CN103418358A - Chromatography media of composite ligand - Google Patents
Chromatography media of composite ligand Download PDFInfo
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- CN103418358A CN103418358A CN2012101481781A CN201210148178A CN103418358A CN 103418358 A CN103418358 A CN 103418358A CN 2012101481781 A CN2012101481781 A CN 2012101481781A CN 201210148178 A CN201210148178 A CN 201210148178A CN 103418358 A CN103418358 A CN 103418358A
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Abstract
The invention relates to a chromatography media of a composite ligand, and the chromatography media is prepared by taking agarose gel as a main media and employing a double-time suspension method. The inner core and the outer shell of a media is modified by two different ligands, and thus a media with the inner core modified by an anion ligand (such as diethylaminoethyl) and the outer shell modified with a cation ligand (such as sulfo group) is obtained, or a media with the inner core modified by an cation ligand and the outer shell modified by an anion ligand is obtained. The media with the inner core and the outer shell respectively modified by two different ligands is capable of adsorbing molecules carrying different positive and negative electric charges during separation, so that the same media is capable of realizing separation and purification of a sample carrying different charges, and is beneficial to separation and purification of samples in production.
Description
Technical field
The present invention relates to a kind of chromatographic media with compound aglucon, it is characterized in that having on same medium the modification of inside and outside two kinds of different aglucons, and present the distribution of shell core formula, belong to the chromatographic separation technology field.
Background technology
In the bio-separation field, the existence that the development of separating and purifying technology all is fixed against separating medium mostly develops.Separating and purifying technology is only to take certain theory to instruct the separation and purification process as basis, and whole process all be take separating medium as basis.If the appearance of novel separating medium is arranged, just may drive isolation technics and make breakthroughs, therefore, prepare one of main goal in research that efficient separation and purification medium is the bio-separation field.
Along with improving constantly that Separation of Proteins is required, promoted further developing of chromatographic media.Even-grained cross-linked polysaccharides is common separating medium, as cross-linked agarose gel, commercial Sepharose Fast Flow, the Sepharose High Performance etc. that have Amersham Biosciences to produce, this class medium not only intensity is applicable to, and granularity has improved its efficiency in the separation and purification stage uniformly.Artificial synthetic macroporous polymer is also a more common class chromatographic media, be characterized in medium except this little diffusion hole is arranged, also has large through hole, so not only greatly accelerated mass transfer, also increased flow velocity, business-like product is the Poros series that PerSeptive Biosystems company releases in 1989 the earliest.The Fractogel EMD series feeler type adsorbent of Merck company has strengthened the interaction of active aglucon and protein by increasing spacerarm and effective functional group.The development of chromatographic technique, promoted the exploitation of chromatographic media, makes the development of new chromatographic media become the key of separation science.
The character of chromatographic media is mainly determined by its physical and chemical performance, its physical characteristic is mainly the medium internal pore structure, as porous, super big hole, through hole, core-shell structure etc.: its chemical characteristic depends primarily on the aglucon of its finishing, as: anion aglucon, cation aglucon, affinity ligand, hydrophobic aglucon etc.The aglucon of medium not only determines the kind of medium, and determines its adsorption property, and different aglucons represents chromatographic media of different nature.Normally a kind of functional group of Ion Exchange Medium (active aglucon) is attached on polymer backbone through ehter bond by the length hydrocarbon chain, has obtained respectively accordingly cation or anionite.The density of aglucon has a great impact the absorption of protein, and before ligand density reaches its extreme value, adsorption capacity increases with the increase of ligand density, and after the density that oversteps the extreme limit, adsorption capacity is not subject to the impact of ligand density substantially.The kind of aglucon also affects its adsorption capacity to protein simultaneously.Usually for the medium of same type, strong sun (the moon) ion-exchanger is stronger to the binding ability of albumen than weak sun (the moon) ion-exchanger, needs stronger salinity to carry out wash-out simultaneously, and it is selective also higher.But the power of medium absorption does not represent the quality of its adsorption effect, strong ion-exchanger may with combined with protein is too strong, the generation Irreversible Adsorption makes wash-out be not easy to carry out, and affects yield.The ion-exchange chromatography medium of exploitation is all single aglucon at present, or is anion aglucon chromatographic media, or is cation aglucon chromatographic media, there is no the chromatographic media of anion and the compound aglucon of cation.Therefore, compound aglucon medium prepared by the present invention has two kinds of opposite charges simultaneously,, demonstrates unique performance under certain condition, and the development of novel chromatographic media and the exploitation of new separation technology from now on had to directive function.
Summary of the invention
The present invention relates generally to a kind of chromatographic media with compound aglucon, take Ago-Gel as main matrix, prepare medium by secondary suspension emulsion process, and it is carried out to the modification of two kinds of different aglucons, kernel and shell are modified with anion and two kinds of different aglucons of cation respectively.Mainly comprise two media, a kind of is that kernel is that anion aglucon (as the diethylin ethyl) is modified, and shell is the medium that cation aglucon (as sulfonic group) is modified; Another kind is that shell is that anion aglucon (as the diethylin ethyl) is modified, and kernel is the medium that cation aglucon (as sulfonic group) is modified.
