CN103788209B - A kind of preparation method of Prolastin - Google Patents
A kind of preparation method of Prolastin Download PDFInfo
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- CN103788209B CN103788209B CN201210421389.8A CN201210421389A CN103788209B CN 103788209 B CN103788209 B CN 103788209B CN 201210421389 A CN201210421389 A CN 201210421389A CN 103788209 B CN103788209 B CN 103788209B
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- 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
- C07K14/8125—Alpha-1-antitrypsin
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Abstract
The invention provides a kind of exhaust components produced from cold ethanol partition method separated plasma albumen---prepare the method for Prolastin component I V precipitation, the raw material adopted is exhaust components, production cost is low, improves the comprehensive utilization ratio of blood resource, has obvious economic benefit; In purge process, adopt PEG precipitation, zinc chloride precipitation, hydroxyapatite chromatography and hydrophobic chromatography two-step chromatography, can obtain the α 1-PI of high purity and high specific activity; Adopt the pasteurization method with high mature, simplicity and economy to carry out inactivation of virus in process of production, ensure that the security of goods.
Description
Technical field
The present invention relates to a kind of preparation method of Prolastin.
Background technology
Congenital α1-antitrypsin deficiency is one of modal lethality heredopathia in world wide; feature is dropped to serum Prolastin level; the Prolastin Prolastin concentration lacked in the individual blood plasma of severe patient is only the 15-20% of normal people; owing to losing the protection of Prolastin; elastoser Progressive symmetric erythrokeratodermia in alveolar structure destroys, thus makes patient that emophysematous danger increase occur in one's early years.Prolastin is as a kind of human plasma goods just occurred the eighties in 20th century, be mainly used in because congenital alpha1-antitrypsin lacks the lifelong substitute treatment of the emphysema patient caused and the control of pulmonary inflammation disease, as chronic obstructive emphysema, pulmonary cystic fibrosis, acute lung injury, adult respiratory distress syndrome etc.It can also with Antithrombin III conbined usage, treatment disseminated intravascular coagulation, have wide practical use clinically.
Prolastin replacement therapy method in 1988 is corrected α1-antitrypsin deficiency and is obtained American health institutions approval, clinical application result for many years shows: the Prolastin congenital alpha1-antitrypsin shortage property emphysema patient being injected weekly to 60mg/kg carries out supplement therapy, good treatment and preventive effect is had to emophysematous burst, have no side effect after patient's life-time service Prolastin, this illustrates that the alternative medicine that congenital alpha1-antitrypsin lacks is proved, and it is respond well, there is good market potential.Since the Prolastin Prolastin of Bayer company production in 1987 gets permission listing, the goods of existing four companies achieve the listing approval of U.S. FDA.Publication date is 2011.06.15, publication number is that the PCT application " preparing the method for Prolastin " of CN102099369A discloses its way of purifying in the solution of one hydrophobic interaction chromatography (HIC) self-contained Prolastin, this method provide a kind of higher rate of recovery, purity and cause of disease clearance rate.
Because Prolastin derives from human plasma substantially, raw material sources are limited, so need a kind of cost low, more can make full use of the manufacture method of blood plasma simultaneously, meet the raising blood plasma utilization ratio of clinical disease Man's Demands and maximum possible to greatest extent.
Summary of the invention
The object of the invention is to provide a kind of low cost, the Prolastin manufacture method of blood plasma utilization ratio can be improved.For realizing this object, the present invention by the following technical solutions.
Prepare a method for Prolastin, it is characterized in that comprising following steps:
(1) take component I V precipitation, add the water for injection of 3-6 times (weightmeasurement ratio), control ph is between 5.0-6.0, and 18-25 DEG C is stirred 0.5-1.5 hour, and solid-liquid separation, abandons supernatant liquor; Described component IV is precipitated as the exhaust components that cold ethanol partition method separated plasma albumen produces.
