CN101279243A - Mixing mode expanded adsorbent bed medium and method for producing the same - Google Patents
Mixing mode expanded adsorbent bed medium and method for producing the same Download PDFInfo
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- CN101279243A CN101279243A CNA2008100618140A CN200810061814A CN101279243A CN 101279243 A CN101279243 A CN 101279243A CN A2008100618140 A CNA2008100618140 A CN A2008100618140A CN 200810061814 A CN200810061814 A CN 200810061814A CN 101279243 A CN101279243 A CN 101279243A
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- 238000002156 mixing Methods 0.000 title claims description 14
- 239000003463 adsorbent Substances 0.000 title abstract description 5
- 238000004519 manufacturing process Methods 0.000 title description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 47
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 36
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 33
- 239000004005 microsphere Substances 0.000 claims abstract description 33
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 claims abstract description 24
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims abstract description 23
- YHMYGUUIMTVXNW-UHFFFAOYSA-N 1,3-dihydrobenzimidazole-2-thione Chemical compound C1=CC=C2NC(S)=NC2=C1 YHMYGUUIMTVXNW-UHFFFAOYSA-N 0.000 claims abstract description 18
- AFOSIXZFDONLBT-UHFFFAOYSA-N divinyl sulfone Chemical compound C=CS(=O)(=O)C=C AFOSIXZFDONLBT-UHFFFAOYSA-N 0.000 claims abstract description 17
- 230000004913 activation Effects 0.000 claims abstract description 16
- 238000005859 coupling reaction Methods 0.000 claims abstract description 15
- 239000011159 matrix material Substances 0.000 claims abstract description 15
- 229910001870 ammonium persulfate Inorganic materials 0.000 claims abstract description 12
- 230000008929 regeneration Effects 0.000 claims abstract description 12
- 238000011069 regeneration method Methods 0.000 claims abstract description 12
- 239000000725 suspension Substances 0.000 claims abstract description 12
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 claims abstract description 11
- 239000000203 mixture Substances 0.000 claims abstract description 7
- 230000008878 coupling Effects 0.000 claims abstract description 6
- 238000010168 coupling process Methods 0.000 claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 239000008367 deionised water Substances 0.000 claims description 40
- 229920002678 cellulose Polymers 0.000 claims description 38
- 239000001913 cellulose Substances 0.000 claims description 38
- 238000005406 washing Methods 0.000 claims description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 31
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 22
- 238000000967 suction filtration Methods 0.000 claims description 22
- 229910021641 deionized water Inorganic materials 0.000 claims description 20
- 229920000297 Rayon Polymers 0.000 claims description 13
- 239000002245 particle Substances 0.000 claims description 13
- 238000009835 boiling Methods 0.000 claims description 11
- 239000000872 buffer Substances 0.000 claims description 11
- 239000003292 glue Substances 0.000 claims description 11
- 235000011187 glycerol Nutrition 0.000 claims description 11
- 239000000843 powder Substances 0.000 claims description 11
- 238000012216 screening Methods 0.000 claims description 11
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 11
- UONOETXJSWQNOL-UHFFFAOYSA-N tungsten carbide Chemical compound [W+]#[C-] UONOETXJSWQNOL-UHFFFAOYSA-N 0.000 claims description 8
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical group O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 5
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 3
- 229910001220 stainless steel Inorganic materials 0.000 claims description 3
- 239000010935 stainless steel Substances 0.000 claims description 3
- 239000004408 titanium dioxide Substances 0.000 claims description 3
- DAPOMFMFTBUAKL-UHFFFAOYSA-N 2-ethylpyridine-3-thiol Chemical compound CCC1=NC=CC=C1S DAPOMFMFTBUAKL-UHFFFAOYSA-N 0.000 claims description 2
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 125000004432 carbon atom Chemical group C* 0.000 claims description 2
