CN1068887C - Synthesis of affinity film medium using cellulose as substrate - Google Patents
Synthesis of affinity film medium using cellulose as substrate Download PDFInfo
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- CN1068887C CN1068887C CN97101243A CN97101243A CN1068887C CN 1068887 C CN1068887 C CN 1068887C CN 97101243 A CN97101243 A CN 97101243A CN 97101243 A CN97101243 A CN 97101243A CN 1068887 C CN1068887 C CN 1068887C
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Abstract
The present invention relates to a synthesizing method using cellulose as a substrate, which is characterized in that epoxypropyl methacrylate or acrylic acid epoxy-propanol phosphate are used for carry out a self polymerization reaction to obtain a polymer; then, the polymer and cellulose have a grafting reaction; finally, a ring opening reaction carried out to obtain a compatible medium whose active groups are aldehyde groups or amino groups. The self polymerization reaction and the grafting reaction are carried out in a neutral medium, reaction temperature is from 50 to 100 DEG C, and the ring opening reaction temperature of the amino groups is from 40 to 80 DEG C. The compatible medium prepared by the method of the present invention enhances the bonding capacity of the cellulose, and has wide application prospect on the aspects of the large-scale separation and purification of biotic products, the immunotherapy of human bodies, etc.
Description
The present invention relates to a kind of is the synthetic method of the affinity film medium of matrix with the Mierocrystalline cellulose.It specifically is the synthetic complex media of matrix and glytidyl methacrylate copolymerized grafting with the Mierocrystalline cellulose, on this basis open loop oxidation or open loop connect different lengths spacerarm as amino, quadrol, hydrazine etc., but the affinity media of synthetic biological aglucon that can the connecting band reactive group.
Along with the development of biotechnology, people deepen continuously to the understanding of biological products in recent years, and demand constantly increases, and following, the various analytical separation methods that are used for the biological products purifying have obtained significant progress.Wherein, affinity chromatography is because the important means that specificity is good, purifying multiple advantages of higher has become biomacromolecules such as analysing protein nucleic acid.At present, the composition that can be used for doing affinity media has agarose, dextran, Mierocrystalline cellulose, silicon limb, polyacrylamide, sintered glass and various high molecular polymers etc.Wherein, agarose is to use the earliest, most widely used normal pressure chromatographic media, its structure homogeneous, non-specific adsorption is extremely low, be extraordinary chromatographic media material, in the following several years, agarose is difficult to be replaced by other matrix as the dominant position of affinity chromatography carrier.But agarose is a kind of gel-like substance, even carry out crosslinkedly, the pressure that can bear is also very limited.Colloid bearers such as dextran are difficult to satisfy the requirement of mass preparation too like this.Jaosoo J.C " Agr.Foodchem.19 (1971) 581 " has studied the relation of Sephadex G150 gel column high flow rate and pressure drop, increase flow velocity, pressure increases thereupon, but flow velocity reduces during to a certain limit, and pressure drop reduces, and this shows that gel has produced irreversible deformation, increase the post height, knee point is that permissible velocity of flow reduces, and visible gel-like matrix is difficult to amplify produces, and can not satisfy the mass preparation requirement.Another kind of common chromatographic media silica gel, because of its good mechanical property, the proof pressure height, especially be common in the high-effective affinity chromatography, but the silicon hydroxyl on silica gel surface has part ion character, make that silica gel surface non-specific adsorption is strong, and silica gel tolerance pH scope is narrower, has limited the application of silica gel in affinity chromatography.
