CN101012271A - Method of recovering protein from discarded matter of preparing alginate microcapsule - Google Patents
Method of recovering protein from discarded matter of preparing alginate microcapsule Download PDFInfo
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- CN101012271A CN101012271A CN 200710010200 CN200710010200A CN101012271A CN 101012271 A CN101012271 A CN 101012271A CN 200710010200 CN200710010200 CN 200710010200 CN 200710010200 A CN200710010200 A CN 200710010200A CN 101012271 A CN101012271 A CN 101012271A
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Abstract
The invention discloses a recycling method of protein from waste of alginate microcapsule, which comprises the following steps: separating microcapsule from gelified solution; condensing gel liquid with protein; desalting; adding capsule-breaking liquid in the microcapsule; separating the protein from waste of alginate microcapsule in the stewing or ultrasonic condition through salting-out sedimenting method; purifying further; condensing; obtaining the product.
Description
Technical field
The invention belongs to the bioseparation field of engineering technology, relate to albumen sepn, purification technique, specially refer to from the waste of preparation alginates microcapsule and reclaim proteic method.
Background technology
Protein, polypeptide drug (especially genetically engineered drug and antibody drug) have that toxic side effect is little, target spot is clear and definite, with strong points, good effect, characteristics such as take effect rapidly, is mainly used in major diseases such as treatment tumour, acquired immune deficiency syndrome (AIDS), cardiovascular and cerebrovascular disease.Yet, since such medicine generally have the transformation period short, easily by problems such as enzyme liberating in the body, make it in the oral administration process, be subjected to great restriction; In addition, the cost of this series products of domestic production is high at present, if produce the 1g protein drug, cost needs 6500-40000 yuan, and its major cause is that acquisition means is limited, is difficult to scale operation etc.; And if more surprising from external introduction protein drug price, the Rituxan that introduced in 2005, the price that 400mg/ props up is approximately 21000 yuans, one the courses of treatment 8 pin, about 170,000 yuan; The monoclonal antibody medicine Herceptin of treatment mammary cancer, the price that 250mg/ props up is approximately 15000 yuans.Therefore, the common method that addresses the above problem is at present utilized " drug delivery (DDS) " exactly, prolongs these medicines in vivo working lipe, reduces the using dosage of medicine.
Lalgine is that a class is nontoxic, good biocompatibility, biodegradable natural macromolecular material, can realize the embedding to active substance under mild conditions, is particularly suitable for wrapping and carries very responsive albumen, polypeptide drugs of condition such as temperature, pH values.At present, preparation particle diameter commonly used less than the method for the alginates microcapsule of 500 μ m adopt more emulsion process (CN1850058, CN1374338), electrostatic method (CN1555784) and spray method etc.Though these methods all possess certain industrial scale, and the particle size range of Zhi Bei alginates microcapsule all can satisfy the actual needs of different way of administration separately, but above method all can the generating unit division aspect and irregular microcapsule or fragment, and all there is certain size distribution in the microcapsule that make, cause these drug-loading microcapsules can not all satisfy strict demand as the vivo medicine-feeding preparation; Simultaneously, owing to the alginates microcapsule that particle diameter is less are limited to the carrying capacity of medicine, and has a bigger pore texture, cause in preparation and carry in the process of protein medicaments alginates microcapsule, still have diffuse in a large number in the gelating soln system and do not reach that protein drug in the alginates microcapsule that preparation requires needs to separate again, purifying, so that recycling, this not only can improve the target that DDS acts in vivo, also will reduce the production cost of DDS greatly.
Summary of the invention
The purpose of this invention is to provide a kind of proteic method that from the waste for preparing the alginates microcapsule, reclaims, the alginates microcapsule that do not meet the preparation requirement are at first broken capsule to be handled, the broken capsule liquid of gained is saltoutd, precipitation is with the physiological saline solution after-filtration and through gel chromatography, promptly obtain the protein solution that reclaims, after testing protein solution purity 〉=95% that should reclaim.