(2) step (1) gained precipitation is collected, add 8-12 doubly (weightmeasurement ratio) water for injection, add Tris ground caustic that final concentration is 0.02M-0.08M, final concentration is the sodium-chlor of 0.005M-0.015M, obtain reaction solution, the pH value controlling reaction solution, between 9.0-9.5, stirs 1-3 hour at 18-25 DEG C;
(3) in step (2) gained reaction solution, add the PEG4000 that final concentration is 10%-15%, adjusted to ph is between 4.8-5.6, and 18-25 DEG C is stirred 0.5-1.5 hour, and solid-liquid separation, gets supernatant liquor;
(4) in step (3) gained supernatant liquor, adding liquor zinci chloridi, is 0.005-0.010M to the final concentration of zinc chloride in supernatant liquor, stirs 1-3 hour, solid-liquid separation with sodium hydroxide adjust pH to 7.0-8.0,18-25 DEG C, gets zinc chloride precipitation;
(5) step (4) gained zinc chloride is precipitated, be that 0.01-0.02M, pH value are between 6.5-7.0 by concentration, be after the phosphate buffered saline buffer dissolving of 0.01MEDTA containing concentration, loading, carry out hydroxyapatite column: be first that 0.01-0.02M, the pH value phosphate buffered saline buffer between 6.5-7.0 washs by concentration, and then be that 0.04-0.10M, the pH value phosphate buffered saline buffer between 6.5-7.0 carries out wash-out by concentration, and collect hydroxyapatite chromatography elutriant;
(6) in hydroxyapatite chromatography elutriant, ammonium sulfate is added, after being 0.8-1.2M to final concentration, passed through phenyl Sepharose hydrophobic chromatography post, be that 0.04-0.10M, pH value are between 6.5-7.0 by concentration again, be the phosphate buffered saline buffer washing of the ammonium sulfate of 0.8-1.2M containing concentration, and collection penetrate peak;
(7) hydrophobic chromatography is penetrated peak and collect the film bag ultrafiltration and concentration of liquid 10Kd molecular retention amount to protein content 1-2%, add protective material, carry out pasteurization (hatching 10 hours for 60 DEG C), described protective material is salt, sugar, amino acid or its mixture;
(8) protective material is removed in ultrafiltration again, is concentrated into protein content 2-5%, adds freeze-dried excipient and stablizer, Sterile Filtration, packing, freeze-drying, obtain Prolastin finished product.
Further, described step (1) control ph scope, between 5.0-6.0, is preferably 5.8.
Further, the pH value range of described step (3), between 4.8-5.6, is preferably 5.2.
Further, in described step (4), zinc chloride is 0.005-0.010M at the final concentration of supernatant liquor, and preferred final concentration is 0.006M.
Further, the adsorption medium that in described step (5), hydroxyapatite column is used is hydroxyapatite, preferably uses Macro-PrepCeramicHydroxyapatiteTypeI.
Further, in described step (5), the balance liquid of hydroxyapatite chromatography post is concentration is 0.01M-0.02M, the pH value phosphate buffered saline buffer between 6.5-7.0, is preferably the phosphate buffered saline buffer of concentration 0.02M, pH value 6.8.
Further, the hydrophobic chromatography post medium in described step (6) is phenyl Sepharose, preferably uses Phenyl-Sepharose6FastFlowHS.
Further, the balance liquid of the hydrophobic chromatography post in described step (6) is concentration is that 0.04-0.10M, pH value are between 6.5-7.0, it is the phosphate buffered saline buffer of the ammonium sulfate of 0.8-1.2M containing concentration, being preferably that concentration is 0.1M, pH value is 6.8, is the phosphate buffered saline buffer of the ammonium sulfate of 1M containing concentration.
Further, in described step (7), the protective material of pasteurization is sucrose and the agent of potassium acetate hybrid protection,
Sucrose content controls at 40%-60%, and potassium acetate content controls at 4%-6%.
The invention provides a kind of exhaust components produced from cold ethanol partition method separated plasma albumen---prepare the method for Prolastin component I V precipitation, the raw material adopted is exhaust components, production cost is low, improves the comprehensive utilization ratio of blood resource, has obvious economic benefit; In purge process, adopt PEG precipitation, zinc chloride precipitation, hydroxyapatite chromatography and hydrophobic chromatography two-step chromatography, can obtain the α 1-PI of high purity and high specific activity; Adopt the pasteurization method with high mature, simplicity and economy to carry out inactivation of virus in process of production, ensure that the security of goods.
Embodiment
Now in conjunction with the embodiments, the present invention is described further.
1. remove low ph value solubility foreign protein: take 25Kg component I V and precipitate, add 125L water for injection, adjusted to ph is stir after 1 hour at 5.80,18-25 DEG C, and press filtration, abandons supernatant, gets precipitation.
2. extract proteins: obtained precipitation is added 250L water for injection, add 1500gTris alkali to make its final concentration be 0.05M, add 146g sodium-chlor and make its final concentration be 0.01M, control the pH value of reaction solution between 9.0-9.5, stir 2 hours, temperature controls at 18-25 DEG C.
