CN102786577B - Simple device for preparing plasma protein products through batch adsorption, and method thereof - Google Patents
Simple device for preparing plasma protein products through batch adsorption, and method thereof Download PDFInfo
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- CN102786577B CN102786577B CN201210252349.5A CN201210252349A CN102786577B CN 102786577 B CN102786577 B CN 102786577B CN 201210252349 A CN201210252349 A CN 201210252349A CN 102786577 B CN102786577 B CN 102786577B
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Abstract
The invention relates to the fields of the medical and biological technologies, and discloses a simple device for preparing plasma protein products through batch adsorption, and a method thereof. The device comprises an upper end sealing cover (1), a reaction bottle (2), a lower end sealing cover (3), a filter cloth sheet (4) and an openmouthed cover (5). The method is characterized in that two ends of the reaction bottle (2) are sealed through the upper end sealing cover (1), the filter cloth sheet (4) and the lower end sealing cover (3); the addition of a material in the reaction bottle (2) is realized through opening the lower end sealing cover (3); a liquid component in the reaction bottle (2) is filtered by the filter cloth sheet (4) through replacing the upper end sealing cover (5) by the openmouthed cover (5) and through pressurizing the middle part of the reaction bottle (2); and the whole batch adsorption preparation process comprising medium balancing, plasma protein adsorption, other protein washing and target plasma protein eluting is carried out in the reaction bottle (2). The device and the method simplify equipment and operations required by the preparation of the plasma proteins through the batch adsorption, and can simply realize the efficient separation of various plasma proteins. Same-batch experiments enable the screening of various conditions of the batch-adsorption preparation technology to be completed by utilizing a plurality of the devices.
Description
Technical field
The present invention relates to medical biotechnology field, is easy device and method that plasma protein products is prepared in a kind of batch of formula absorption.
Background technology
The formula of criticizing absorption is plasma proteins industry appellation sanctified by usage, it is one of method of preparing plasma protein products, detailed process refers to: blood plasma or its compositions derived therefrom are rich in multiple plasma proteins, under certain condition blood plasma or its compositions derived therefrom with together with medium with special groups, fully mix, and object plasma proteins is combined with medium, filter out afterwards medium, then carry out as washing, wash-out subsequent disposal, thereby isolate the process of object plasma proteins.This preparation method typically used both domestic and external is exactly the preparation for Prothrombin Complex Concent-.
Along with understanding in depth and the exploitation of specific binding novel medium of existing dielectric behavior, the formula absorption of criticizing is applied in the preparation of multiple plasma proteins with the comparatively simple and direct advantage of its treating processes, but, with regard to laboratory study, plasma proteins is prepared in the formula absorption of criticizing does not have supporting device and corresponding method thereof so far, this situation has limited batch application of formula absorption method in plasma protein products separation and Extraction, has also hindered relevant plasma protein products in addition and criticize formula absorption method preparation technology's research.
Summary of the invention
In view of the foregoing, the object of the present invention is to provide a kind of batch of formula absorption to prepare easy device and the method for plasma protein products, can carry out simply plasma proteinss such as Prothrombin Complex Concent-, PROTEIN C, albumen Z, Protein S, blood coagulation factor VIIIs, effectively extract and separate by these apparatus and method, can complete comparison and screening with batch kinds of processes condition simultaneously.
Technical scheme:
The present invention is easy device and the method that plasma protein products is prepared in a kind of batch of formula absorption.
Described device comprises: upper end closed cover, reaction flask, lower end closed cover, filter cloth sheet and uncovered lid five parts.Described reaction flask be two ends not only can be airtight but also can opening, the resilient cylindrical plastic of middle portion or quality of rubber materials container; Described upper end closed cover and lower end closed cover be with uncovered lid comparatively speaking, refer to that the circular surface of lid is completely airtight; Described uncovered lid refers to that there is circular hole the circular surface centre of lid; Described uncovered cap surface Circularhole diameter is not less than 3/4 of circular lid diameter; Sealing and the unlatching of the airtight and opening of described reaction flask by closed cover or be replaced by uncovered lid and realize; Described reaction flask upper end open disposes upper end closed cover and uncovered lid; Described reaction flask lower ending opening configuration lower end closed cover; Between described reaction flask upper end open place and upper end closed cover and uncovered lid, there is filter cloth sheet; Described filter cloth sheet filter opening footpath, depending on media particle size, should be less than 1/3 of media particle size.