- 238000001179 sorption measurement Methods 0.000 abstract description 22
- 230000002209 hydrophobic effect Effects 0.000 abstract description 11
- 230000003993 interaction Effects 0.000 abstract description 5
- 238000000926 separation method Methods 0.000 abstract description 4
- 239000002131 composite material Substances 0.000 abstract description 3
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- 102000009123 Fibrin Human genes 0.000 abstract 2
- 108010073385 Fibrin Proteins 0.000 abstract 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 abstract 2
- 230000009471 action Effects 0.000 abstract 2
- 229950003499 fibrin Drugs 0.000 abstract 2
- AFOLOMGWVXKIQL-UHFFFAOYSA-O petunidin Chemical compound OC1=C(O)C(OC)=CC(C=2C(=CC=3C(O)=CC(O)=CC=3[O+]=2)O)=C1 AFOLOMGWVXKIQL-UHFFFAOYSA-O 0.000 abstract 2
- 229930015717 petunidin Natural products 0.000 abstract 2
- 235000006384 petunidin Nutrition 0.000 abstract 2
- 230000006698 induction Effects 0.000 abstract 1
- 230000007246 mechanism Effects 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 50
- 239000000243 solution Substances 0.000 description 25
- 238000010521 absorption reaction Methods 0.000 description 9
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 8
- 239000005864 Sulphur Substances 0.000 description 8
- 230000003068 static effect Effects 0.000 description 8
- 238000004587 chromatography analysis Methods 0.000 description 6
- 210000002969 egg yolk Anatomy 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
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- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 229920006395 saturated elastomer Polymers 0.000 description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 239000005557 antagonist Substances 0.000 description 3
- 239000007979 citrate buffer Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
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- UAVKJJWZLWPSMK-UHFFFAOYSA-N 3-ethyl-1h-benzimidazole-2-thione Chemical compound C1=CC=C2NC(=S)N(CC)C2=C1 UAVKJJWZLWPSMK-UHFFFAOYSA-N 0.000 description 1
- CDNHLXOFELOEOL-UHFFFAOYSA-N 3-methyl-1h-benzimidazole-2-thione Chemical compound C1=CC=C2N(C)C(S)=NC2=C1 CDNHLXOFELOEOL-UHFFFAOYSA-N 0.000 description 1
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 1
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- 238000002649 immunization Methods 0.000 description 1
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Abstract
The invention discloses a mixed model expanded adsorbent bed medium and a preparation method thereof. In the composition of medium, matrix is a composite microsphere with fibrin/inorganic weighting agent, and petunidin is sulfonyl group and mercapto benzimidazole. The composite microsphere with fibrin/inorganic weighting agent is acquired by inverse suspension thermal regeneration and is taken as the matrix to mix divinyl sulfone, soda buffer solution and DMSO for activation, then swab-off activation matrix, the mercapto benzimidazole and sodium hydroxide containing ammonium persulfate are mixed for coupling, thus acquiring the mixed model expanded adsorbent bed medium taking the mercapto benzimidazole and the sulfonyl group as the petunidin. The novel expanded bed adsorbing medium developed by the invention combines with a plurality of petunidin-protein interaction mechanisms such as hydrophobic interaction, charge induction action, thiophilic action, etc, to form mixed model adsorption, which has the advantages of large adsorption capacity, high selectivity, etc., thus being used for high efficient separation and purification of bioactivators such as antibody, etc.
Description
Technical field
The present invention relates to a kind of mixing mode expanded bed adsorbing medium and preparation method thereof, is aglucon with mercaptobenzimidazole and sulfuryl, belongs to Expanded Bed Adsorption isolated protein technology in the chromatography.
Background technology
Antibody is a kind of important biological technology products, is widely used in immunization therapy, prevention from suffering from the diseases and medical diagnosis.Isolation technics at antibody has obtained certain progress in recent years, but also needs constantly additional and perfect.
