CN101792490A - Method for recycling albumin in Cohn's fraction IV precipitate - Google Patents
Method for recycling albumin in Cohn's fraction IV precipitate Download PDFInfo
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Abstract
The invention discloses a method for recycling albumin in Cohn's fraction IV precipitate. By adopting the method, albumin can be fully separated from other proteins in fraction IV through selecting pH value, alcohol concentration, acidity and alkalinity and temperature, 110-120g of albumin can e prepared from per kilogram of fraction IV, and the total extraction rate of albumin in blood plasma is improved to over 93 percent.
Description
Technical field
The present invention relates to the separation method of blood product, specifically is to adopt cold ethanol method albuminous method of Separation and Recovery from CohnShi component I V precipitation.
Background technology
Cold ethanol technology separated plasma albumen since creating the forties in 20th century, has had the history in 60 years so far.Cohn 6 methods that Cohn professor and leader's thereof development teams is founded have become the isolating classical way of albumin, are the bases of general in the world plasma albumin separating technology.Employed subsequently various albumin separation method is a foundation with the pH value in Cohn 6 methods, temperature, alcohol concn, ionic strength and 5 parameters of protein concentration still, by different parameters combination, can carry out the separation of other multiple protein goods.
Residual in the CohnShi component I V precipitation have a small amount of albumin.From human blood component I V precipitation, separate albuminous method and use cold ethanol method and heat ethanol methods substantially.Cold ethanol method is at low temperatures, as solvent, utilizes albumin with the different of other foreign protein crystallization condition albumin to be separated with other foreign protein with ethanol." Chinese medicine company " 2003 the 12nd the 04th phases of volume " from CohnShi component I V precipitation, reclaiming albuminous low temperature process ", on the basis of cold ethanol method, improve, controlled temperature, alcohol concn, potential of hydrogen, extract residual albumin in the component I V precipitation, every kg of component IV precipitates about recyclable albumin 50g.
Heat ethanol methods is that component I V precipitation is mixed with protein liquid, heating is solidified the foreign protein sex change in the protein liquid, makes solid-liquid separation by pressure filtration again, and the filtrate ultrafiltration and concentration is obtained the albumin raw product, as CN1660898A, CN1660903A disclosed method, but the rate of recovery is not high yet.
Summary of the invention
Purpose of the present invention provides a kind of and reclaim albuminous method from CohnShi component I V precipitation, this method is on the basis of cold ethanol method, select the parameters combination of suitable temperature, alcohol concn, potential of hydrogen, make albumin and other albumen sepn, improve the albumin rate of recovery greatly.
The inventive method may further comprise the steps:
A. get component I V precipitation, add 8 ± 0.5 times of component I V precipitation weight 0～2 ℃, the NaCl solution of weight concentration 0.75 ± 0.03% is after the stirring and dissolving, with acetic acid/sodium-acetate buffer and/or NaHCO
3The aqueous solution is adjusted pH value to 6.90 ± 0.10, and stirring became reaction solution in 5-7 hour;
B. adjust reacting liquid pH value to 5.70 ± 0.05 with acetic acid/sodium-acetate buffer, be cooled to-0.5 ± 0.5 ℃, spraying adds volumetric concentration greater than 60% ethanol, and reaction solution reduces temperature gradually to-4.5 ± 0.5 ℃, and amount of alcohol added is that the alcoholic acid volumetric concentration reaches 40% in the reaction solution, after ethanol adds, reacting liquid pH value is adjusted at 5.85-5.90, continues to stir 1～3 hour, leave standstill more than 8 hours, with reaction solution centrifuging, get filtrate then;
C. filtrate to be adjusted to the pH value be 4.70 ± 0.10 with containing 0.4 ± 0.1mol/L acetic acid and volumetric concentration 40 ± 1.0% alcoholic acid acetic acid-alcohol mixed solutions, stirred 1～3 hour, leave standstill more than 8 hours at-9.0 ± 1.0 ℃, filter, the collecting precipitation thing is rough albumin;
D. with rough albumin purifying, concentrated, Pasteur's inactivation of virus, can be mixed with the injection human serum albumin.