Separate a lot of of medium though use commodity agarose, silica gel etc. in recent years in the document, traditional sucroses such as simple agarose, silica gel have been left in research as medium, seek the direction that complex media becomes affinity media research.Use affinity media to have following requirement in the mass preparation: (1). cost is low, and (2) bear elevated pressures, (3) high affine capacity, (4) degrees of expansion is low, and (5) compression is indeformable.Mierocrystalline cellulose is as the affinity media good mechanical property, and the chemical stability height is a raw material the most cheap and easy to get in all chromatographic medias, and Mierocrystalline cellulose is the first-selected affinity media that scale operation is amplified.But cellulose media itself also has very big defective, this drawbacks limit its application in affinity chromatography.Mainly cause by cellulosic structure.Mierocrystalline cellulose is to connect into straight chain by many β-D-glucose with the 1-4 glycosidic link, parallel to each other between straight chain, interchain glucose hydroxyl can form hydrogen bond, and this very orderly structure is cellulosic crystallizing field, and the glycosidic linkage that leaves this crystallizing field then forms noncrystalline domain.Crystallizing field and noncrystalline domain are responsive especially to the physico chemical factor of affinity chromatography preparation, cellulosic derivatization reaction generally occurs in the low or noncrystalline domain of crystallization degree hinge, make that like this aglucon that connects is quite inhomogeneous on microcosmic, and activable group content makes simple cellulose media be difficult to obtain the affine effect of ideal far below agarose.
The purpose of this invention is to provide a kind of is the affinity film medium and the synthetic method of matrix with the Mierocrystalline cellulose.Affinity media by this method preparation has improved cellulosic bonding capacity greatly.The Mierocrystalline cellulose complex media can be made film very easily, film medium have expand little, permeability good, withstand voltage height, advantage such as not yielding.Film medium is easy to load into axial columns and radial column, and axial columns is suitable for small-scale separation and purification, but radial column is suitable for large-scale separation and purification owing to have characteristics such as linear amplification, flow velocity be fast.This method will have broad application prospects at aspects such as the extensive separation and purification of biological product and human immunity treatments.
For achieving the above object, the present invention Mierocrystalline cellulose and glytidyl methacrylate copolymerized grafting, but make derived hydroxy groups few in the Mierocrystalline cellulose and a macromolecular chain bonding, each monomer in this macromolecular chain contains the active functional group that can react, can improve cellulosic aglucon capacity greatly, and can make active functional group content controlled by changing polymerizing condition and bonding conditions.Activity group content 1.0-2.5mmol/g (medium) has solved the Mierocrystalline cellulose low problem of capacity of can deriving itself.Because cellulose surface is assembled polymer substance is arranged simultaneously, Mierocrystalline cellulose swelling degree is reduced, because Mierocrystalline cellulose complex media system film is convenient, and the film medium permeability of making is good, flow velocity is fast, is suitable for doing the extensive separation of biomacromolecule again.The method of the synthetic affinity media of the invention described above, it is characterized in that adopting and carry out autohemagglutination with glytidyl methacrylate or acrylic acid epoxy propyl ester and obtain polymkeric substance, carry out graft reaction with Mierocrystalline cellulose again, last ring-opening reaction, obtain active group and be aldehyde radical or be amino affinity media, autohemagglutination and graft reaction carry out in neutral medium, and temperature of reaction is 50～100 ℃, and the ring-opening reaction temperature is 40～80 ℃.Simultaneously, in autohemagglutination and graft reaction, to add initiator, Diisopropyl azodicarboxylate for example, n-Butyl Lithium or ammonium persulphate and Sulfothiorine, the initiator add-on be reaction monomers heavy 1～5%.Ring-opening reaction should be controlled under certain pH value and carry out in addition, and the pH value is 8～12 when the ring-opening reaction thing is hexanediamine.Specifically, the present invention with the Mierocrystalline cellulose be matrix affinity media preparation by following three the step carry out:
1. glytidyl methacrylate or acrylic acid epoxy propyl ester autohemagglutination become a certain size polymkeric substance;
2. Mierocrystalline cellulose and polymkeric substance carry out graft reaction;
3. the preparation of the affinity media of different interval arm lengths.