Conventional ion-exchange chromatography and hydrophobic interaction chromatography can binding antibodies, but specificity and selectivity are relatively poor, and separating step is many, influences the activity yield of antibody.Protein A affinity chromatography medium can selective absorption antibody, but medium costs an arm and a leg, and aglucon is easily by proteasome degradation in the feed liquid, and it is low to reuse number of times, and running cost is high, limits its large-scale application.Mixed mode chromatography (Mixed-modechromatography) is a kind of novel biochemical isolation technics of development in recent years, in conjunction with two kinds and two or more aglucon-protein interaction patterns, can significantly improve adsorption capacity and adsorptive selectivity, wherein salt tolerance parent sulphur chromatography is an important directions.From Porath (Porath et al.FEBS letters in 1985,1985,185:306) etc. first Application parent's sulphur chromatography (thiophilic chromatography) has been isolated since the antibody from serum, parent's sulphur chromatography is aspect protein purification, and particularly there have been many application the antibody purification aspect.The sulphur of sulfuryl is a short of electricity subarea in parent's sulphur aglucon, can be used as electron acceptor, and the sulphur in the thioether that closes on is then for the electronics enrichment region, as electron donor.This special construction can antagonist molecule conserved region some electron donors-acceptor site produce specific effect, improve the selectivity of antibody absorption.Yet traditional close sulphur chromatography must just can effectively adsorb antibody under certain density ammonium sulfate or sodium sulphate, is unfavorable for that antibody separates, and limits its scale and uses, and seeking thiophilicity mixed mode aglucon novel, salt tolerant is development trend.
(Expanded Bed Adsorption, EBA) technology is formed at the nineties in 20th century to Expanded Bed Adsorption, is a kind of Separation of Solid and Liquid, the concentrated and novel protein separating and purifying technology of initial stage purifying among an operating unit of collecting.EBA can directly catch target product from cell liquid, cell homogenates liquid or tissue extract, reduced the operating unit number, has shortened the processing time, has saved production cost, is a new unit operations that occurs in recent ten years.Therefore, the present invention is an Expanded Bed Adsorption matrix with cellulose/inorganic weighting agent complex microsphere, sulfuryl of introducing after the divinylsulfone activation and the mercaptobenzimidazole after the coupling are as its major function aglucon, formed a kind of novel thiophilicity mixing mode expanded adsorbent bed medium, can be used for extensive Expanded Bed Adsorption separation antibody, become a kind of efficient integrated novel antibodies separating technology, reported in literature is not seen in correlative study.
Summary of the invention
The purpose of this invention is to provide a kind of is the mixing mode expanded bed adsorbing medium and preparation method thereof of aglucon with mercaptobenzimidazole and sulfuryl.
With mercaptobenzimidazole and sulfuryl is the mixing mode expanded bed adsorbing medium of aglucon: the composition mesostroma of medium is cellulose/inorganic weighting agent complex microsphere, aglucon is the sulfuryl of divinylsulfone activation back introducing and the mercaptobenzimidazole after the coupling, and structural group becomes:
The skeleton of described cellulose/inorganic weighting agent complex microsphere is a cellulose, and weighting agent is titanium dioxide, stainless steel powder, nickel powder or tungsten carbide powder, and the mass percentage content that weighting agent accounts for wet bulb is 16%～61%; The particle diameter of cellulose/inorganic weighting agent complex microsphere is 50～250 μ m.Aglucon is 2-sulfydryl-1-R-benzimidazole, and R is the short chain alkanes of 0-2 carbon atom.Aglucon density is 28～58 μ mol/ml media.