Corresponding said apparatus, described method concrete operations are as follows:
(1) balanced medium
Filter cloth sheet is positioned over reaction flask upper end open, and upper end closed cover sealed upper end opening is inverted reaction flask.Take adsorption medium, the amount of taking is determined according to plasma volume, pours in reaction flask, and then add appropriate balance liquid from lower ending opening, carries out balanced medium.Balance is complete, and lower end closed cover sealing lower ending opening, is just putting reaction flask, and upper end closed cover is replaced by uncovered lid, outside filter cloth sheet middle portion is exposed to, then is inverted reaction flask, and to the pressurization of reaction flask middle part, balance liquid leaches by filter cloth sheet.
(2) plasma proteins absorption
Just putting reaction flask, the uncovered lid in upper end open place is replaced by upper end sealing cover, is inverted reaction flask, opens lower end sealing cover, adds blood plasma, sealing lower ending opening, and now reaction flask is in two ends air-tight state.Fixation reaction bottle, in temperature control shaking table, regulates shaking table temperature and rotating speed, carries out plasma proteins absorption.Adsorb completely, just putting reaction flask, upper end closed cover is replaced by uncovered lid, then is inverted reaction flask, to the pressurization of reaction flask middle part, leaches blood plasma.
(3) medium washing
Just putting reaction flask, changing uncovered lid is upper end closed cover, is inverted reaction flask, opens lower ending opening, adds washings, closed lower end opening.Fixation reaction bottle, in temperature control shaking table, regulates temperature and rotating speed, washing foreign protein.Complete, leach washings by aforesaid operations.
(4) plasma proteins wash-out
Just putting reaction flask, changing uncovered lid is upper end closed cover, is inverted reaction flask, opens lower ending opening, adds elutriant, closed lower end opening.Fixation reaction bottle, in temperature control shaking table, regulates temperature and rotating speed, wash-out target protein.Complete, leach elutriant by aforesaid operations, be object plasma proteins enriched material.
Beneficial effect:
The invention has the beneficial effects as follows, provide a kind of batch of formula absorption to prepare the easy device and method of plasma protein products, by experiment, can complete simply and effectively Prothrombin Complex Concent-, PROTEIN C, albumen Z, Protein S, the separation of blood coagulation factor VIII etc., wherein prothrombin in Prothrombin Complex Concent-, VII, IX, X yield can reach respectively: 92.26%, 70.86%, 97.05%, 82.40%, specific activity can reach respectively simultaneously: 0.95IU/mg, 0.63IU/mg, 1.05IU/mg, 0.76IU/mg, higher than the requirement of plasma thromboplastin component specific activity >=0.3IU/mg in " Chinese Pharmacopoeia " (2010 editions) Prothrombin Complex Concent-, PROTEIN C yield and specific activity are respectively 92.20%, 1.04IU/mg, Protein S yield and specific activity are respectively 81.00%, 1.06IU/mg, albumen Z yield and specific activity are respectively 88.18%, 4.37 μ g/mg (by albumen Z cubage), blood coagulation factor VIII yield and specific activity are respectively 61.30%, 1.02IU/mg.In addition, utilize multiple these devices, can realize batch same batch of comparison of formula absorption kinds of processes condition, the screening of preparing plasma proteins processing condition for criticizing formula absorption provides and has provided powerful support for.
Brief description of the drawings:
Fig. 1 is device appearance schematic diagram.
Fig. 2 is device decomposing schematic representation.
Fig. 3 is filter cloth sheet vertical view.
The uncovered lid vertical view of Fig. 4.
In figure: 1. upper end closed cover; 2. reaction flask; 3. lower end closed cover; 4. filter cloth sheet; 5. uncovered lid.
Embodiment
By reference to the accompanying drawings and embodiment the present invention is elaborated, but embodiments of the present invention are not limited to this.
