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CN1179355A - Process for preparing protein adsorbent and using method thereof - Google Patents

Process for preparing protein adsorbent and using method thereof Download PDF

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Publication number
CN1179355A
CN1179355A CN 96120043 CN96120043A CN1179355A CN 1179355 A CN1179355 A CN 1179355A CN 96120043 CN96120043 CN 96120043 CN 96120043 A CN96120043 A CN 96120043A CN 1179355 A CN1179355 A CN 1179355A
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China
Prior art keywords
adsorbent
silica gel
protein
gel particle
resin
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Pending
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CN 96120043
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Chinese (zh)
Inventor
朱玉含
朱文瑾
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Individual
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Individual
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Priority to CN 96120043 priority Critical patent/CN1179355A/en
Publication of CN1179355A publication Critical patent/CN1179355A/en
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  • Peptides Or Proteins (AREA)

Abstract

A protein adsorbent is prepared from silica gel particles through chemical expansion of bores in silica gel particles in a certain granularity and coating a layer of resin "812" film over the surface of silica gel particles. Its application method includes that said adsorbent is directly put in the liquid to be adsorbed to directly adsorb the protein in the liquid, or said adsorbent is loaded in column tube to form a chromatographic column to adsorb protein in liquid. Its advantages are simple preparing process, easy operation and application, and high adsorption rate.