The preparation method of mixing mode expanded bed adsorbing medium: at first cellulose viscose glue and inorganic weighting agent are fully mixed, the quality percentage composition of inorganic weighting agent in mixture is 16%～61%, the pump oil that adds 5 times of cellulose viscose qualities, control rotating speed 500～800rpm, ball is made in the regeneration of anti-phase suspension heat, the boiling water washing, the cellulose/inorganic weighting agent complex microsphere of screening 50～250 μ m particle diameters is as matrix; Secondly sodium carbonate buffer and the dimethyl sulfoxide (DMSO) with cellulose/inorganic weighting agent complex microsphere, divinylsulfone, 0.1～0.5M pH12 mixes, the volume that divinylsulfone adds is 18%～36% of cellulose/inorganic weighting agent complex microsphere volume, activation is 4 hours in 25 ℃ of following 180rpm shaking tables, suction filtration spends deionised water and obtains activated substrate; Then the mercaptoethyl pyridine of the two key moles of activated substrate, 3 times and 0.2～0.7M sodium hydroxide solution of containing the 25mg/ml ammonium persulfate are mixed coupling 8h in 60 ℃ of following 180rpm shaking tables; At last microballoon is joined and contain 1%H
2O
2With soaked 2 hours in the solution of 1% glycerine, the deionized water washing, obtaining with mercaptobenzimidazole and sulfuryl is the mixing mode expanded bed adsorbing medium of aglucon.The course of reaction schematic diagram is:
The present invention fully combines multiple aglucon-protein interaction principles such as hydrophobic interaction, electric charge inducing action and close sulphur effect, forms the absorption of special mixed mode, has that adsorption capacity is big, the selectivity advantages of higher.Simultaneously, novel mixed mode meter aglucon is coupled on cellulose/inorganic weighting agent composite expansion bed substrate, has formed novel Expanded Bed Adsorption medium, can be used for the scale of protein such as antibody, efficient separation and purification.
Description of drawings
The static adsorption isotherm (different pH) of Fig. 1 mixed mode medium absorption Yolk antibody;
The static adsorption isotherm of Fig. 2 mixed mode medium absorption Yolk antibody (different salinity, pH=7).
The specific embodiment
The invention will be further described by the following examples:
300rpm in the 500ml round-bottomed flask mixes with 50g cellulose viscose glue and 40g tungsten-carbide powder, add 300g pump oil, the 700rpm rotating speed, ball is made in the regeneration of anti-phase suspension heat, the boiling water washing, deionized water washing 3～5 times, the cellulose/inorganic weighting agent complex microsphere of screening 50～250 μ m particle size is as matrix; Sodium carbonate buffer and the 1.5ml dimethyl sulfoxide (DMSO) of the complex microsphere that 10ml is drained, 3.6ml divinylsulfone, 9ml 0.25M pH12 join in the conical flask, activation is 4 hours in 25 ℃ of following 180rpm shaking tables, suction filtration is removed mixed liquor, spends deionised water, drains.Add the 2-mercaptobenzimidazole of 3 times of two key moles and the 0.5M sodium hydroxide solution that 10ml contains the 25mg/ml ammonium persulfate, coupling reaction is 8 hours in 60 ℃ of following 180rpm shaking tables, and suction filtration is removed mixed liquor, spends deionised water, drains.Medium joins 100ml and contains 1%H
2O
2With soaked 2 hours in the solution of 1% glycerine, the deionized water washing obtains hydrophobic electric charge induced type thiophil expanded bed adsorbing medium, the medium wet true density is 1.81g/ml, aglucon density is 58 μ mol/ml.
300rpm in the 500ml round-bottomed flask mixes with 50g cellulose viscose glue and 40g tungsten-carbide powder, add 300g pump oil, the 700rpm rotating speed, ball is made in the regeneration of anti-phase suspension heat, the boiling water washing, deionized water washing 3～5 times, the cellulose/inorganic weighting agent complex microsphere of screening 50～250 μ m particle size is as matrix; Sodium carbonate buffer and the 1.5ml dimethyl sulfoxide (DMSO) of the complex microsphere that 10ml is drained, 0.9ml divinylsulfone, 9ml 0.5M pH12 join in the conical flask, activation is 4 hours in 25 ℃ of following 180rpm shaking tables, suction filtration is removed mixed liquor, spends deionised water, drains.Add the 2-mercaptobenzimidazole of 3 times of two key moles and the 0.7M sodium hydroxide solution that 10ml contains the 25mg/ml ammonium persulfate, coupling reaction is 8 hours in 60 ℃ of following 180rpm shaking tables, and suction filtration is removed mixed liquor, spends deionised water, drains.Medium joins 100ml and contains 1%H
2O
2With soaked 2 hours in the solution of 1% glycerine, the deionized water washing obtains hydrophobic electric charge induced type thiophil expanded bed adsorbing medium, the medium wet true density is 1.81g/ml, aglucon density is 28 μ mol/ml.