Embodiment 1: Prothrombin Complex Concent-preparation experiment
(1) balanced medium
Place filter cloth sheet 4 in reaction flask 2 upper end open places, with closed cover 1 sealed upper end opening, be inverted reaction flask 2.Take 0.15g DEAE-Sephedex A50 medium, measure 80ml balance liquid, lower ending opening place is poured in reaction flask 2 successively, and described balance liquid is the sodium citrate solution of pH6.8, containing the NaCl of 0.05mol/L.After mixing, 4～8 DEG C of conditions are carried out DEAE-Sephedex A50 balanced medium.After 24h, closed cover 3 seals lower ending opening, is just putting reaction flask 2, and upper end closed cover 1 is replaced by uncovered lid 5, is inverted reaction flask 2, reaction flask 2 middle part pressurizations, and balance liquid leaches by filter cloth sheet 4; Just putting reaction flask 2, uncovered lid 5 is replaced by upper end closed cover 1, is inverted reaction flask 2, opens lower ending opening, then adds 80ml balance liquid to DEAE-Sephedex A50 balanced medium, after 24h, leaches by aforesaid operations.
(2) absorption of prothrombin, VII, IX, X
Get 100ml Healthy People pooled plasma, lower ending opening is poured reaction flask 2 into, and closed cover 3 seals lower ending opening, and fixation reaction bottle 2 is in temperature control shaking table, and regulating temperature is 4 DEG C, and rotating speed is 200rpm, absorption 40min.Adsorb completely, changing upper end closed cover 1 be uncovered lid 5, leaches mixed slurry.
(3) foreign protein washing
Get respectively 50ml washings, described washings is the sodium citrate solution of pH7.0, containing the NaCl of 0.1mol/L, and by adsorption process operation, washing DEAE-Sephedex A50 medium 2 times, each 20min, leaches washings.
(4) wash-out of prothrombin, VII, IX, X
Get respectively 25ml elutriant, described elutriant is the sodium citrate solution of pH7.2, containing the NaCl of 2.4mol/L, and by adsorption process operation, wash-out 2 times, each 15min.
Collect elutriant, obtain Prothrombin Complex Concent-enriched material, the yield of its prothrombin, VII, IX, X and specific activity are in table 1.
Yield and the specific activity of four kinds of main thrombin of table 1 Prothrombin Complex Concent-enriched material
| Type | Yield (%) | Specific activity (IU/mg) |
| Prothrombin | 92.26 | 0.95 |
| Proconvertin | 70.86 | 0.63 |
| Plasma thromboplastin component | 97.05 | 1.05 |
| Factor X | 82.40 | 0.76 |
Embodiment 2: PROTEIN C separation and purification experiment
(1) balanced medium
Take 0.3g DEAE-Sephedex A50 medium, get respectively 300ml balance liquid, described balance liquid is pH7.0 sodium citrate solution, containing the NaCl of 0.05mol/L.2 balances of DEAE-Sephedex A50 medium are operated by (1) in embodiment 1.
(2) absorption of PROTEIN C
Get 100ml Healthy People pooled plasma; Shaking table temperature is 4 DEG C, and rotating speed is 250rpm, 50min.Operated the absorption of PROTEIN C by (2) in embodiment 1.
(3) foreign protein washing
Get respectively 150ml washings, described washings is pH7.4 sodium citrate solution, containing the NaCl of 0.1mol/L.2 washings of foreign protein, each 30min are operated by (3) in embodiment 1.
(4) wash-out of PROTEIN C
Get respectively 50ml elutriant, described elutriant is the sodium citrate solution of pH7.4, containing 1.5mol/L NaCl.2 wash-outs of PROTEIN C are operated by (4) in embodiment 1, each 20min.
Collect elutriant, obtain PROTEIN C enriched material, its PROTEIN C yield and specific activity are in table 2.
Yield and the specific activity of PROTEIN C in table 2 PROTEIN C enriched material
| Type | Yield (%) | Specific activity (IU/mg) |
| PROTEIN C | 92.20 | 1.04 |
Embodiment 3: Protein S separation and purification experiment
(1) balanced medium
Take 0.2g DEAE-Sephedex A50 medium, get respectively 120ml balance liquid, described balance liquid is pH7.0 sodium citrate solution, containing the NaCl of 0.075mol/L.2 balances of DEAE-Sephedex A50 medium are operated by (1) in embodiment 1.
(2) absorption of Protein S
Get 100ml Healthy People pooled plasma; Shaking table temperature is 4 DEG C, and rotating speed is 230rpm, 40min.Operated the absorption of Protein S by (2) in embodiment 1.
(3) foreign protein washing
Get respectively 40ml washings, described washings is the sodium citrate solution of pH7.0, containing the NaCl of 0.1mol/L.2 washings of foreign protein, each 30min are operated by (3) in embodiment 1.
