocity 3.2L/h, eluting temperature is controlled at 2-8 ℃.In the monitoring automatically down of UV-light 280nm wavelength, write down the chromatography collection of illustrative plates simultaneously, selective collection contains the component that a 1-AT is a main component.
(3) Sephacryl S-200 gel permeation chromatography
Get Sephacryl S-200 gel 5L, after reaching temperature equilibrium, room temperature placement 2h adorns post, with 15mM, the sodium phosphate buffer balance of pH7.0 is washed post, 10 times of column volumes of balance liquid consumption, a 1-AT component of collecting after with chromatography with 1M NaOH solution transfers to pH7.2, is splined on the good Sephacryl5-200 post (7.5x90cm) of balance, with the flow velocity wash-out of identical damping fluid with 1.8L/h, eluting temperature is controlled at 2-8 ℃.Simultaneously in the monitoring automatically down of UV-light 280nm wavelength, record chromatography collection of illustrative plates, the component of optionally collecting a 1-AT.
(4) desalination and concentration by ultrafiltration
Go into ultrafilter through a of chromatography purification 1-AT solution pump, with pyrogen-free water for injection 550L ultrafiltration desalination, the ultra-filtration membrane molecular weight cut-off is 100KD, is concentrated to protein content 12%.
(5) pasteurization
From ultra-fine filter, pump protein concentrated solution, add pyrogen-free water for injection and be diluted to protein content 2%, add 55mM glycine and 25% maltose protective material as pasteurization.Protein solution is in 60 ± 0.5 ℃ of insulations 10 hours, the virus in the deactivation plasma protein products.
(6) freeze-drying packing
The a 1-AT protein liquid vacuum-freeze-dry of Pasteur's deactivation, water content is controlled at below 3%, and lyophilized powder changes product to be checked storehouse over to according to 500mg albumen/bottle is aseptic subpackaged.
(7) dry heat sterilization/full inspection packing
Dried frozen aquatic products is put 80 ℃ of heat rooms and is placed 60 hours deactivation goods virus, changes stockyard over to and examines packing entirely.Finished product detection result such as following table 1:
Table 1
| Test item | Method | Measured value | Conclusion |
| Protein content | Kjeldahl determination | 2.08％ | Qualified |
| Purity | Electrophoretic method | 98.8％ | Qualified |
| Purity | The HPLC method | 96.4％ | Qualified |
| Molecular weight | The SDS-PAGE electrophoresis | 54.8KD | Qualified |
| Iso-electric point | Isoelectric focusing electrophoresis | PI4.58 | Qualified |
| Telling test | Immune double diffusion method | Product and AHS form precipitation line, do not produce precipitation line with the negative control product | Qualified |
| Active | Trypsinase suppresses method | 720±30IU/mg | Qualified |
Embodiment 2
(1) FIV precipitation pre-treatment
Take by weighing Cohn method component precipitation FIV 3kg, add the sodium acetate buffer solution of 50L 30mM, spend the night in 2～8 ℃ of stirrings, add 3kg diatomite, stirred 1 hour at 2～8 ℃, the pressure filtration of sample introduction sheet frame filter, temperature is controlled at 4 ℃, add perlite 1.5kg in the filtrate, 2-8 ℃ is stirred 3h, transfers pH value of solution 6.2, the pressure filtration of sample introduction sheet frame filter with 1M HCl, temperature is controlled at 4 ℃, and getting supernatant liquor is chromatography upper prop liquid.
(2) DEAE-Sepharose Fast Flow anion-exchange chromatography
Take out DEAF-Sepharose Fast Flow gel, room temperature is placed to reach temperature equilibrium, gets 6L gel dress post, and with 30mM, the sodium acetate buffer solution equilibria of pH6.0 is washed post, 12 times of column volumes of balance liquid consumption.Upper prop liquid 10L is splined on the good DEAE-Sepharose Fast Flow post (9.5x110cm) of balance, use 30mM, the sodium acetate buffer solution of pH6.0 is washed post, wash 10 times of column volumes of post liquid consumption, flow velocity 2.0L/h, with pH6.0, the citric acid one sodium citrate buffer solution wash-out of 20mM, flow velocity 4.5L/h, eluting temperature is controlled at 2-8 ℃.In the monitoring automatically down of UV-light 280nm wavelength, write down the chromatography collection of illustrative plates simultaneously, selective collection contains the component that a 1-AT is a main component.