300rpm in the 500ml round-bottomed flask mixes with 50g cellulose viscose glue and 80g tungsten-carbide powder, add 310g pump oil, the 800rpm rotating speed, ball is made in the regeneration of anti-phase suspension heat, the boiling water washing, deionized water washing 3～5 times, the cellulose/inorganic weighting agent complex microsphere of screening 50～250 μ m particle size is as matrix; Sodium carbonate buffer and the 1.5ml dimethyl sulfoxide (DMSO) of the complex microsphere that 10ml is drained, 1.8ml divinylsulfone, 9ml 0.1M pH12 join in the conical flask, activation is 4 hours in 25 ℃ of following 180rpm shaking tables, suction filtration is removed mixed liquor, spends deionised water, drains.Add the 2-mercaptobenzimidazole of 3 times of two key moles and the 0.2M sodium hydroxide solution that 10ml contains the 25mg/ml ammonium persulfate, coupling reaction is 8 hours in 60 ℃ of following 180rpm shaking tables, and suction filtration is removed mixed liquor, spends deionised water, drains.Medium joins 100ml and contains 1%H
2O
2With soaked 2 hours in the solution of 1% glycerine, the deionized water washing obtains hydrophobic electric charge induced type thiophil expanded bed adsorbing medium, the medium wet true density is 2.40g/ml, aglucon density is 41 μ mol/ml.
300rpm in the 500ml round-bottomed flask mixes with 50g cellulose viscose glue and 60g tungsten-carbide powder, add 300g pump oil, the 800rpm rotating speed, ball is made in the regeneration of anti-phase suspension heat, the boiling water washing, deionized water washing 3～5 times, the cellulose/inorganic weighting agent complex microsphere of screening 50～250 μ m particle size is as matrix; Sodium carbonate buffer and the 1.5ml dimethyl sulfoxide (DMSO) of the complex microsphere that 10ml is drained, 3.6ml divinylsulfone, 9ml 0.25M pH12 join in the conical flask, activation is 4 hours in 25 ℃ of following 180rpm shaking tables, suction filtration is removed mixed liquor, spends deionised water, drains.Add the 2-mercaptobenzimidazole of 3 times of two key moles and the 0.5M sodium hydroxide solution that 10ml contains the 25mg/ml ammonium persulfate, coupling reaction is 8 hours in 60 ℃ of following 180rpm shaking tables, and suction filtration is removed mixed liquor, spends deionised water, drains.Medium joins 100ml and contains 1%H
2O
2With soaked 2 hours in the solution of 1% glycerine, the deionized water washing obtains hydrophobic electric charge induced type thiophil expanded bed adsorbing medium, the medium wet true density is 2.11g/ml, aglucon density is 56 μ mol/ml.
Embodiment 5
300rpm in the 500ml round-bottomed flask mixes with 50g cellulose viscose glue and 12g nickel powder, add 300g pump oil, the 700rpm rotating speed, ball is made in the regeneration of anti-phase suspension heat, the boiling water washing, deionized water washing 3～5 times, the cellulose/inorganic weighting agent complex microsphere of screening 50～250 μ m particle size is as matrix; Sodium carbonate buffer and the 1.5ml dimethyl sulfoxide (DMSO) of the complex microsphere that 10ml is drained, 3.6ml divinylsulfone, 9ml 0.25M pH12 join in the conical flask, activation is 4 hours in 25 ℃ of following 180rpm shaking tables, suction filtration is removed mixed liquor, spends deionised water, drains.Add the 2-mercaptobenzimidazole of 3 times of two key moles and the 0.5M sodium hydroxide solution that 10ml contains the 25mg/ml ammonium persulfate, coupling reaction is 8 hours in 60 ℃ of following 180rpm shaking tables, and suction filtration is removed mixed liquor, spends deionised water, drains.Medium joins 100ml and contains 1%H
2O
2With soaked 2 hours in the solution of 1% glycerine, the deionized water washing obtains hydrophobic electric charge induced type thiophil expanded bed adsorbing medium, the medium wet true density is 1.42g/ml, aglucon density is 57 μ mol/ml.
