CN1313488C - Method for retrieving albumin form constituent deposition 123 - Google Patents
Method for retrieving albumin form constituent deposition 123 Download PDFInfo
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- CN1313488C CN1313488C CNB2004100776678A CN200410077667A CN1313488C CN 1313488 C CN1313488 C CN 1313488C CN B2004100776678 A CNB2004100776678 A CN B2004100776678A CN 200410077667 A CN200410077667 A CN 200410077667A CN 1313488 C CN1313488 C CN 1313488C
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Abstract
The present invention relates to a method for recovering residual albumin from precipitates 123 of discarded components for preparing human plasma protein. The method of the present invention comprises: precipitates 123 of components are sufficiently leached by leaching liquid prepared from distilled water, alcohol and dodecahydrote disodium, so entrainment albumin enters a solution; protein liquid whose volume is reduced is then obtained by ultrafiltration and concentration; the concentration of ethanol in the protein liquid is adjusted, and a protective agent is added to the protein liquid; the protein liquid is heated so as to cause the denaturation of hybridprotein; a crude product of albumin is finally obtained by pressurization and filtration. The present invention effectively recovers entrainment albumin in precipitates of components by changing pH values, temperatures, protein concentration, ionic strength and ethanol concentration and by heat treatment combined with pressurization and filtration technology. The present invention overcomes the defects of small treating capacity of chromatography columns, large equipment investment for centrifugal separation of solids and liquids and high energy consumption of traditional recovery methods. The present invention effectively increases the total recovery yield of albumin by more than 85%. Moreover, the production cost is reduced, and the production profit is increased.
Description
Technical field
The present invention relates to a kind of discarded component and precipitate the residual albuminous method of recovery 123 from the preparation human plasma protein fraction.
Background technology
Albumin is the highest protein of content in the human plasma, and albumin is at home and abroad widely applied as treatment and nutritious supplementary.At present, the albuminous traditional method of separation and purification is the cold ethanol fractionation precipitation technology that professor E.J.Cohn foundes from human plasma.This technology is by effectively controlling five factors of protein precipitation: pH value, temperature, protein concentration, ionic strength and alcohol concn precipitate the different proteins in the blood plasma step by step, its general step is at first to be settled out deposited components 123, and then be settled out deposited components 4, being settled out deposited components 5 at last is the albumin raw product; Also can be settled out deposited components 1234 by single step reaction, be settled out deposited components 5 then and obtain the albumin raw product.
Yet, because it is a lot of to influence the factor of albumin precipitation separation, the albumin in the blood plasma often has a large amount of losses in fractionation precipitation, and its rate of loss is generally 40%, the part albumin is entrained in the component precipitation 123, also has the part albumin to be entrained in the component precipitation 4; And adopt the albumin loss of single step reaction deposited components 1234 just more.Therefore, reclaiming albumin from the component precipitation is the work with practical significance.And traditional albuminous method that reclaims from the component precipitation generally is to adopt the method that changes deposition condition and column chromatography, optionally adsorbs albumin from the component precipitation, thereby reaches the purpose of recovery.But this tradition recovery method exists the treatment capacity of chromatography column less, the facility investment of centrifugation solid-liquid is big and the higher shortcoming of energy consumption.
Summary of the invention
The objective of the invention is at the problems referred to above, provide a kind of and from component precipitation 123, reclaim albuminous method, can solve the deficiencies in the prior art part.
The present invention utilizes the abundant drip washing component precipitation 123 of the leacheate of certain proportioning; its albumin of carrying secretly is entered in the solution; obtain the protein liquid that volume reduces by ultrafiltration and concentration then; adjust alcohol concn and interpolation protective material in the protein liquid; heat treated makes the foreign protein sex change, obtains albuminous raw product by pressure filtration at last.
Technical scheme of the present invention is as follows: reclaim albuminous method from component precipitation 123, in turn include the following steps:
A, with leacheate drip washing component repeatedly precipitation 123, wherein, leacheate is 890～1088: 185～227 by distilled water, alcohol and disodium hydrogen phosphate by weight: 1 is formulated, PH is 7.4～7.5, temperature is-5.5～-4.5 ℃, and the leacheate consumption is 8 a liters/kg of component precipitation 123; Alcohol density is 800 grams per liters.