(3) Sephacryl S-200 gel permeation chromatography
Get Sephacryl S-200 gel 5L, after reaching temperature equilibrium, room temperature placement 2h adorns post, with 30Mm, the sodium acetate buffer solution equilibria of pH7.2 is washed post, 10 times of column volumes of balance liquid consumption, a 1-AT component of collecting after with chromatography with 1M NaOH solution transfers to pH7.2, is splined on the good Sephacryl5-200 post (7.5x90cm) of balance, with the flow velocity wash-out of identical damping fluid with 1.5L/h, eluting temperature is controlled at 2-8 ℃.Simultaneously in the monitoring automatically down of UV-light 280nm wavelength, record chromatography collection of illustrative plates, the component of optionally collecting a 1-AT.
(4) desalination and concentration by ultrafiltration
Go into ultrafilter through a of chromatography purification 1-AT solution pump, with pyrogen-free water for injection 550L ultrafiltration desalination, the ultra-filtration membrane molecular weight cut-off is 100KD, is concentrated to protein content 12%.
(5) pasteurization
From ultra-fine filter, pump protein concentrated solution, add pyrogen-free water for injection and be diluted to protein content 2%, add 55mM glycine and 32% maltose protective material as pasteurization.Protein solution is in 60 ± 0.5 ℃ of insulations 10 hours, the virus in the deactivation plasma protein products.
(6) freeze-drying packing
The a 1-AT protein liquid vacuum-freeze-dry of Pasteur's deactivation, water content is controlled at below 3%, and lyophilized powder changes product to be checked storehouse over to according to 500mg albumen/bottle is aseptic subpackaged.
(7) dry heat sterilization/full inspection packing
Dried frozen aquatic products is put 80 ℃ of heat rooms and is placed 72 hours deactivation goods virus, changes stockyard over to and examines packing entirely.
Claims (10)
1. the method for a separation and purification alpha1-antitrypsin from human plasma component FIV precipitation is characterized in that may further comprise the steps:
(A) blood plasma precipitation pre-treatment: get the FIV precipitation and add lysate and be diluted to protein content 2%～5%, pH8.0～8.5, ionic strength 0.09-0.12, temperature is controlled at 2～8 ℃ of stirrings and spends the night, and the ratio according to 0.5%～15% (W/V) adds the silicate auxiliary agent and stirs 1h, solid-liquid separation at 2-8 ℃, get supernatant liquor, ratio (W/V) according to 0.5%-15% adds the silicate auxiliary agent once more at 2-8 ℃ of stirring 3h, and solid-liquid separation is got supernatant liquor sample introduction chromatography column;
(B) negatively charged ion gel chromatography: get gel, room temperature is adorned post after placing and reaching temperature equilibrium, with the buffer solution elution balance, pH6.0～6.5, equal amount is 8～12 times of column volume, and supernatant liquor is splined on the good chromatography column of balance, uses buffer solution elution, pH6.0～6.5, temperature is controlled at 2～8 ℃, washes 8～10 times of column volumes of post, in the monitoring automatically down of UV-light 280nm wavelength, record chromatography collection of illustrative plates is optionally collected to contain the component that a 1-AT is a main component;
(C) gel permeation chromatography: get gel, room temperature is adorned post after placing and reaching temperature equilibrium, with the damping fluid balance, the component of the a1-AT that collects behind the chromatography is transferred to pH 7.0-7.5, be splined on the good Sephacryl5-200 post of balance, with identical buffer solution elution, simultaneously in the monitoring automatically down of UV-light 280nm wavelength, record chromatography collection of illustrative plates, the component of optionally collecting a 1-AT;
(D) desalination and concentration by ultrafiltration;
(E) Pasteur's inactivation of virus: protein concentrate liquid is added the dilution of pyrogen-free water for injection, add protective material, in 60 ± 0.5 ℃ of insulations 8～12 hours, the virus in the deactivation plasma protein products;
(F) freeze-drying packing: a 1-AT protein liquid of Pasteur's deactivation changes the Freeze Drying Equipment vacuum-freeze-dry over to through Sterile Filtration;
(G) dry heat sterilization: the freeze-dried powder of packing is positioned in the heat room and is incubated, and inactivation of viruses changes stockyard over to and examines packing entirely.
2. method according to claim 1 is characterized in that: the lysate of described steps A is the phosphate buffered saline buffer of 8～18mM or the acetate buffer of 30～35mM; The auxiliary agent that adds in the lysate is diatomite, perlite, and the consumption of additive is that per 100 milliliters of lysates add 0.5 gram-15 grams.
3. method according to claim 1 is characterized in that: the solid-liquid separation in the described steps A adopts high speed centrifugation to separate or sheet frame pressure filtration, and separation temperature is controlled at 4 ℃, and supernatant liquor is adjusted to pH5.5-6.5 after the solid-liquid separation.