In above-mentioned 1,2 reactions, reaction medium is a neutral aqueous solution, and wherein cellulosic content is 1.5～2.5%, and the content of glytidyl methacrylate or acrylic acid epoxy propyl ester is 3～10%, control reaction temperature is 50～100 ℃, and the autohemagglutination graft reaction time was advisable with 0.5～3 hour.Will add initiator and bring out reaction in reaction process, the initiator of the anionic polymerisation of employing is Diisopropyl azodicarboxylate, ends butyllithium or ammonium persulphate and Sulfothiorine, and the initiator add-on is 1～5% of a reaction monomers amount.Amino ring-opening reaction and is carried out under certain pH value under 40～80 ℃, and concrete operating process technology is routinely carried out.Below by example technology of the present invention is given to illustrate further:
The preparation of example 1 complex media
Self-chambering is in agitator, temperature are taken into account 250ml mouth flask with condenser, add water 100ml, be warming up to 80 ℃ and add 2 gram Mierocrystalline celluloses, stir, add the 5ml glytidyl methacrylate, after stirring 5 minutes, add the mixing solutions (about 10ml) that contains ammonium persulphate and each 0.5 gram of Sulfothiorine, isothermal reaction 2 hours, stopped reaction is reduced to after the room temperature with a large amount of washings, uses the remaining organism of acetone or alcohol flush away cellulose surface again, in vacuum drying oven, dry the synthetic complex media that has active epoxy group.The content of active epoxy group is 1.0～2.5mmol/g (dried medium) in the complex media.
Example 2 has affinity media synthetic of 4 atomic separation arms
Place the hydrochloric acid soln of pH=0.5 with example 1 prepared epoxy type complex media, evenly stir, make the abundant open loop of epoxy become vicinal hydroxyl groups, general room temperature needs 3 days, can not detect the epoxy in the medium, stopped reaction is filtered, the water thorough washing joins 1.5% sodium iodate (NalO of new preparation then to neutral
4) in 30 minutes, filter, massive laundering is washed, the oven dry of system film.Film medium is loaded on connects aglucon experiment in the pillar.With the phosphoric acid buffer balance of pillar with 0.1mol/lpH=7.6, getting a certain amount of protein is dissolved in the level pad, room temperature or 4 ℃ of last samples circulated 4 hours or the longer time, washing to A280 with level pad does not have absorption, borate buffer balance with 0.1mol/lpH=8.2, with glycine ethyl ester hydrochloride solution passivation 1 hour, add a certain amount of NaBH again
4Or NaCHBH
3The reduction carbonnitrogen bond, reduced 12 hours or the longer time, borate buffer by 0.1mol/lpH=8.2 successively then, 0.2mol/lpH=2.3 glycine solution, 0.1mol/lpH=7.6 contain the phosphoric acid buffer of 1mol/lNaCl, use the phosphoric acid buffer balance of 0.1mol/lpH=7.6 at last, synthetic brachium is the affinity media of 4 atoms.
Example 3: brachium is affinity media synthetic of 11 atoms
With prepared epoxy type complex media 3 grams of example 1, add in the 150ml water, stir strongly to being uniformly dispersed, be warming up to 80 ℃ after, add ammoniacal liquor 10～30ml, control pH value is 8～12, refluxes 1～2 hour, suction filtration is to filtrate neutrality.Do film dress post, the borate buffer balance of 0.1mol/l pH=8.2 was with a certain amount of 0.25% glutaraldehyde solution circulation 2 hours, by the 2mol/l acetum, being washed till A280 with pure water does not have absorption, and with the phosphoric acid buffer balance of 0.1mol/l pH=7.6, bonding aglucon step is with example 1.Synthetic brachium is the affinity media of 11 atoms.
Example 4: brachium is affinity media synthetic of 18 atoms
Restrain with example 1 prepared epoxy type complex media 3, add in the 150ml water, violent stirring is to being uniformly dispersed, be warming up to 80 ℃ after, add the quadrol solid that is equivalent to 1～5 times of epoxy content, control pH value is 8～12, reacted 1～2 hour, and took out washing, do film oven dry dress post to neutral, with glutaraldehyde activation and connect the aglucon step with example 1, synthetic brachium is the affinity media of 18 atoms.