In steps A, NaCl solution preferably adds at twice, adds 2～4 times of component I V precipitation weight for the first time, and reaction solution stirs into pasty state, adds remaining NaCl solution again.
The ethanol volumetric concentration that spraying adds is preferably 75～95%.
The inventive method is by selecting pH value, alcohol concn, potential of hydrogen and temperature, albumin in the component I V precipitation is fully separated with other albumen, per kilogram component I V precipitation can make albumin 110～120 grams, and albuminous total extraction yield is brought up to more than 93%.
Embodiment
One, component I V is sedimentary thaws and dissolves
Take out component I V precipitation from freezer below-30 ℃, weighing is placed in the clean stainless steel reaction jar, pumps into 3 times of component I V weight 0～2 ℃ in retort, the NaCl solution of 0.75wt%, and stirring and dissolving, reaction solution becomes pasty state.
After the dissolving, reacting liquid pH value is transferred to 6.90 ± 0.10, and stirred 5～7 hours under this pH condition fully, stirring velocity is 1400～1500rpm.
Add 5 times of component I V weight 0-2 ℃ again, the NaCl solution of 0.75wt% stirs.
Two, other albumen of precipitate and separate
Spraying adds the acetic acid/sodium-acetate buffer of pH value 4.0 in reaction solution, and adding speed 150～200ml/min transfers to 5.70 ± 0.05 with reacting liquid pH value, reacting liquid temperature is reduced to-0.5 ± 0.5 ℃.
Spraying adds 95% (v/v) ethanol, and reaction solution is cooled to-4.5 ± 0.5 ℃ gradually, and amount of alcohol added is that the alcoholic acid volumetric concentration is 40% in the reaction solution.
After ethanol adds, continue to stir 15 minutes, sampling and measuring pH value should be 5.85-5.90, if not in this scope, correct with the NaOH solution of 0.5mol/L or the acetic acid of pH value 4.0/sodium-acetate buffer, continue to stir 1.5 hours, leave standstill more than 8 hours at-4.5 ± 0.5 ℃.
With reaction solution centrifuging, get filtrate.
Three, albuminous precipitation
Filtrate transfers to alcohol concn 40% (v/v) with 50% (v/v) ethanol, continue to stir 15 minutes, repetition measurement pH value, regulating the pH value with 0.5mol/L NaOH solution or pH value 4.0 acetic acid/sodium-acetate buffer is 4.70 ± 0.10, restir 1.5 hours leaves standstill more than 8 hours at-9.0 ± 1.0 ℃.
With reaction solution centrifuging, the collecting precipitation thing is rough albumin.
Rough albumin in time with 0～2 ℃ of water for injection dissolving, is stored in the freezer below-30 ℃.
Four, rough albuminous purifying
After rough albumin is closed batch, extraordinarily go into 0～2 ℃ water for injection by rough albumin weight 6～8, dissolve in the stainless steel reaction jar, solvent temperature is controlled between 0～2 ℃.
Fully after the dissolving, the water for injection of adding 0-2 ℃ is to 9～11 times of rough albumin weight.
Start whipping appts, stirring velocity is controlled at 75～105 revolutions per seconds, regulate pH value to 4.60 ± 0.10, spraying adds the ethanol of-15 ℃ 65% (v/v), alcohol concn to 12 in solution ± 0.20% (v/v), and alcoholic acid adding speed begins to be 500ml/min, when alcohol concn reaches 5% (v/v) in the solution, alcoholic acid adding speed is 1000ml/min, and when alcohol concn reached 10% (v/v) in the solution, alcoholic acid adding speed was 1500ml/min.
After ethanol adds, continue to stir 1 hour, press 0.5% of solution weight and add the good diatomite of pre-treatment, keep solution temperature, leave standstill more than 8 hours at-2.5 ± 0.5 ℃.
With carrying out Depth Filtration through 400 * 400 good sheet frame filters of pre-treatment, the liquid that begins to leach turns back to be treated in the filtering retort of deep layer originally, begin to collect behind the no opalescence of liquid clarification to be leached, clarifying filtrate is transferred in another clean retort.
Stir filtrate, with on average≤speed of 500ml/min adds the NaHCO of 1mol/L to the filtrate spraying
3Solution is 5.10 ± 0.05 until filtrate pH value.