For achieving the above object, the present invention carries out according to following technical scheme:
The coagulant liquid that contains the alginates microcapsule that carry protein medicaments that 1) will prepare, according to the preparation actual needs, adopt a kind of or more than one arbitrary combination in method of sieving, centrifugal settling method and the cyclonic separation method to isolate liquid phase, meet the microcapsule that preparation requires and do not reach the microcapsule that preparation requires;
2), adopt a kind of or more than one arbitrary combination in ultrafiltration, dialysis and the gel chromatography method that it is concentrated and desalting treatment to step 1) gained liquid;
3) to the alginates microcapsule that preparation requires that do not reach of step 1) gained, according to microcapsule: broken capsule liquid weight ratio is 1: 20～1: 50 a ratio, after adding concentration is the broken capsule liquid of 0.05～0.2mol/L, leave standstill 8～24h, wherein broken capsule liquid can be a kind of or more than one arbitrary combination in Citrate trianion, phosphoric acid salt, EGTA and the lactic acid salt;
4) to the centrifugal 5～20min under 1000～10000rpm of step 3) gained liquid elder generation, the gained supernatant liquor leave standstill or ultransonic condition under carry out salt precipitation after, centrifugal 5～20min under 1000～10000rpm again, what the employed salt of wherein saltouing can be in the ammonium sulfate of 30%～100% saturation ratio, sodium sulfate (potassium), sodium-chlor (potassium), the sal epsom is a kind of;
5) with gained precipitation in the step 4), after the damping fluids dissolving with a kind of or more than one arbitrary combination among the PBS of the Tris-HCl of physiological saline, 0.05～0.2mol/L and 0.05～0.2mol/L, again through the filtering with microporous membrane of 0.45～0.22 μ m;
6) with gel chromatography column on the liquid of step 5) gained, moving phase select for use with step 6) in identical damping fluid during dissolution precipitation, flow velocity with 0.1～1mL/min, detect and collect proteic absorption peak, wherein the medium of gel chromatography is looked and is reclaimed proteic different in kind, can select a kind of in dextran, agarose or the polyacrylamide of corresponding granular size for use;
7) liquid that step 6) is collected adopts a kind of or more than one arbitrary combination in ultrafiltration, dialysis and the gel chromatography method to concentrate and desalting treatment.
Effect of the present invention and benefit be adopt that step is simple, method that easy handling and industry are amplified directly separates, reclaims albumen from carry protein medicaments alginates microcapsule, improve the utilization ratio of protein medicaments, greatly reduced the production cost of protein medicaments transfer system.This method equally also has the advantages such as biological activity that good reproducibility, cost are low, do not destroy protein drug.
Embodiment
Below in conjunction with technical scheme, the calcium alginate microcapsule that carries BSA for preparing with spray method is an example, is described in detail embodiments of the present invention.
Embodiment 1:
1) carries the Preparation of Calcium Alginate Microcapsule of BSA: the sodium alginate of 2.2% (w/v) is dissolved in the 45mL deionized water, adds the BSA solution of 5mL, 60mg/ml again, stir.Adjustments of gas speed is 0.7m
3/ h, liquid velocity are 13mL/min, adopt spray method, and above-mentioned raw materials liquid is sprayed into 400mLCaCl
2In the solution, continue to stir 0.5h.
2) separation of different-grain diameter scope microcapsule: with the CaCl that contains the alginates microcapsule that obtains in the step 1)
2At first (sieve aperture: 0.111mm) sub-sieve separates solution, the solid phase of damming I through 130 orders; Liquid phase I centrifugal 10min separation under 1000rpm obtains solid phase II then; This moment, liquid phase II was by 0.45 μ m membrane filtration, obtained solid phase III and liquid phase III.
3) the concentrating of liquid phase III, desalination step 2): the method that adopts ultrafiltration is to step 2) in liquid phase III concentrate and desalination, the molecular cut-off value of employing film is 10kD in the ultra-filtration process.
4) solid phase I and III place beaker the broken capsule of solid phase I and III step 2): get step 2), add broken capsule liquid (sodium citrate solution of 0.2mol/L) with the ratio of microcapsule and broken capsule liquid 1: 30 (g/mL), leave standstill 12h under 4 ℃.
5) the saltouing of BSA in the broken capsule liquid of step 4) gained: at first with the broken capsule liquid centrifugal 10min under 8000rpm of step 4) gained, the solid ammonium sulfate that in the liquid phase IV that obtains, adds 40%～85% saturation ratio, after leaving standstill 12h under 4 ℃, centrifugal 20min under 8000rpm; Again with the centrifugal post precipitation that obtains of 0.9% NaCl solution dissolving, by 0.45 this lysate of μ m water film filtering (liquid phase V).
6) gel chromatography of step 5) gained liquid phase V: step 5) gained liquid phase V is added to uses phosphoric acid buffer (0.1mol/L, contain 0.2mol/L NaCl, pH 7.4) the Sephadex G-75 chromatography column that balance is good (on the Φ 1.6cm * 55cm) in advance, flow velocity with 0.5mL/min carries out gel chromatography, carry out wash-out with balance liquid, ultraviolet 280nm detects and collects the albumen absorption peak.
7) the concentrating of the liquid collected of step 6), desalination: the liquid that adopts the method for ultrafiltration that step 6) is collected concentrates, desalting treatment, and adopting the molecular cut-off value of film in the ultra-filtration process is 10kD.