Description

A kind of preparation of protein adsorbent and using method
The invention belongs to the absorption of proteins agent, is to be the method for feedstock production adsorbent and the using method of this adsorbent with the inorganic rigid silica gel particle.
At present, the natural biological chemical pharmacy extracts the biological activity protein of trace, as the human chorionic gonadotrophin HCG in pregnant woman's urine or people's dirty from the device of animal the hematuria liquid; Hinder patient again and urinate extraction erythropoietin EPO; From people's urine, extract macrophage-colony stimulating factor M-CSF.Some of them product such as HCG product divide added value less, and treating capacity is big, need to require to have high yield, low cost in extraction, purge process.The extracting method of domestic routine adopts adsorbents such as hydroxyapatite, and often adsorption efficiency is low, does not reach the requirement of the high yield of large-scale production.The lot of domestic and international people attempts to study the outstanding adsorbent of high efficiency, low cost now, and most of adsorbent has a lot of limitation.The one, to the specificity of adsorbent; The 2nd, adsorbance is undesirable.So be difficult to obtain bigger economic benefit.In addition, some biogenetic products such as people urinate EPO, and its content is atomic, and added value is but quite high, and supply falls short of demand in the international market.Yet its urine source is limited.Therefore, adsorbent efficiently must be arranged, otherwise can't carry out large-scale economical production.
The object of the present invention is to provide a kind of preparation method and using method of protein adsorbent.Its preparation method is that each silica gel particle with 5-40 μ m drops in Na2CO3 aqueous solution, makes the adsorption hole aperture that each particle surface gathers be enlarged to 200-400A ° after stirring a period of time at a certain temperature.Again the silica gel particle through reaming is immersed in the 812 resin methanol solutions in proportion, leaving standstill certain hour after the stirring filters and drains, the silica gel particle that will coat resin bed more places in the reactor, and select for use ethyl acetate and ethylenediamine as reaction dissolvent and crosslinking agent, back insulation certain hour heats up, naturally cool to normal temperature again, respectively through running water and distilled water rinsing, gained white silica gel particle is this adsorbent after the drying.
The protein adsorption amount of adsorbent of the present invention is every gram adsorbents adsorb protein 10-12mg.The using method of adsorbent of the present invention is divided into two kinds, and the one, directly in solution such as nutrient solution that will adsorb or urine, add a certain amount of adsorbent.Its addition is decided according to the concentration of protein, after stirring natural sedimentation, discards protein-free supernatant, uses NaCl solution washing adsorbent then, behind the distilled water wash-out, collects eluent, promptly is desired reactive protein crude product.Another kind of using method is with adsorbent of the present invention, be suspended in the NaCl aqueous solution, pack in the column jeckets such as glass, lucite or stainless steel, after peristaltic pump or constant-flux pump compacting, required concentrated solution is pumped in the column jecket with peristaltic pump or constant-flux pump, pump into volume and decide on the concentration of the protein of the amount of adsorbent and solution.Then use the NaCl aqueous equilibrium, use the distilled water wash-out after the balance, the volume of reactive protein solution only is 20 to 1/50th of an original volume under the wash-out.This method thickening efficiency is very high.Its distinctive feature is that the pyrogen lipopolysaccharides is not adsorbed, and can when the NaCl aqueous equilibrium pyrogen be eliminated.This is very suitable for resembling the very high bacterium of pyrogen content (Escherichia coli) nutrient solution.Adsorbent can overcome the deficiencies in the prior art effectively with method preparation of the present invention and use.
Preparation method's of the present invention technical characterictic is:
A kind of preparation of protein adsorbent and using method are that micron-sized each rigidity material particle aggregation forms by granularity, each rigidity material of adsorbent is that diameter is the silica gel of 5-40 μ m, utilize the method for chemical reaction to make each adsorption hole aperture of gathering on each silica gel particle surface be 200-400A °, and coat 812 resin beds at each silica gel particle outer surface, the resin film layer average thickness is at least 0.1 μ m, prepares this protein adsorbent and it is characterized in that:
A, surperficial reaming: it is the 3-8%Na2CO3 aqueous solution that 10 kilograms of silica gel particles of 5-40 μ m are put into 50l concentration, after stirring 12-24 hour under 40-50 ℃ the temperature conditions, each adsorption hole aperture that each silica gel particle surface gathers is promptly expanded to 200-400A °;
B, resin-coated film: it is in the 812 resin methanol solutions of 2-9% that the silica gel particle after the reaming is immersed concentration, the relative scale of the two is that 812 required resin methanol solutions of per kilogram silica gel particle are 3000-8000ml, after evenly stirring, left standstill 1-2 hour, refilter and drain, the all coated one deck resin film of each silica gel particle surface this moment, the silica gel particle that is coated with resin is put into autoclave, and select for use ethyl acetate and ethylenediamine as reaction dissolvent and crosslinking agent, be needed protein adsorbent in reaction under the 100-130 ℃ of temperature after 3-4 hour.
The addition of ethyl acetate and ethylenediamine is respectively:
The addition of ethyl acetate and ethylenediamine is respectively per kilogram silica gel particle 5-15ml and 0.5-4ml, each silica gel particle after the moulding is cooled off, use running water and distilled water rinsing 2-4 time more respectively, the protein adsorbent of oven dry back gained is white in color, and is its finished product.