Filtrate is cooled to-3.5 ± 0.5 ℃, adds 95% ethanol (v/v), and alcohol concn in the filtrate is adjusted to 40% (v/v), adds ethanol process and is cooled to-9.0 ± 1.0 ℃ simultaneously, continues to stir 1.5 hours down at-9.0 ± 1.0 ℃, leaves standstill more than 8 hours.
Centrifuging, control centrifugal speed 50～55L/ platform/hour, centrifugally go out liquid temp-7.5 ± 1.0 ℃.Filtration finishes, the promptly refining albumin of collecting precipitation thing.
Refining albumin is dissolved with 0～2 ℃ of water for injection, adds 0.5% diatomite in solution, stirs 30 minutes, uses through 400 * 400 good sheet frame filters of pre-treatment and carries out Depth Filtration.
Adjust filtrate pH value to 7.25 ± 0.05.
Above-mentioned filtrate is carried out twice concentrate, being concentrated into albumin content for the first time is 105g/L, dialyses to residual ethanol＜0.025% (v/v) with water for injection, carries out being concentrated into albumin content the second time to 210g/L again.
It is 0.165mmol/gPr that solution after concentrating is mixed with Sodium octoate content, and protein content is 195g/L, Na
+Content is 150mmol/L, the work in-process of pH value 6.80 ± 0.20.
Work in-process carry out 60.0 ± 0.5 ℃, and Pasteur's inactivation of virus of 10 hours becomes the injection human serum albumin.
Following table is enumerated 3 batches of product technology parameters that adopt aforesaid method to produce.
| Product batch number | ??1 | ??2 | ??3 |
| Total protein concentration (gram) in the component I V precipitation | ??4501.8 | ??4788.0 | ??4398.3 |
| Albumin total amount (gram) in the component I V precipitation that sampling analysis calculates | ??801.32 | ??799.60 | ??818.08 |
| Separate the rough albumin weight (gram) that obtains | ??617.29 | ??626.59 | ??626.04 |
| Rough albumin purity (%) | ??94.7 | ??94.0 | ??94.0 |
| Rough albumin separation yield (albumin total amount * 100% in the component I V precipitation that rough albumin weight/sampling analysis calculates) | ??77.03 | ??78.36 | ??76.53 |
| Refining albumin weight (gram) behind the purifying | ??507.33 | ??517.58 | ??536.27 |
| Refining albumin purity (%) | ??98.7 | ??98.4 | ??98.5 |
| The refining albumin rate of recovery (albumin total amount * 100% in the component I V precipitation that refining albumin weight/sampling analysis calculates) | ??63.31 | ??64.73 | ??65.55 |
| Product batch number | ??1 | ??2 | ??3 |
| Every kg of component IV precipitation can get albumin (gram) | ??112.7 | ??108.1 | ??121.9 |
| Whole albumin (gram) of the extraction of every liter of human blood | ??30.3 | ??29.8 | ??30.7 |
| The total extraction yield of albumin (%) in the blood plasma | ??93.24 | ??93.98 | ??93.30 |
Claims (3)
1. reclaim albuminous method in the CohnShi component I V precipitation, it is characterized in that, may further comprise the steps successively:
A. get component I V precipitation, add 8 ± 0.5 times of component I V precipitation weight 0-2 ℃, the NaCl solution of weight concentration 0.75 ± 0.03% is after the stirring and dissolving, with acetic acid/sodium-acetate buffer and/or NaHCO
3The aqueous solution is adjusted pH value to 6.90 ± 0.10, stirs 5-7 hour;
B. adjust reacting liquid pH value to 5.70 ± 0.05 with acetic acid/sodium-acetate buffer, be cooled to-0.5 ± 0.5 ℃, spraying adds volumetric concentration greater than 60% ethanol, and reaction solution reduces temperature gradually to-4.5 ± 0.5 ℃, and amount of alcohol added is that the alcoholic acid volumetric concentration is 40% in the reaction solution, after ethanol adds, reacting liquid pH value is adjusted at 5.85-5.90, continues to stir 1～3 hour, leave standstill more than 8 hours, with reaction solution centrifuging, get filtrate then;
C. filtrate to be adjusted to the pH value be 4.70 ± 0.10 with containing 0.4 ± 0.1mol/L acetic acid and volumetric concentration 40 ± 1.0% alcoholic acid solution, stirred 1～3 hour, leave standstill more than 8 hours at-9.0 ± 1.0 ℃, filter, the collecting precipitation thing is rough albumin;
D. with rough albumin purifying, concentrate, Pasteur's inactivation of virus and be mixed with the injection human serum albumin.