The result is as follows for the experiment gained:
The CaCl that contains BSA
2The rate of recovery of BSA is 81.38% in the solution; The rate of recovery of BSA in the microcapsule is 58.48%.The BSA purity of the two all can reach more than 98%.
Embodiment 2:
1) carries the Preparation of Calcium Alginate Microcapsule of BSA: the sodium alginate of 2.2% (w/v) is dissolved in the 45mL deionized water, adds the BSA solution of 5mL, 80mg/ml again, stir.Adjustments of gas speed is 0.7m
3/ h, liquid velocity are 13mL/min, by the spray gun of atomizer, above-mentioned raw materials liquid are sprayed into 500mL CaCl
2In the solution, continue to stir 0.5 h.
2) separation of different-grain diameter scope microcapsule: with the CaCl that contains the alginates microcapsule that obtains in the step 1)
2At first (sieve aperture: 0.100mm) sub-sieve separates solution, the solid phase of damming I through 150 orders; Liquid phase I centrifugal 10min separation under 1000rpm obtains solid phase II then; This moment, liquid phase II was by 0.45 μ m membrane filtration, obtained solid phase III and liquid phase III.
3) the concentrating of liquid phase III, desalination step 2): the method that adopts ultrafiltration is to step 2) in liquid phase III concentrate and desalination, the molecular cut-off value of employing film is 10kD in the ultra-filtration process.
4) solid phase I and III place beaker the broken capsule of solid phase I and III step 2): get step 2), add broken capsule liquid (sodium citrate solution of 0.2mol/L) with the ratio of microcapsule and broken capsule liquid 1: 30 (g/mL), leave standstill 12h under 4 ℃.
5) the saltouing of BSA in the broken capsule liquid of step 4) gained: at first with step 2) the broken capsule liquid centrifugal 10min under 8000rpm of gained, the solid ammonium sulfate that in the liquid phase IV that obtains, adds 60%～85% saturation ratio, ultrasonic 3min under 250W, 20kHz, leave standstill 1h after, centrifugal 20min under 8000rpm; Dissolve the precipitation of centrifugal gained again with 0.9% NaCl solution, and by 0.45 this lysate of μ m water film filtering (liquid phase V).
6) gel chromatography of step 5) gained liquid phase V: step 5) gained liquid phase V is added to uses phosphoric acid buffer (0.1mol/L, contain 0.2mol/L NaCl, pH 7.4) the Sephadex G-75 chromatography column that balance is good (on the Φ 1.6cm * 55cm) in advance, flow velocity with 0.5mL/min carries out gel chromatography, carry out wash-out with balance liquid, ultraviolet 280nm detects and collects the albumen absorption peak.
7) the concentrating of the liquid collected of step 6), desalination: the solution that adopts the method for ultrafiltration that step 6) is collected concentrates, desalting treatment, and adopting the molecular cut-off value of film in the ultra-filtration process is 10kD.
The result is as follows for the experiment gained:
The CaCl that contains BSA
2The rate of recovery of BSA is 81.38% in the solution; The rate of recovery of BSA in the microcapsule is 50.15%, and the operating time has shortened 42.31%.The BSA purity of the two all can reach more than 98%.
Claims (3)
1. one kind is reclaimed proteic method from the waste of preparation alginates microcapsule, it is characterized in that this method follows these steps to carry out:
The coagulant liquid that contains the alginates microcapsule that 1) will prepare, actual requirement according to pharmaceutical preparation, adopt a kind of or more than one arbitrary combination in method of sieving, centrifugal settling method and the cyclonic separation method, isolate liquid phase, meet the microcapsule of preparation requirement and do not reach the microcapsule that preparation requires;
2), adopt a kind of or more than one arbitrary combination in ultrafiltration, dialysis and the gel chromatography method that it is concentrated and desalting treatment to step 1) gained liquid;
3) to the alginates microcapsule that preparation requires that do not reach of step 1) gained, according to microcapsule: broken capsule liquid weight ratio is 1: 20～1: 50 a ratio, after adding concentration is the broken capsule liquid of 0.05～0.2mol/L, leave standstill 8～24h, wherein broken capsule liquid is a kind of in Citrate trianion, phosphoric acid salt, EGTA and the lactic acid salt;
4) to the centrifugal 5～20min under 1000～10000rpm of step 3) gained liquid elder generation, the gained supernatant liquor leave standstill or ultransonic condition under carry out salt precipitation after, centrifugal 5～20min under 1000～10000rpm again, the employed salt of wherein saltouing are a kind of in the ammonium sulfate, sodium sulfate (potassium), sodium-chlor (potassium), sal epsom of 30%～100% saturation ratio;
5) centrifugation gets the precipitation in the step 4), after the damping fluids dissolving with a kind of or more than one arbitrary combination among the PBS of the Tris-HCl of physiological saline, 0.05～0.2mol/L and 0.05～0.2mol/L, again through the filtering with microporous membrane of 0.45～0.22 μ m;
6) with gel chromatography column on the liquid of step 5) gained, moving phase select for use with step 6) in identical damping fluid during dissolution precipitation, flow velocity with 0.1～1mL/min, detect and collect proteic absorption peak, the proteic different in kind of the visual recovery of the medium of gel chromatography is selected a kind of in dextran, agarose or the polyacrylamide of corresponding granular size for use;
7) liquid that step 6) is collected adopts a kind of or more than one arbitrary combination in ultrafiltration, dialysis and the gel chromatography method to concentrate and desalting treatment.