Embodiment 6
300rpm in the 500ml round-bottomed flask mixes with 50g cellulose viscose glue and 20g stainless steel powder, add 300g pump oil, the 700rpm rotating speed, ball is made in the regeneration of anti-phase suspension heat, the boiling water washing, deionized water washing 3～5 times, the cellulose/inorganic weighting agent complex microsphere of screening 50～250 μ m particle size is as matrix; Sodium carbonate buffer and the 1.5ml dimethyl sulfoxide (DMSO) of the complex microsphere that 10ml is drained, 3.6ml divinylsulfone, 9ml 0.25M pH12 join in the conical flask, activation is 4 hours in 25 ℃ of following 180rpm shaking tables, suction filtration is removed mixed liquor, spends deionised water, drains.Add the 2-mercaptobenzimidazole of 3 times of two key moles and the 0.5M sodium hydroxide solution that 10ml contains the 25mg/ml ammonium persulfate, coupling reaction is 8 hours in 60 ℃ of following 180rpm shaking tables, and suction filtration is removed mixed liquor, spends deionised water, drains.Medium joins 100ml and contains 1%H
2O
2With soaked 2 hours in the solution of 1% glycerine, the deionized water washing obtains hydrophobic electric charge induced type thiophil expanded bed adsorbing medium, the medium wet true density is 1.71g/ml, aglucon density is 54 μ mol/ml.
Embodiment 7
300rpm in the 500ml round-bottomed flask mixes with 50g cellulose viscose glue and 15g titanium dioxide, add 300g pump oil, the 700rpm rotating speed, ball is made in the regeneration of anti-phase suspension heat, the boiling water washing, deionized water washing 3～5 times, the cellulose/inorganic weighting agent complex microsphere of screening 50～250 μ m particle size is as matrix; Sodium carbonate buffer and the 1.5ml dimethyl sulfoxide (DMSO) of the complex microsphere that 10ml is drained, 3.6ml divinylsulfone, 9ml 0.25M pH12 join in the conical flask, activation is 4 hours in 25 ℃ of following 180rpm shaking tables, suction filtration is removed mixed liquor, spends deionised water, drains.Add the 2-mercaptobenzimidazole of 3 times of two key moles and the 0.5M sodium hydroxide solution that 10ml contains the 25mg/ml ammonium persulfate, coupling reaction is 8 hours in 60 ℃ of following 180rpm shaking tables, and suction filtration is removed mixed liquor, spends deionised water, drains.Medium joins 100ml and contains 1%H
2O
2With soaked 2 hours in the solution of 1% glycerine, the deionized water washing obtains hydrophobic electric charge induced type thiophil expanded bed adsorbing medium, the medium wet true density is 1.39g/ml, aglucon density is 58 μ mol/ml.
Embodiment 8
300rpm in the 500ml round-bottomed flask mixes with 50g cellulose viscose glue and 40g tungsten-carbide powder, add 300g pump oil, the 700rpm rotating speed, ball is made in the regeneration of anti-phase suspension heat, the boiling water washing, deionized water washing 3～5 times, the cellulose/inorganic weighting agent complex microsphere of screening 50～250 μ m particle size is as matrix; Sodium carbonate buffer and the 1.5ml dimethyl sulfoxide (DMSO) of the complex microsphere that 10ml is drained, 3.6ml divinylsulfone, 9ml 0.25M pH12 join in the conical flask, activation is 4 hours in 25 ℃ of following 180rpm shaking tables, suction filtration is removed mixed liquor, spends deionised water, drains.Add the 2-sulfydryl-1-methyl-benzimidazole of 3 times of two key moles and the 0.5M sodium hydroxide solution that 10ml contains the 25mg/ml ammonium persulfate, coupling reaction is 8 hours in 60 ℃ of following 180rpm shaking tables, and suction filtration is removed mixed liquor, spends deionised water, drains.Medium joins 100ml and contains 1%H
2O
2With soaked 2 hours in the solution of 1% glycerine, the deionized water washing obtains hydrophobic electric charge induced type thiophil expanded bed adsorbing medium, the medium wet true density is 1.81g/ml, aglucon density is 51 μ mol/ml.