2. according to the described method of claim 1, it is characterized in that in steps A, NaCl solution adds at twice, add 2～4 times of component I V precipitation weight for the first time, reaction solution stirs into pasty state, adds remaining NaCl solution again.
3. according to the described method of claim 1, it is characterized in that in step B, the ethanol volumetric concentration that spraying adds is 75～95%.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102311497A (en) * | 2011-08-22 | 2012-01-11 | 西安回天血液制品有限责任公司 | Method for recovering albumin from component IV deposition by low temperature ethanol method |
| CN102552906A (en) * | 2012-01-11 | 2012-07-11 | 西安回天血液制品有限责任公司 | Productive technology of intravenous injection human immunoglobulin |
| CN103333240A (en) * | 2013-07-22 | 2013-10-02 | 北海开元生物科技有限公司 | Method for reclaiming human albumin from component IV precipitate |
| CN104558154A (en) * | 2015-01-05 | 2015-04-29 | 深圳市卫光生物制品股份有限公司 | Method for extracting human albumin from component IV-2 sediment |
| CN106800583A (en) * | 2015-11-26 | 2017-06-06 | 上海洲跃生物科技有限公司 | It is a kind of instant without the cryodesiccant human fibrinogen preparation technology for separating out |
| CN113980118A (en) * | 2021-12-13 | 2022-01-28 | 广东卫伦生物制药有限公司 | Serum albumin degreasing method |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SK278348B6 (en) * | 1990-02-14 | 1996-12-04 | Ivan Stepanek | Preparation method of blood albumin |
| CN1305903C (en) * | 2004-12-22 | 2007-03-21 | 三九集团湛江开发区双林药业有限公司 | Method for retrieving albumin from deposited components I, II, III, IV |
| CN1313488C (en) * | 2004-12-29 | 2007-05-02 | 三九集团湛江开发区双林药业有限公司 | Method for retrieving albumin form constituent deposition 123 |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102311497A (en) * | 2011-08-22 | 2012-01-11 | 西安回天血液制品有限责任公司 | Method for recovering albumin from component IV deposition by low temperature ethanol method |
| CN102311497B (en) * | 2011-08-22 | 2013-05-22 | 西安回天血液制品有限责任公司 | Method for recovering albumin from component IV deposition by low temperature ethanol method |
| CN102552906A (en) * | 2012-01-11 | 2012-07-11 | 西安回天血液制品有限责任公司 | Productive technology of intravenous injection human immunoglobulin |
| CN102552906B (en) * | 2012-01-11 | 2013-11-06 | 西安回天血液制品有限责任公司 | Productive technology of intravenous injection human immunoglobulin |
| CN103333240A (en) * | 2013-07-22 | 2013-10-02 | 北海开元生物科技有限公司 | Method for reclaiming human albumin from component IV precipitate |
| CN103333240B (en) * | 2013-07-22 | 2015-01-28 | 北海开元生物科技有限公司 | Method for reclaiming human albumin from component IV precipitate |
| CN104558154A (en) * | 2015-01-05 | 2015-04-29 | 深圳市卫光生物制品股份有限公司 | Method for extracting human albumin from component IV-2 sediment |
| CN104558154B (en) * | 2015-01-05 | 2016-10-12 | 深圳市卫光生物制品股份有限公司 | A kind of method extracting human albumin from component IV-2 precipitates |
| CN106800583A (en) * | 2015-11-26 | 2017-06-06 | 上海洲跃生物科技有限公司 | It is a kind of instant without the cryodesiccant human fibrinogen preparation technology for separating out |
| CN113980118A (en) * | 2021-12-13 | 2022-01-28 | 广东卫伦生物制药有限公司 | Serum albumin degreasing method |
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