(4) wash-out of Protein S
Get respectively 25ml elutriant, described elutriant is the sodium citrate solution of pH7.0, containing the NaCl of 1.6mol/L.2 wash-outs of Protein S are operated by (4) in embodiment 1, each 15min.
Collect elutriant, obtain Protein S enriched material, its Protein S yield and specific activity are in table 3.
Yield and the specific activity of Protein S in table 3 Protein S enriched material
| Type | Yield (%) | Specific activity (IU/mg) |
| Protein S | 81.00 | 1.06 |
Embodiment 4: albumen Z separation and purification experiment
(1) balanced medium
Take 0.25g DEAE-Sephedex A50 medium, get respectively 150ml balance liquid, described balance liquid is pH7.0 sodium citrate solution, containing the NaCl of 0.075mol/L.2 balances of DEAE-Sephedex A50 medium are operated by (1) in embodiment 1.
(2) absorption of albumen Z
Get 100ml Healthy People pooled plasma; Shaking table temperature is 4 DEG C, and rotating speed is 220rpm, 40min.Operated the absorption of albumen Z by (2) in embodiment 1.
(3) foreign protein washing
Get respectively 100ml washings, described washings is the sodium citrate solution of pH7.0, containing the NaCl of 0.1mol/L.2 washings of foreign protein, each 25min are operated by (3) in embodiment 1.
(4) wash-out of albumen Z
Get respectively 30ml elutriant, described elutriant is the sodium citrate solution of pH7.0, containing the NaCl of 1.6mol/L.2 wash-outs of albumen Z are operated by (4) in embodiment 1, each 15min.
Collect elutriant, obtain albumen Z enriched material, its albumen Z yield and specific activity are in table 4.
Yield and the specific activity of albumen Z in table 4 albumen Z enriched material
| Type | Yield (%) | Specific activity (μ g/mg) |
| Albumen Z | 88.18 | 4.37 |
(1) the synthetic CGA-15 balanced medium of self-control
Take the synthetic CGA-15 medium of 10g self-control, get respectively 200ml balance liquid, described balance liquid is the sodium citrate solution of pH6.5.Room temperature condition, has operated 3 balances of CGA-15 medium by (1) in embodiment 1.
(2) absorption of blood coagulation factor VIII
Get 80ml Healthy People pooled plasma; Shaking table temperature is 20 DEG C, and rotating speed is 220rpm, 30min.Operated the absorption of blood coagulation factor VIII by (2) in embodiment 1.
(3) foreign protein washing
Get respectively 100ml balance liquid, operated 2 washings of foreign protein, each 15min by (3) in embodiment 1.
(4) wash-out of blood coagulation factor VIII
Get respectively 40ml elutriant, described elutriant is pH7.0, the solution of 0.6mol/L concentration NaCl.2 wash-outs of blood coagulation factor VIII are operated by (4) in embodiment 1, each 20min.
Collect elutriant, obtain blood coagulation factor VIII enriched material, its blood coagulation factor VIII yield and specific activity are in table 5.
Yield and the specific activity of table 5 blood coagulation factor VIII enriched material blood coagulation factor VIII
| Type | Yield (%) | Specific activity (IU/mg) |
| Blood coagulation factor VIII | 61.30 | 1.02 |
The different elution requirement screening experiments of embodiment 6 Protein S
(1) balanced medium
Get 9 covering devices, take respectively 9 parts of 0.2g DEAE-Sephedex A50 media, get respectively 9 portions of 120ml balance liquids, described balance liquid is pH6.8 sodium citrate solution, containing the NaCl of 0.05mol/L.2 balances of DEAE-Sephedex A50 medium in 9 covering devices are operated by (1) in embodiment 1.
(2) absorption of Protein S
Get 9 parts of 120ml Healthy People pooled plasmas; Shaking table temperature is 4 DEG C, and rotating speed is 220rpm, 50min.9 covering devices are all fixed on same shaking table, have operated the absorption of Protein S by (2) in embodiment 1.
(3) foreign protein washing
Get respectively 9 parts of 40ml washingss, described washings is the sodium citrate solution of pH7.2, containing the NaCl of 0.1mol/L.Complete respectively 2 washings of foreign protein, each 20min by (3) operation in embodiment 1.