The technical characterictic of using method of the present invention is:
A kind of preparation of protein adsorbent and using method are that micron-sized each rigidity material particle aggregation forms by granularity, each rigidity material grains of adsorbent is that diameter is the silica gel of 5-40 μ m, utilize the method for chemical reaction to make each adsorption hole aperture that each silica gel particle surface gathers be 200-400A °, and coat 812 resin beds at each silica gel particle outer surface, the resin film layer average thickness is at least 0.1 μ m, the using method of this protein adsorbent be divided into direct input contain adsorb in the protein solution or make in the dress this adsorbent chromatographic column adsorb, it is characterized in that ratio in every gram adsorbents adsorb protein 10-12mg
A, directly in being adsorbed nutrient solution or urine, add this adsorbent, stir after 20-30 minute, make the adsorbent natural subsidence, survey supernatant and do not have protein, abandoning supernatant, the adsorbent of collection is used PH2.0-4.5 distilled water wash-out after using the 2-20%NaCl solution washing again, collecting eluent, promptly is desired reactive protein crude product;
B, adsorbent of the present invention is suspended in the 10%NaCl aqueous solution, the glass of packing into, in lucite or the stainless steel column jecket, after peristaltic pump or constant-flux pump compacting, required concentrated solution is pumped in the column jecket with pump, then using the 2-20%NaCl aqueous equilibrium, is the distilled water wash-out of 2.0-4.5 with pH value after the balance, is that the 2%-5% of original volume is advisable with the volume of reactive protein solution under the wash-out.
Embodiments of the invention are respectively:
One, preparation method's embodiment:
Embodiment 1, takes by weighing one kilogram 10 microns silica gel particle, adds 5%812 resin methanol solutions, after stirring, places 1 hour.Filter then and drain.The silica gel particle of coating resin is put into autoclave, add ethyl acetate 8.5ml, ethylenediamine 1.2ml is warming up to 127 ℃, is incubated 4 hours.After the cooling, the silica gel particle that has coated is taken out, use running water and distilled water rinsing three times respectively, get white adsorbent finished product, encapsulation after the oven dry.
Embodiment 2, take by weighing two kilograms 30 microns silica gel, add 8%812 resin methanol solutions, after stirring, place 2 hours, and filtration is drained.The silica gel of coating resin is put into autoclave, add ethyl acetate 17ml, ethylenediamine 4ml is warming up to 130 ℃, is incubated 4 hours.After the cooling, the silica gel particle that has coated is taken out, use running water and distilled water rinsing four times respectively, get white adsorbent finished product, encapsulation after the oven dry.
Two, the embodiment of using method
Embodiment 1, and the people urinates the thick extraction of M-CSF
Add 50 grams adsorbent of the present invention in per 10 liters of urine, 0-4 ℃ is stirred after 30 minutes down, leaves standstill 15 minutes, and supernatant has not been measured protein, and abandoning supernatant is collected the adsorbent of adsorbed proteins.Place the long-pending 8%NaCl aqueous wash medium of decaploid to wash in adsorbent.Use 500ml PH3.7 aqueous solution wash-out again, can obtain the M-CSF crude product.Per 10 liters of urine can contain the crude product of 80 μ gM-CSF.
Embodiment 2, and the people urinates the thick extraction of HCG
One kilogram of adsorbent diameter 100mm that packs into, in the high 1000mm lucite tube, filter is equipped with in the lower end outlet.Use the 5%NaCl aqueous equilibrium.The pregnant woman urinates 100 kilograms, injects continuously, and effluent color and former urine perusal indistinction have not also detected HCG, treat that urine all flows out after, use 10 liters of washings of the 5%NaCl aqueous solution after, use PH3.8 aqueous solution wash-out again, 5 liters of collected volume.Protein total recovery 85%, the activity yield 95% of HCG.Overall process is done 8 hours.
Embodiment 3, and the people urinates the thick extraction of EPO
One kilogram of adsorbent diameter 80mm that packs into, in the high 500mm stainless steel column jecket, the column jecket lower end is sintered microporous filter plate.Be continuously pumped into aplastic amenia people's urine with constant-flux pump with the 200ml/min flow velocity, flow out in the liquid and should not contain EPO.After last sample finishes, pump into the 5%NaCl aqueous equilibrium.The protein that goes out to be adsorbed with PH3.9 aqueous solution wash-out again.24 times of cycles of concentration, protein yield 89%, activity yield 92%.
The substantive distinguishing features that the present invention has and the remarkable technological progress that obtains are: do suction with this silica gel particle Attached dose or be filled in and do chromatographic column in the column jecket, because the resin that coats with chemical method at each silica gel particle surface Film shields the strong adsorption site of each particle surface fully, and become the hydrophilic absorption surface and be weak hydrophobic state, Can select adsorbed proteins and get rid of impurity such as salt, pigment and carbohydrate in the solution. Our experiments show that, This adsorbent is urinated HCG, EPO, M-CSF, CHO, cell culture fluid TPO to the people, Protein adsorption in the bioactivators such as the interferon of Bacillus coli expression is respond well. Other front institute State the range of application that two kinds of usings method of this adsorbent have also been widened this adsorbent greatly, not only use very Convenient, and decrease the cost of extract. It is higher to have adsorption rate, and adsorbance is big and cost is low Etc. advantage. Be a kind ofly can adsorb biological activity protein, also can as microorganism, live in the cell culture fluid The adsorbent of property material.