3.PEG precipitates: add 30KgPEG4000 at reaction solution and make its concentration reach 12%, and be 5.2,18-25 DEG C with hydrochloric acid adjustment pH and stir 1 hour, press filtration, obtains 238L supernatant.
4. zinc chloride precipitation: add 14.28L0.1M liquor zinci chloridi in 238L supernatant, adjust pH to 7.5 with sodium hydroxide, make its final concentration be 0.006M, 18-25 DEG C and stir 1 hour, centrifuging and taking precipitates.
5. hydroxyapatite chromatography: the precipitation phosphate buffered saline buffer of the 0.02MpH6.8 containing 0.01MEDTA dissolves, the hydroxyapatite chromatography post balanced by the phosphate buffered saline buffer in advance with 0.02MpH6.8, wash 3 column volumes, then the phosphate buffered saline buffer wash-out of 0.04-0.1MpH6.8 is selected, collect elution peak, all process temperatures control to carry out under 18-25 DEG C of condition, collect the elutriant 150L containing α 1-PI.
6. hydrophobic chromatography: add 19.8Kg ammonium sulfate in 150L hydroxyapatite chromatography elutriant, its concentration is made to be 1M, stirring at room temperature was filtered after 15 minutes, with the phenyl Sepharose hydrophobic chromatography post that the phosphate buffered saline buffer containing 1M ammonium sulfate 0.1MpH6.8 balances on filtrate, afterwards with balance liquid washing, collect and penetrate peak 180L altogether.
7. the film bag of the elutriant 10Kd molecular retention amount of inactivation of virus: 180L carries out ultrafiltration and concentration, remove ammonium sulfate, dialyzate is 0.01M phosphate buffered saline buffer (pH7.4,0.05MNaCl), obtain 9L concentrated solution altogether, add 10Kg sucrose and 1Kg potassium acetate, fully dissolve rear 60 DEG C of water-bath 10hr and carry out Pasteur's inactivation of virus.
8. ultrafiltration, preparation, Sterile Filtration, packing, freeze-drying: sucrose and potassium acetate are removed in ultrafiltration again, and dialyzate is 10mM phosphate buffered saline buffer (pH7.4,0.05MNaCl, 0.15M N.F,USP MANNITOL), under room temperature, concentrated solution protein content is adjusted to 2%, Sterile Filtration, packing, freeze-drying is preserved.Obtain the goods that 86 bottles of specifications are 1g/ bottle altogether.
Finished product result detects
Claims (9)
1. prepare a method for α 1 ?proteinase inhibitor, it is characterized in that comprising following steps:
(1) take component IV to precipitate, add 3 ?the water for injection of 6 times of weightmeasurement ratios, control ph 5.0 ?between 6.0,18 ?25 DEG C stir 0.5 ?1.5 hours, solid-liquid separation, abandons supernatant liquor; Described component IV is precipitated as the exhaust components that cold ethanol partition method separated plasma albumen produces;
(2) step (1) gained precipitation is collected, add 8 ?12 times of weightmeasurement ratio waters for injection, add final concentration be 0.02M ?the Tris ground caustic of 0.08M, final concentration be 0.005M ?the sodium-chlor of 0.015M, obtain reaction solution, the pH value controlling reaction solution 9.0 ?between 9.5,18 ?stir at 25 DEG C 1 ?3 hours;
(3) add in step (2) gained reaction solution final concentration be 10% ?15% PEG4000, adjusted to ph 4.8 ?between 5.6,18 ?25 DEG C stir 0.5 ?1.5 hours, solid-liquid separation, gets supernatant liquor;
(4) in step (3) gained supernatant liquor, liquor zinci chloridi is added, to the final concentration of zinc chloride in supernatant liquor be 0.005 ?0.010M, with sodium hydroxide adjust pH to 7.0 ?8.0,18 ?25 DEG C stir 1 ?3 hours, solid-liquid separation, gets zinc chloride precipitation;
(5) step (4) gained zinc chloride is precipitated, with concentration be 0.01 ?0.02M, pH value 6.5 ?between 7.0, and after dissolving containing the phosphate buffered saline buffer that concentration is 0.01MEDTA, loading, carry out hydroxyapatite column: first with concentration be 0.01 ?0.02M, pH value 6.5 ?phosphate buffered saline buffer between 7.0 wash, and then with concentration be 0.04 ?0.10M, pH value 6.5 ?phosphate buffered saline buffer between 7.0 carry out wash-out, and collect hydroxyapatite chromatography elutriant; The balance liquid of described hydroxyapatite chromatography post to be concentration be 0.01M ?0.02M, pH value 6.5 ?phosphate buffered saline buffer between 7.0;
(6) in hydroxyapatite chromatography elutriant, ammonium sulfate is added, to final concentration be 0.8 ?after 1.2M, passed through phenyl Sepharose hydrophobic chromatography post, again with concentration be 0.04 ?0.10M, pH value 6.5 ?between 7.0, containing quality be 0.8 ?1.2M ammonium sulfate phosphate buffered saline buffer washing, collect penetrate peak; Described hydrophobic chromatography post medium is phenyl Sepharose; The balance liquid of hydrophobic chromatography post to be concentration be 0.04 ?0.10M, pH value 6.5 ?between 7.0, containing concentration be 0.8 ?the phosphate buffered saline buffer of 1.2M ammonium sulfate;
(7) hydrophobic chromatography is penetrated the film bag ultrafiltration and concentration of collecting liquid 10Kd molecular retention amount in peak to protein content 1 ?2%, add protective material, carry out pasteurization, hatch 10 hours for 60 DEG C, described protective material is salt, sugar, amino acid or its mixture;
(8) protective material is removed in ultrafiltration again, be concentrated into protein content 2 ?5%, add freeze-dried excipient and stablizer, Sterile Filtration, packing, freeze-drying, obtain α 1 ?proteinase inhibitor finished product.