Comparative example 1
With the comparison of the QHprotein A adsorption column of the technology of the present invention preparation with the ZETAFINITY-10 protein A adsorption column of U.S. CUNO company production
(1) experiment material and method
The bSA that uses in the experiment is pure as electrophoresis, and IgG is that immunity is pure, all available from magnificent biochemical reagents company.Used pump is Cole Parmer model 7518-10, and detector is GILSONmodel 112 UV-detector, detects wavelength 254 (in)/280 (out), Sensitivity (AUFS) 1.0, KNUER meter record instrument, chart speed 1.0mm/ branch.752C type ultraviolet-visible pectrophotometer (Shanghai second analytical instrument factory product).Determination of protein concentration: the A280 uv detection method, with bSA normal concentration curve determination.
(2) QH protein A post and the brief introduction of ZETA-10 protein A post
QH protein A post: film medium is synthetic by technological method of the present invention, and column dimension is 8 * 20mmI.D., and medium-weight 1.04g, flow are the 4ml/ timesharing, and pressure is 10psi before the post.The pillar shell is a synthetic glass, ZETA-10protein A post: medium is of a size of 38 * 10mmI.D. for being matrix synthetic affinity media with the Mierocrystalline cellulose, can adorn medium 0.9～1.0g, the pillar shell is plastics, when flow is 3ml/min, post is pressed and is increased, and pillar very easily breaks.
(3) to the comparison of people lgG maximal absorptive capacity
People lgG is dissolved in the 0.01mol/pH=7.5 phosphoric acid buffer, and concentration 10.6mg/ml gets the 4ml upper prop, circulated 10 minutes, and, be eluted to baseline with 0.2mol/l pH-2.3 glycine buffer with level pad flushing registering instrument baseline, collect elutriant, survey concentration and volume.Two post applied sample amounts and treatment process are identical, and its result is that it divides dried medium of 32.0mg/g and the dried medium of 12.0mg/g respectively to people lgG maximum adsorption capacity when adopting QH protein A post and ZETA-10 protein A post.
(4) comparison of maximum non-special loading capacity
BSA is dissolved in the 0.01mol/l pH=7.5 phosphoric acid buffer, concentration 1mg/ml, get 4ml and go up sample circulation 10 minutes, with level pad flushing registering instrument baseline, be eluted to baseline with pH=7.5mol/lNaCl, collect elutriant, survey concentration and volume, two post applied sample amounts and treatment process are identical, and its result is for adopting QH protein A post and ZETA-10 protein A post, and its maximum non-special loading capacity is respectively dried medium of 1.30mg/g and the dried medium of 2.11mg/g.
Comparative example 2
Be used for the comparison of the protein A film adsorption column and the proteinA agarose adsorption column of immunoadsorption therapy.
The immunosorption therapy is by interior originality, exogenesis virulence factor in antigen-antibody immune response or the physics chemical action removal human blood, purifies the blood, thereby reaches the purpose for the treatment of some doubtful piebald horse illness.In the last few years, immunoadsorption therapy became the important branch of blood purification technology gradually, caused the concern of medical circle day by day.Be used for clinical therapeutic efficacy now and be preferably A albumen.At present, had commodity selling what be that Sweden Gambro company produces is the A protein immunization adsorption column of matrix with the agarose.It is 62.5ml that this post contains the agar amount, and A protein binding ability is a 20mgIgG/ml agar, and shell is bundled into the cylindrical of 50 * 40mm by acrylate, and plasma perfusion speed is 15～35ml/ branch, is that the commodity immunoabsorbent column of matrix yet there are no report at present with the Mierocrystalline cellulose.Having synthesized spacerarm length with technological method of the present invention is the Protein A immunoabsorbent column of 18 atoms.The pillar form is divided two kinds, and a kind of is axial columns, is of a size of 14 * 60mm, I.D, dress medium 12g, plasma perfusion speed is 20～80ml/ branch, A protein binding ability is the dried medium of 32mg lgG/g, another kind of cylindricality formula is a radial column, and it is to adopt the spiral membrane module structure, liquid by the post circumferential flux to the post core, be of a size of 20 * 60mmI.D., interior dress medium 13g, plasma perfusion speed is 20～200ml/ branch, A protein binding ability is constant.The human normal plasma is by above-mentioned A protein film adsorption column, and the lgG in the absorption gets off with glycine=HCL buffer solution elution of pH=2.3, does mass spectrum inspection purity altogether, and the result shows, adopts the protein A film adsorption column specificity of technology preparation of the present invention better.ProteinA film adsorption column is through the past bacterium, the former processing of reducing phlegm and internal heat, and clinical experimentation on animals effect is better.