4. method according to claim 1, it is characterized in that: step B intermediate ion displacement chromatography filler is a DEAF-Sepharose Fast Flow gel, elutriant is phosphate buffered saline buffer or the acetate buffer of 30～35mM or the citrate buffer of 12～25mM of 8～18mM, and flow rate control is at 2.0～4.5L/h.
5. method according to claim 1, it is characterized in that: the gel permeation chromatography filler is a Sephacryl S-200 gel among the step C, elutriant is phosphate buffered saline buffer or the acetate buffer of 30～35mM or the citrate buffer of 12～25mM of 8～18mM, pH7.0-7.5, elution amount is 8-12 a times of column volume, flow rate control is at 1.5～3.5 times of column volumes per hour, and eluting temperature is controlled at 2-8 ℃.
6. method according to claim 1 is characterized in that: described step D will go into ultrafilter through a of chromatography purification 1-AT solution pump, and with pyrogen-free water for injection ultrafiltration desalination, the ultra-filtration membrane molecular weight cut-off is 100KD.
7. method according to claim 1, it is characterized in that: step e is diluted to protein content and is controlled at 1.5%-2.5%, pH6.4-6.8.
8. method according to claim 1 is characterized in that: Pasteur's disinfectant protective material is glycine and the agent of maltose hybrid protection in the step e, and glycine content is controlled at 45-55mM, and maltose content is controlled at 25%-35%.
9. method according to claim 1 is characterized in that: the degerming membrane pore size 0.22um that Sterile Filtration described in the step F is adopted, the vacuum-freeze-dry water content is controlled at below 3%.
10. method according to claim 1 is characterized in that: the holding temperature of freeze-dried products is 80 ℃ among the step G, and soaking time is 60～72 hours.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102180966A (en) * | 2011-01-28 | 2011-09-14 | 哈尔滨派斯菲科生物制药股份有限公司 | Method for producing human alpha1-antitrypsin in large scale |
| CN102993298A (en) * | 2012-12-14 | 2013-03-27 | 成都蓉生药业有限责任公司 | Method for preparing alpha 1-antitrypsin |
| CN106279405A (en) * | 2016-09-23 | 2017-01-04 | 中国药科大学 | A kind of method that Cohn component four blood plasma functional protein purifies |
| CN107022025A (en) * | 2017-04-17 | 2017-08-08 | 深圳职业技术学院 | A kind of purification of alpha1Antitryptic method |
| CN107163138A (en) * | 2017-03-28 | 2017-09-15 | 深圳市卫光生物制品股份有限公司 | A kind of antitryptic isolation and purification methods of human plasma protein fraction α 1 |
| CN107569680A (en) * | 2016-07-05 | 2018-01-12 | 武汉生物制品研究所有限责任公司 | A kind of 1 antitryptic freeze-dried compositions of people α and preparation method thereof |
| EP2945962B1 (en) | 2013-01-18 | 2019-04-17 | Prothera Biologics, Inc. | Methods for isolating blood products from an inter-alpha inhibitor protein-depleted blood product material |
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| CN100562523C (en) * | 2006-07-26 | 2009-11-25 | 河北大安制药有限公司 | The preparation method of alpha1-antitrypsin |
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| CN102180966A (en) * | 2011-01-28 | 2011-09-14 | 哈尔滨派斯菲科生物制药股份有限公司 | Method for producing human alpha1-antitrypsin in large scale |
| CN102180966B (en) * | 2011-01-28 | 2013-04-03 | 哈尔滨派斯菲科生物制药股份有限公司 | Method for producing human alpha1-antitrypsin in large scale |
| CN102993298A (en) * | 2012-12-14 | 2013-03-27 | 成都蓉生药业有限责任公司 | Method for preparing alpha 1-antitrypsin |
| EP2945962B1 (en) | 2013-01-18 | 2019-04-17 | Prothera Biologics, Inc. | Methods for isolating blood products from an inter-alpha inhibitor protein-depleted blood product material |
| CN107569680A (en) * | 2016-07-05 | 2018-01-12 | 武汉生物制品研究所有限责任公司 | A kind of 1 antitryptic freeze-dried compositions of people α and preparation method thereof |
| CN106279405A (en) * | 2016-09-23 | 2017-01-04 | 中国药科大学 | A kind of method that Cohn component four blood plasma functional protein purifies |
| CN107163138A (en) * | 2017-03-28 | 2017-09-15 | 深圳市卫光生物制品股份有限公司 | A kind of antitryptic isolation and purification methods of human plasma protein fraction α 1 |
| CN107022025A (en) * | 2017-04-17 | 2017-08-08 | 深圳职业技术学院 | A kind of purification of alpha1Antitryptic method |
| CN112250757A (en) * | 2020-11-13 | 2021-01-22 | 广东深蓝生物科技有限公司 | Method for extracting and purifying protein in porcine plasma |
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