2. as claimed in claim 1 a kind of prepare α 1 ?the method of proteinase inhibitor, it is characterized in that: described step (1) control ph scope 5.0 ?between 6.0.
3. as claimed in claim 1 a kind of prepare α 1 ?the method of proteinase inhibitor, it is characterized in that: the pH value range of described step (3) 4.8 ?between 5.6.
4. as claimed in claim 1 a kind of prepare α 1 ?the method of proteinase inhibitor, it is characterized in that: in described step (4) zinc chloride the final concentration of supernatant liquor be 0.005 ?0.010M.
5. as claimed in claim 1 a kind of prepare α 1 ?the method of proteinase inhibitor, it is characterized in that: the adsorption medium that in described step (5), hydroxyapatite column is used is hydroxyapatite.
6. as claimed in claim 1 a kind of prepare α 1 ?the method of proteinase inhibitor, it is characterized in that: in described step (5), the balance liquid of hydroxyapatite chromatography post is the phosphate buffered saline buffer of concentration 0.02M, pH value 6.8.
7. as claimed in claim 1 a kind of prepare α 1 ?the method of proteinase inhibitor, it is characterized in that: the hydrophobic chromatography post medium in described step (6) use Phenyl ?Sepharose6FastFlowHS.
8. as claimed in claim 1 a kind of prepare α 1 ?the method of proteinase inhibitor, it is characterized in that: the balance liquid of the hydrophobic chromatography post in described step (6) is concentration is 0.1M, pH value is 6.8, is the phosphate buffered saline buffer of the ammonium sulfate of 1M containing concentration.
9. as claimed in claim 1 a kind of prepare α 1 ?the method of proteinase inhibitor; it is characterized in that: in described step (7), the protective material of pasteurization is sucrose and the agent of potassium acetate hybrid protection; sucrose content control 40% ?60%, potassium acetate content control 4% ?6%.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201210421389.8A CN103788209B (en) | 2012-10-29 | 2012-10-29 | A kind of preparation method of Prolastin |
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| CN201210421389.8A CN103788209B (en) | 2012-10-29 | 2012-10-29 | A kind of preparation method of Prolastin |
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| CN103788209B true CN103788209B (en) | 2016-02-24 |
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| IL267923B2 (en) * | 2018-08-02 | 2023-06-01 | Grifols Worldwide Operations Ltd | Composition comprising highly-concentrated alpha-1 proteinase inhibitor and method for obtaining thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020082214A1 (en) * | 1997-06-10 | 2002-06-27 | Erwin Mattes | Alpha 1-antitrypsin preparation as well as a method for producing the same |
| CN101134776A (en) * | 2007-07-31 | 2008-03-05 | 上海新兴医药股份有限公司 | Method for preparing alpha1-antitrypsin |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020082214A1 (en) * | 1997-06-10 | 2002-06-27 | Erwin Mattes | Alpha 1-antitrypsin preparation as well as a method for producing the same |
| CN101134776A (en) * | 2007-07-31 | 2008-03-05 | 上海新兴医药股份有限公司 | Method for preparing alpha1-antitrypsin |
Non-Patent Citations (1)
| Title |
|---|
| α1-蛋白酶抑制剂产品介绍;ARALAST NP;《中国新特药网药讯》;20111208;全文 * |
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