Embodiment 9
300rpm in the 500ml round-bottomed flask mixes with 50g cellulose viscose glue and 40g tungsten-carbide powder, add 300g pump oil, the 700rpm rotating speed, ball is made in the regeneration of anti-phase suspension heat, the boiling water washing, deionized water washing 3～5 times, the cellulose/inorganic weighting agent complex microsphere of screening 50～250 μ m particle size is as matrix; Sodium carbonate buffer and the 1.5ml dimethyl sulfoxide (DMSO) of the complex microsphere that 10ml is drained, 3.6ml divinylsulfone, 9ml 0.25M pH12 join in the conical flask, activation is 4 hours in 25 ℃ of following 180rpm shaking tables, suction filtration is removed mixed liquor, spends deionised water, drains.Add the 2-sulfydryl-1-ethyl-benzimidazole of 3 times of two key moles and the 0.5M sodium hydroxide solution that 10ml contains the 25mg/ml ammonium persulfate, coupling reaction is 8 hours in 60 ℃ of following 180rpm shaking tables, and suction filtration is removed mixed liquor, spends deionised water, drains.Medium joins 100ml and contains 1%H
2O
2With soaked 2 hours in the solution of 1% glycerine, the deionized water washing obtains hydrophobic electric charge induced type thiophil expanded bed adsorbing medium, the medium wet true density is 1.81g/ml, aglucon density is 49 μ mol/ml.
Embodiment 10
The Staticadsorption experiment of mixed mode medium (aglucon density is 58 μ mol/ml) absorption Yolk antibody is at first carried out balance with medium with the citrate buffer solution of pH5; Accurately take by weighing the 0.4g medium behind the suction filtration respectively in 25ml band plug conical flask, add the cushioning liquid of different Yolk antibody (IgY) concentration of 8ml; Conical flask is placed shaking bath, 25 ℃ of following 180rpm vibration 8h; Take out the concentration that supernatant is measured IgY after the balance; The amount and the solution residual concentration of adhesion protein tried to achieve in horizontal calculation according to material, draws static adsorption isotherm, and is 167.3mg/ml according to the saturated extent of adsorption that the Langmuir equation model obtains medium.Adopt same quadrat method, the static adsorption isotherm when having measured pH4 and pH8, medium is respectively 101.9 and 114.9mg/ml to the static saturated adsorption capacity of IgY under pH4 and 8.The result shows, the adsorption capacity height of medium antagonist, and very responsive to the pH variation, embody typical mixed mode absorption characteristics.
Embodiment 11
The Staticadsorption experiment of mixed mode medium (aglucon density is 58 μ mol/ml) absorption Yolk antibody, at first configuration contains the product pH5 citrate buffer solution of 0.2M ammonium sulfate; Medium is carried out balance with buffer solution; Accurately take by weighing the 0.4g medium behind the suction filtration respectively in 25ml band plug conical flask, add the cushioning liquid of the different Yolk antibodies of 8ml (IgY) concentration; Conical flask is placed shaking bath, 25 ℃ of following 180rpm vibration 8h; Take out the concentration that supernatant is measured IgY after the balance; The amount and the solution residual concentration of adhesion protein tried to achieve in horizontal calculation according to material, draws static adsorption isotherm, and is 144.5mg/ml according to the saturated extent of adsorption that the Langmuir equation model obtains medium.Adopt same quadrat method, measure the static adsorption isotherm of IgY in the citrate buffer solution of the pH7 contain 0.6M ammonium sulfate, the static saturated adsorption capacity of IgY is 171.4mg/ml.The result shows, the adsorption capacity height of medium antagonist, and insensitive to the salinity variation, embody typical mixed mode absorption characteristics.
Claims (5)
1. mixing mode expanded bed adsorbing medium, the composition mesostroma that it is characterized in that medium is cellulose/inorganic weighting agent complex microsphere, and aglucon is the sulfuryl introduced of divinylsulfone activation back and the mercaptobenzimidazole after the coupling, and structural group becomes:
2. a kind of mixing mode expanded bed adsorbing medium according to claim 1, the skeleton that it is characterized in that described cellulose/inorganic weighting agent complex microsphere is a cellulose, weighting agent is titanium dioxide, stainless steel powder, nickel powder or tungsten carbide powder, and the mass percentage content that weighting agent accounts for wet bulb is 16%～61%; The particle diameter of cellulose/inorganic weighting agent complex microsphere is 50～250 μ m.