(4) wash-out of Protein S
Get respectively 9 parts of 30ml elutriants, described elutriant composition is in table 6.Complete respectively 2 wash-outs of Protein S by (4) operation in embodiment 1, each 15min.
Collect respectively elutriant, obtain Protein S enriched material, its Protein S yield and specific activity are in table 7.
Table 69 kind of Protein S elutriant
Yield and the specific activity of Protein S in table 7 Protein S enriched material
| Elution requirement type | Yield (%) | Specific activity (IU/mg) |
| One | 80.55 | 0.63 |
| Two | 63.01 | 0.57 |
| Three | 64.33 | 0.63 |
| Four | 58.61 | 0.45 |
| Five | 44.53 | 0.43 |
| Six | 42.16 | 0.41 |
| Seven | 87.26 | 0.84 |
| Eight | 67.21 | 0.59 |
| Nine | 53.58 | 0.61 |
Claims (2)
1. one kind batch of formula is adsorbed the easy device of preparing plasma protein products, it is characterized in that: comprise upper end closed cover, reaction flask, lower end closed cover, filter cloth sheet and uncovered lid five parts, described reaction flask is that two ends not only can be airtight but also can opening, the resilient cylindrical plastic of middle portion or quality of rubber materials container, the airtight sealing by closed cover of described reaction flask realizes, the opening of described reaction flask is opened by closed cover and closed cover is replaced by uncovered lid and realizes, described uncovered lid is the middle round-meshed bottle cap of the circular surface of lid, described filter cloth sheet is middle 1～3 metafiltration cloth, rigid plastics ring or the fixing disc-shaped structure of rubber ring for edge, described filter cloth sheet is embedded in the middle of reaction flask upper end open and upper end closed cover or uncovered lid.
2. utilize the easy device of claim 1 to prepare a method for plasma protein products, it is characterized in that: sealed at both ends by lower end closed cover, upper end closed cover and filter cloth sheet realization response bottle; Be inverted reaction flask and open lower end closed cover, the interpolation of realization response vial material; Just putting reaction flask, upper end closed cover is replaced by uncovered lid, then is inverted reaction flask to the pressurization of reaction flask middle part, and in reaction flask, liquid component leaches by filter cloth sheet; Balanced medium, plasma proteins absorption, foreign protein washing, whole batch of formula absorption preparation process of object plasma proteins wash-out are all carried out in reaction flask; Reaction flask completes plasma proteins absorption, medium washing and plasma proteins elution process in shaking table.
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| CN201210252349.5A CN102786577B (en) | 2012-07-20 | 2012-07-20 | Simple device for preparing plasma protein products through batch adsorption, and method thereof |
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| CN102786577B true CN102786577B (en) | 2014-07-09 |
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| CN111965303A (en) * | 2020-07-25 | 2020-11-20 | 山东泰邦生物制品有限公司 | Device for batch type adsorption separation test and using method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3560066B2 (en) * | 1993-12-17 | 2004-09-02 | 三菱ウェルファーマ株式会社 | Method for fractionating plasma protein |
| CN2911683Y (en) * | 2006-07-11 | 2007-06-13 | 北京丰杰华信科学仪器有限公司 | Gel chromatographic column |
| CN101444757A (en) * | 2007-08-14 | 2009-06-03 | 米利波尔有限公司 | Porous sorptive medium, sorption device containing the medium and chromatography system |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3560066B2 (en) * | 1993-12-17 | 2004-09-02 | 三菱ウェルファーマ株式会社 | Method for fractionating plasma protein |
| CN2911683Y (en) * | 2006-07-11 | 2007-06-13 | 北京丰杰华信科学仪器有限公司 | Gel chromatographic column |
| CN101444757A (en) * | 2007-08-14 | 2009-06-03 | 米利波尔有限公司 | Porous sorptive medium, sorption device containing the medium and chromatography system |
Non-Patent Citations (2)
| Title |
|---|
| Contributions to the Optimal Use of Human Blood.11. The Large-Scale Preparation of Prothrombin Complex.A Comparison between Two Methods Using the Anion Exchangers DEAE-Cellulose DE 52 and DEAE-Sephadex A-50;J. HEYST等;《Vox Sang》;19730831;第114页第6段，第116页图2，第117页4-7段 * |
| 人凝血酶原复合物分离纯化工艺的研究进展;邱家山等;《生物技术通报》;20060430(第2期);第18页右栏，图1 * |
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