B, with the elutant ultrafiltration and concentration collected to protein content more than 3%;
C, add cold ethanol to alcohol concn 8.0 ± 0.5% (V/V) in concentrated solution, and add the Sodium octoate protective material, the consumption of Sodium octoate is 0.10～0.16 grams per liter human plasma;
D, adjustment PH6.45～6.55, and with protein liquid constant temperature thermal treatment 40～90 minutes, thermostat temperature was 68.0 ± 0.5 ℃;
Cooling as early as possible after E, thermal treatment finish is adjusted PH4.2～4.3, and finally is cooled to-1.0～1.0 ℃ when being cooled to below 30 ℃;
F, stir more than one hour, and add the diatomaceous ratio adding of 15～30 grams diatomite mixing by every liter of human plasma amount;
G, filter with containing behind the diatomaceous washing lotion pre-treatment pressure filter protein liquid pumped in the pressure filter, collect filtrate and clarification filtration, wherein washing lotion is 90～110: 6.5～8.5 by distilled water, alcohol and sodium-chlor by weight: 1 is formulated, temperature is-1.0～1.0 ℃, and the content of diatomite in washing lotion is 3～8% (W/V);
H, filtrate adjusting PH6.5～7.0 and ultrafiltration and concentration promptly reclaim and obtain albuminous raw product to protein content 3～4%.
Above-mentioned A go on foot described leacheate by distilled water, alcohol and disodium hydrogen phosphate be by weight 989: 206: 1 formulated.
The consumption that above-mentioned C goes on foot described Sodium octoate is 0.12 grams per liter human plasma.
Above-mentioned G go on foot described washing lotion by distilled water, alcohol and sodium-chlor be by weight 102: 7.6: 1 formulated.
The present invention is by changing pH value, temperature, protein concentration, ionic strength and alcohol concn, adopt the albumin of carrying secretly in the combined with heat treatment pressure filtration technology efficient recovery component precipitation, overcome the shortcoming that the chromatography column treatment capacity is little in traditional recovery method, the facility investment of centrifugation solid-liquid is big and energy consumption is high, effectively improving albuminous total yield reaches more than 85%, and reduced production cost, improved productive profit.
Embodiment
Be that example is set forth recovery method of the present invention now with the component precipitation 123 that reclaims 1500 liters of blood plasma preparations.
Through the deposited components 123 of 1500 liters of blood plasma, be weighed as 210 kilograms through preparing.With the leacheate drip washing precipitation of preparation, the leacheate consumption is that every kilogram of precipitation is used 8 liters, the proportioning of leacheate approximately: 713 liters of distilled water, 186 liters of alcohol, disodium hydrogen phosphate 721 grams, PH7.4～7.5, temperature-5.5～-4.5 ℃.
1630 liters of the elutants of collecting are with concentrated about 200 liters of the protein liquid that obtains of the ultrafilter of NMWL10000 specification.Add cold ethanol in the concentrated solution and reach alcohol concn 8.0 ± 0.5%, and add 180 gram Sodium octoate protective materials.Add sodium-acetate-acetate buffer solution and adjust PH6.45～6.55, and protein liquid is put into thermostat temperature is 68.0 ± 0.5 ℃ constant temperature water tank thermal treatment 40 minutes.Cooling as early as possible after thermal treatment finishes adds dilute hydrochloric acid and adjusts PH4.2～4.3 when being cooled to below 30 ℃, finally be cooled to-1.0～1.0 ℃.
Above-mentioned protein liquid was stirred two hours, adds 25 kilograms of diatomite mixings, and with the washing lotion pre-treatment pressure filter that contains diatomite 3% (W/V), washing lotion is restrained formulated by 320 liters of distilled water, 30 liters of alcohol and sodium-chlor 3150, temperature is-1.0～1.0 ℃.After the pressure filter pre-treatment protein liquid is pumped into wherein filtration, the filtrate of collecting is clarified the filter clarification filtration with the A80 type.Filtrate adds the sodium hydrogen carbonate solution of 0.2mol/L and regulates PH6.5～7.0, is that the ultrafilter of NMWL10000 is concentrated with specification, obtains the concentrated solution of protein content 3.2%.
The albuminous yield of present embodiment is about 300 grams.
Claims (8)
1, from component precipitation 123, reclaims albuminous method, it is characterized in that: use by the abundant drip washing component precipitation 123 of the formulated leacheate of distilled water, alcohol and disodium hydrogen phosphate, its albumin of carrying secretly is entered in the solution, obtain the protein liquid that volume reduces by ultrafiltration and concentration then, adjust alcohol concn and interpolation protective material in the protein liquid, heat treated makes the foreign protein sex change, obtains albuminous raw product by pressure filtration at last; Wherein, leacheate is 890～1088: 185～227 by distilled water, alcohol and disodium hydrogen phosphate by weight: 1 is formulated, and PH is 7.4～7.5, and temperature is-5.5～-4.5 ℃, and the leacheate consumption is 8 a liters/kg of component precipitation 123.