3. a kind of mixing mode expanded bed adsorbing medium according to claim 1 is characterized in that aglucon is 2-sulfydryl-1-R-benzimidazole, and R is the short chain alkanes of 0-2 carbon atom.
4. a kind of mixing mode expanded bed adsorbing medium according to claim 1 is characterized in that aglucon density is 28～58 μ mol/ml media.
5. the preparation method of an a kind of mixing mode expanded bed adsorbing medium as claimed in claim 1, it is characterized in that: at first cellulose viscose glue and inorganic weighting agent are fully mixed, the quality percentage composition of inorganic weighting agent in mixture is 16%～61%, the pump oil that adds 5 times of cellulose viscose qualities, control rotating speed 500～800rpm, ball is made in the regeneration of anti-phase suspension heat, the boiling water washing, and the cellulose/inorganic weighting agent complex microsphere of screening 50～250 μ m particle diameters is as matrix; Secondly sodium carbonate buffer and the dimethyl sulfoxide (DMSO) with cellulose/inorganic weighting agent complex microsphere, divinylsulfone, 0.1～0.5M pH12 mixes, the volume that divinylsulfone adds is 18%～36% of cellulose/inorganic weighting agent complex microsphere volume, activation is 4 hours in 25 ℃ of following 180rpm shaking tables, suction filtration spends deionised water and obtains activated substrate; Then the mercaptoethyl pyridine of the two key moles of activated substrate, 3 times and 0.2～0.7M sodium hydroxide solution of containing the 25mg/ml ammonium persulfate are mixed coupling 8h in 60 ℃ of following 180rpm shaking tables; At last microballoon is joined in the solution that contains 1%H2O2 and 1% glycerine and soaked 2 hours, the deionized water washing, obtaining with mercaptobenzimidazole and sulfuryl is the mixing mode expanded bed adsorbing medium of aglucon.
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| CN104603144A (en) * | 2012-09-03 | 2015-05-06 | 株式会社钟化 | Mix-mode antibody affinity separation matrix and purification method using same, and target molecule |
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| CN113289587A (en) * | 2021-05-10 | 2021-08-24 | 苏州君盟生物医药科技有限公司 | Sulfydryl modified magnetic nano-microsphere and preparation method and application thereof |
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| CN104603144A (en) * | 2012-09-03 | 2015-05-06 | 株式会社钟化 | Mix-mode antibody affinity separation matrix and purification method using same, and target molecule |
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| CN106749623B (en) * | 2016-12-09 | 2020-04-10 | 浙江大学 | Method for separating human albumin by expanded bed adsorption based on mixed mode |
| CN107243336A (en) * | 2017-06-27 | 2017-10-13 | 大连理工大学 | A kind of chromatography media and its preparation method and application |
| CN107243336B (en) * | 2017-06-27 | 2019-12-27 | 大连理工大学 | Chromatography medium, preparation method and application thereof |
| CN107413302A (en) * | 2017-09-04 | 2017-12-01 | 重庆希尔康血液净化器材研发有限公司 | A kind of immune absorption material of multiple spot fixed protein A and preparation method thereof |
| CN107413302B (en) * | 2017-09-04 | 2021-03-09 | 重庆希尔康血液净化器材研发有限公司 | Immunoadsorption material for multi-point immobilized protein A and preparation method thereof |
| CN113289587A (en) * | 2021-05-10 | 2021-08-24 | 苏州君盟生物医药科技有限公司 | Sulfydryl modified magnetic nano-microsphere and preparation method and application thereof |
| CN113289587B (en) * | 2021-05-10 | 2023-11-28 | 苏州君盟生物医药科技有限公司 | Sulfhydryl modified magnetic nano microsphere and preparation method and application thereof |
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