2. a kind of proteic method that reclaims from the waste of preparation alginates microcapsule according to claim 1 is characterized in that adopting a kind of or more than one arbitrary combination in method of sieving, centrifugal settling method and the cyclonic separation method to isolate the microcapsule of different-grain diameter scope.
3. a kind of proteic method that from the waste of preparation alginates microcapsule, reclaims according to claim 1, it is characterized in that selecting for use in the ammonium sulfate, sodium sulfate (potassium), sodium-chlor (potassium), sal epsom of 30%～100% saturation ratio a kind of leave standstill or ultransonic condition under the broken capsule supernatant liquor after centrifugal is carried out salt precipitation.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102150767A (en) * | 2010-12-23 | 2011-08-17 | 青岛明月海藻集团有限公司 | Composite edible gel material capable of forming into thermal irreversible gel by means of thermal melting and preparation method thereof |
| CN101831002B (en) * | 2009-03-11 | 2011-11-16 | 中国科学院大连化学物理研究所 | Preparation method of sodium alginate for tissue engineering |
| CN102626399A (en) * | 2012-04-05 | 2012-08-08 | 中国科学院化学研究所 | Calcium alginate microcapsule, preparation method and application thereof |
| CN102053123B (en) * | 2009-11-04 | 2013-09-11 | 中国科学院大连化学物理研究所 | Method for detecting activation of microencapsulated cells |
| CN105734006A (en) * | 2014-12-09 | 2016-07-06 | 中国科学院大连化学物理研究所 | Preparation method of acellular sodium alginate bionic hydrogel |
| CN106492714A (en) * | 2016-10-28 | 2017-03-15 | 华中科技大学 | The preparation and application of calcium alginate coated Nanoscale Iron microsphere |
| CN111562210A (en) * | 2020-06-16 | 2020-08-21 | 北京挑战农业科技有限公司 | Method for detecting viable count in pre-coated forage microecological preparation product |
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2007
- 2007-01-24 CN CNB2007100102005A patent/CN100522990C/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831002B (en) * | 2009-03-11 | 2011-11-16 | 中国科学院大连化学物理研究所 | Preparation method of sodium alginate for tissue engineering |
| CN102053123B (en) * | 2009-11-04 | 2013-09-11 | 中国科学院大连化学物理研究所 | Method for detecting activation of microencapsulated cells |
| CN102150767A (en) * | 2010-12-23 | 2011-08-17 | 青岛明月海藻集团有限公司 | Composite edible gel material capable of forming into thermal irreversible gel by means of thermal melting and preparation method thereof |
| CN102150767B (en) * | 2010-12-23 | 2013-08-28 | 青岛明月海藻集团有限公司 | Composite edible gel material capable of forming into thermal irreversible gel by means of thermal melting and preparation method thereof |
| CN102626399A (en) * | 2012-04-05 | 2012-08-08 | 中国科学院化学研究所 | Calcium alginate microcapsule, preparation method and application thereof |
| CN105734006A (en) * | 2014-12-09 | 2016-07-06 | 中国科学院大连化学物理研究所 | Preparation method of acellular sodium alginate bionic hydrogel |
| CN105734006B (en) * | 2014-12-09 | 2020-02-14 | 中国科学院大连化学物理研究所 | Preparation method of acellular sodium alginate bionic hydrogel |
| CN106492714A (en) * | 2016-10-28 | 2017-03-15 | 华中科技大学 | The preparation and application of calcium alginate coated Nanoscale Iron microsphere |
| CN111562210A (en) * | 2020-06-16 | 2020-08-21 | 北京挑战农业科技有限公司 | Method for detecting viable count in pre-coated forage microecological preparation product |
| CN111562210B (en) * | 2020-06-16 | 2023-01-03 | 北京挑战农业科技有限公司 | Method for detecting number of viable bacteria in pre-coated feed microecological preparation product |
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