2, method according to claim 1, it in turn includes the following steps:
A, with leacheate drip washing component repeatedly precipitation 123;
B, with the elutant ultrafiltration and concentration collected to protein content more than 3%;
C, add cold ethanol to alcohol concn 8.0 ± 0.5% (V/V) in concentrated solution, and add the Sodium octoate protective material, the consumption of Sodium octoate is 0.10～0.16 grams per liter human plasma;
D, adjustment PH6.45～6.55, and with protein liquid constant temperature thermal treatment 40～90 minutes, thermostat temperature was 68.0 ± 0.5 ℃;
Cooling as early as possible after E, thermal treatment finish is adjusted PH4.2～4.3, and finally is cooled to-1.0～1.0 ℃ when being cooled to below 30 ℃;
F, stir more than one hour, and add the diatomaceous ratio adding of 15～30 grams diatomite mixing by every liter of human plasma amount;
G, filter with containing behind the diatomaceous washing lotion pre-treatment pressure filter protein liquid pumped in the pressure filter, collect filtrate and clarification filtration, wherein washing lotion is 90～110: 6.5～8.5 by distilled water, alcohol and sodium-chlor by weight: 1 is formulated, temperature is-1.0～1.0 ℃, and the content of diatomite in washing lotion is 3～8% (W/V);
H, filtrate adjusting PH6.5～7.0 and ultrafiltration and concentration promptly reclaim and obtain albuminous raw product to protein content 3～4%.
3, method according to claim 2, A go on foot described leacheate by distilled water, alcohol and disodium hydrogen phosphate be by weight 989: 206: 1 formulated.
4, method according to claim 2, the consumption that C goes on foot described Sodium octoate is 0.12 grams per liter human plasma.
5, method according to claim 2, G go on foot described washing lotion by distilled water, alcohol and sodium-chlor be by weight 102: 7.6: 1 formulated.
6, method according to claim 2, the D step is adjusted pH value with sodium-acetate-acetate buffer solution.
7, method according to claim 2, the E step is adjusted pH value with dilute hydrochloric acid.
8, method according to claim 2, the H step is adjusted pH value with the sodium hydrogen carbonate solution of 0.2mol/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100776678A CN1313488C (en) | 2004-12-29 | 2004-12-29 | Method for retrieving albumin form constituent deposition 123 |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2004100776678A CN1313488C (en) | 2004-12-29 | 2004-12-29 | Method for retrieving albumin form constituent deposition 123 |
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| CN1660898A CN1660898A (en) | 2005-08-31 |
| CN1313488C true CN1313488C (en) | 2007-05-02 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2511707B1 (en) * | 2009-12-10 | 2017-02-01 | Sysmex Corporation | Method for detection of basic peptide and reagent for detection of basic peptide |
| CN101792490B (en) * | 2010-01-19 | 2012-06-13 | 广东卫伦生物制药有限公司 | Method for recycling albumin in Cohn's fraction IV precipitate |
| CN102662415B (en) * | 2012-05-11 | 2014-03-26 | 青岛海颐天仪器有限公司 | Method for supplementing leacheate during on-line acidic precipitation monitoring |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1120439A (en) * | 1994-08-10 | 1996-04-17 | 美国拜尔公司 | Low temperature albumin fractionation using sodium caprylates as a partitioning agent |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1120439A (en) * | 1994-08-10 | 1996-04-17 | 美国拜尔公司 | Low temperature albumin fractionation using sodium caprylates as a partitioning agent |
Non-Patent Citations (3)
| Title |
|---|
| 人血清白蛋白纯化技术研究进展 路秀玲等，生物工程学报，第18卷第6期 2002 * |
| 人血白蛋白的两种低温乙醇工艺的比较 张勇等，中国医药工业杂志，第27卷第10期 1996 * |
| 人血白蛋白的两种低温乙醇工艺的比较 张勇等，中国医药工业杂志，第27卷第10期 1996;人血清白蛋白纯化技术研究进展 路秀玲等，生物工程学报，第18卷第6期 2002 * |
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Denomination of invention: Method for retrieving albumin form constituent deposition 123 Effective date of registration: 20100803 Granted publication date: 20070502 Pledgee: Zhanjiang commercial bank Limited by Share Ltd Jinxiu branch Pledgor: GUANGDONG SHUANGLIN BIOLOGICAL PHARMACEUTICAL CO., LTD. Registration number: 2010990000834 |
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