By as can be seen above, exceed nearly 2 times with the protein A immunoabsorbent column loading capacity of technology of the present invention preparation than the ZETA-10protein A post of U.S. CUNO company, the loading capacity of unit volume be equivalent to the agarose be matrix protein A adsorption column 1/2nd, but its flow will be apparently higher than agarose column.
The result of above-mentioned example and comparative example shows, technological method synthetic affinity film medium of the present invention has been avoided the weakness of soft matrix such as agarose, has stronger resistance to pressure, medium exists in the post with the form of film, still can stable existence under higher flow velocity, just can finish lock out operation at short notice, macromole flows in the film space in the mode of convection current, can combine very soon with aglucon, improved mass transfer in the post, theoretically, radially have can the big characteristics in linearity side for membrane chromatography, are suitable for large-scale separation and purification.This method will have broad application prospects at aspects such as the extensive separation and purification of biological product and human immunity treatments.
Claims (2)
1. one kind is the synthetic method of the affinity film medium of matrix with the Mierocrystalline cellulose, it is characterized in that it being to adopt to carry out self-polymeric reaction with glytidyl methacrylate or acrylic acid epoxy propyl ester and obtain polymkeric substance, carry out graft reaction with Mierocrystalline cellulose again, carry out ring-opening reaction at last, obtain active group and be aldehyde radical or be amino affinity media, autohemagglutination and graft reaction carry out in neutral medium, temperature of reaction is 50～100 ℃, amino ring-opening reaction temperature is 40～80 ℃, to add the initiator Diisopropyl azodicarboxylate in autohemagglutination and the graft reaction, n-Butyl Lithium or ammonium persulphate and Sulfothiorine, initiator add-on be reaction monomers heavy 1～5%.
2. according to the described synthetic method of claim 1, the pH value is 8～12 when it is characterized in that the ring-opening reaction thing is quadrol.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97101243A CN1068887C (en) | 1997-02-25 | 1997-02-25 | Synthesis of affinity film medium using cellulose as substrate |
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| Application Number | Priority Date | Filing Date | Title |
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| CN97101243A CN1068887C (en) | 1997-02-25 | 1997-02-25 | Synthesis of affinity film medium using cellulose as substrate |
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| CN1191866A CN1191866A (en) | 1998-09-02 |
| CN1068887C true CN1068887C (en) | 2001-07-25 |
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| CN97101243A Expired - Fee Related CN1068887C (en) | 1997-02-25 | 1997-02-25 | Synthesis of affinity film medium using cellulose as substrate |
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| CN107866206B (en) * | 2017-10-31 | 2020-07-31 | 苏州博进生物技术有限公司 | Epoxy activated affinity chromatography medium |
| CN107790108B (en) * | 2017-10-31 | 2020-07-14 | 苏州博进生物技术有限公司 | Affinity chromatography medium using hollow zinc oxide microspheres as rigid matrix |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4788288A (en) * | 1985-07-30 | 1988-11-29 | Air Products And Chemicals, Inc. | Self-and Hydroxyl reactive formaldehyde-free cyclic hemiamidal and hemiamide ketal crosslinking monomers |
| CN1072969A (en) * | 1992-10-05 | 1993-06-09 | 中国科学院广州化学研究所 | The viscose grafting, copolymerization and modification prepares the wool-like fiber method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4788288A (en) * | 1985-07-30 | 1988-11-29 | Air Products And Chemicals, Inc. | Self-and Hydroxyl reactive formaldehyde-free cyclic hemiamidal and hemiamide ketal crosslinking monomers |
| CN1072969A (en) * | 1992-10-05 | 1993-06-09 | 中国科学院广州化学研究所 | The viscose grafting, copolymerization and modification prepares the wool-like fiber method |
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