Claims (3)

1, a kind of preparation of protein adsorbent and using method are that micron-sized each rigidity material particle aggregation forms by granularity, each rigidity material of adsorbent is that diameter is the silica gel of 5-40 μ m, utilize the method for chemical reaction to make each adsorption hole aperture of gathering on each silica gel particle surface be 200-400 , and coat 812 resin beds at each silica gel particle outer surface, the resin film layer average thickness is at least 0.1 μ m, prepares this protein adsorbent and it is characterized in that:
A, surperficial reaming: it is the 3-8%Na2CO3 aqueous solution that 10 kilograms of silica gel particles of 5-40 μ m are put into 50l concentration, after stirring 12-24 hour under 40-50 ℃ the temperature conditions, each adsorption hole aperture that each silica gel particle surface gathers is promptly expanded to 200-400A °;
B, resin-coated film: it is in the 812 resin methanol solutions of 2-9% that the silica gel particle after the reaming is immersed concentration, the relative scale of the two is that 812 required resin methanol solutions of per kilogram silica gel particle are 3000-8000ml, after evenly stirring, left standstill 1-2 hour, refilter and drain, the all coated one deck resin film of each silica gel particle surface this moment, the silica gel particle that is coated with resin is put into autoclave, and select for use ethyl acetate and ethylenediamine as reaction dissolvent and crosslinking agent, be needed protein adsorbent in reaction under the 100-130 ℃ of temperature after 3-4 hour.
2, adsorbent according to claim 1, the addition that it is characterized in that ethyl acetate and ethylenediamine is respectively per kilogram silica gel particle 5-15ml and 0.5-4ml, each silica gel particle after the moulding is cooled off, use running water and distilled water rinsing 2-4 time more respectively, the protein adsorbent of oven dry back gained is white in color, and is its finished product.
3, a kind of preparation of protein adsorbent and using method are that micron-sized each rigidity material particle aggregation forms by granularity, each rigidity material grains of adsorbent is that diameter is the silica gel of 5-40 μ m, utilize the method for chemical reaction to make each adsorption hole aperture that each silica gel particle surface gathers be 200-400A °, and coat 812 resin beds at each silica gel particle outer surface, the resin film layer average thickness is at least 0.1 μ m, the using method of this protein adsorbent be divided into direct input contain adsorb in the protein solution or make in the dress this adsorbent chromatographic column adsorb, it is characterized in that ratio in every gram adsorbents adsorb protein 10-12mg
A, directly in being adsorbed nutrient solution or urine, add this adsorbent, stir after 20-30 minute, make the adsorbent natural subsidence, survey supernatant and do not have protein, abandoning supernatant, the adsorbent of collection is used PH2.0-4.5 distilled water wash-out after using the 2-20%NaCl solution washing again, collecting eluent, promptly is desired reactive protein crude product;
B, adsorbent of the present invention is suspended in the 10%NaCl aqueous solution, the glass of packing into, in lucite or the stainless steel column jecket, after peristaltic pump or constant-flux pump compacting, required concentrated solution is pumped in the column jecket with pump, then using the 2-20%NaCl aqueous equilibrium, is the distilled water wash-out of 2.0-4.5 with pH value after the balance, is that the 2%-5% of original volume is advisable with the volume of reactive protein solution under the wash-out.
CN 96120043 1996-10-14 1996-10-14 Process for preparing protein adsorbent and using method thereof Pending CN1179355A (en)

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Application Number Priority Date Filing Date Title
CN 96120043 CN1179355A (en) 1996-10-14 1996-10-14 Process for preparing protein adsorbent and using method thereof

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Application Number Priority Date Filing Date Title
CN 96120043 CN1179355A (en) 1996-10-14 1996-10-14 Process for preparing protein adsorbent and using method thereof

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CN1179355A true CN1179355A (en) 1998-04-22

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1332718C (en) * 2005-04-26 2007-08-22 广州康盛生物科技有限公司 Silica gel carrier protein A immune absorption material and its preparation method
CN101340959B (en) * 2005-12-20 2011-04-13 帝斯曼知识产权资产管理有限公司 Process for the treatment of an aqueous mixture comprising a dipolar aprotic compound

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1332718C (en) * 2005-04-26 2007-08-22 广州康盛生物科技有限公司 Silica gel carrier protein A immune absorption material and its preparation method
CN101340959B (en) * 2005-12-20 2011-04-13 帝斯曼知识产权资产管理有限公司 Process for the treatment of an aqueous mixture comprising a dipolar aprotic compound

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