Characteristics of the present invention mainly comprise the following aspects:
(1) having realized the modification of two kinds of different aglucons on same medium, is respectively anion and cation.
(2) on same medium, two kinds of different aglucons present the distribution of shell core formula, and controlled.
Main purpose of the present invention is, prepares the medium that a kind of compound aglucon is modified, and can carry out exchange adsorption from the material with different electric charges, is conducive to the lifting of separative efficiency.
The accompanying drawing explanation
Fig. 1: kernel cation aglucon (sulfonic group) is modified, the medium that shell anion aglucon (diethylin ethyl) is modified.
Fig. 2: kernel anion aglucon (diethylin ethyl) is modified, the media particle enlarged drawing that shell cation aglucon (sulfonic group) is modified.
Fig. 3: kernel cation aglucon is modified, the medium that shell anion aglucon is modified, and first to the absorption of pH=4.2BSA solution, the Langmuir isothermal fitted figure of result after then pH=7.6BSA solution being adsorbed.
The specific embodiment
The present invention, by the secondary suspension method, be take Ago-Gel as matrix, prepares inside and outside two kinds of chromatographic medias that different aglucons are modified on same medium.The aglucon method of modifying of medium is as follows:
(1) modification of diethylin ethyl: get 10g through medium crosslinked and reduction, add 20mL 0.5molL
-1DEAE-HCl, sealing is put into 60 ℃ of constant-temperature table preheatings after 10 minutes, by 20mL 3.5molL
-1NaOH add wherein, sealing continues reaction 1 hour, reaction is cooling by it after finishing, by medium with deionized water rinsing to neutral.
(2) sulfonic modification: comprise two processes.
1. pi-allyl activation process: 10g is the crosslinked and medium that reduced adds 0.068gNaBH
4With the 1.627g anhydrous sodium sulfate, add 5mL 4-6molL
-1Sodium hydroxide solution and the allyl glycidyl ether of 4-7mL, at 50 ℃, 150 turn under per minute constant temperature oscillation 9 hours, reaction is washed till neutrality by deionized water by it after finishing.
2. coupling process: by the Ago-Gel of the pi-allyl of the above-mentioned wash clean of 10g activation, add certain density NaOH to regulate pH to 4-6, the Sodium Metabisulfite of quality such as add, 37 ℃ of lower oscillating reactions 23 hours.Reaction is washed till neutrality by deionized water after finishing, and drains.
Example 1
The kernel sulfonic group is modified, and shell diethylin ethyl is modified the preparation flow of medium:
After agarose gel microsphere after crosslinked and reduction is carried out to the sulfonic group modification, according to solid-to-liquid ratio, being 0.2-0.8 mixes the kernel modified and the concentration ratio of cumulative volume (the agarose quality with) mutually for the Ago-Gel of 5%-8%, add in oil phase reactor (the 300mL soybean oil mixes with class of 5-15mL department 80), complete the parcel of medium kernel by suspension method, after crosslinked to it, with anion aglucon (as the diethylin ethyl), the shell Ago-Gel is modified, so just having made shell is that the anion aglucon is modified, kernel is the chromatographic media that the cation aglucon is modified.
Characterization of Adsorption: (1) take concentration as 0.25-2mgmL
-1The bovine serum albumin(BSA) (BSA) of different gradients (pH=4.2 take sodium hydrogen phosphate-citrate buffer solution preparation) is for target protein carries out Static Adsorption, and the calculating adsorption capacity.(2) by adsorbing medium completely in (1), by deionized water, clean up, the centrifugal supernatant that goes, in order to Tris-HCl (pH=7.6,0.01molL
-1) for the BSA solution of the variable concentrations of buffer solution preparation is that target protein carries out Static Adsorption, and calculate adsorption capacity.Known to the variation of protein concentration before BSA solution absorption under different pH and after absorption by comparative medium, this medium has adsorption capacity to the BSA solution with different electric charges.
Example 2
Kernel diethylin ethyl is modified, and the shell sulfonic group is modified the preparation flow of medium:
After agarose gel microsphere after crosslinked and reduction is carried out to the modification of diethylin ethyl anion aglucon, according to solid-to-liquid ratio, being 0.2-0.8 mixes the kernel modified and the concentration ratio of cumulative volume (the agarose quality with) mutually for the Ago-Gel of 5%-8%, add in oil phase reactor (the 300mL soybean oil mixes with class of 5-15mL department 80), complete the parcel of medium kernel by suspension method, after crosslinked to it, with cation aglucon (as sulfonic group), the shell Ago-Gel is modified, so just having made shell is that the cation aglucon is modified, kernel is the chromatographic media that the anion aglucon is modified.
Characterization of Adsorption: (1) take concentration as 0.25-2.0mgmL
-1BSA (pH=7.6, the 0.01molL of different gradients
-1Tris-HCl be buffer solution) for target protein carries out Static Adsorption, and calculate adsorption capacity.(2) by adsorbing medium completely in (1), by deionized water, clean up, the centrifugal supernatant that goes, the BSA solution (positively charged) of the variable concentrations that the sodium hydrogen phosphate-citric acid (pH=4.2) of take prepares as buffer solution carries out Static Adsorption as target protein, and calculates adsorption capacity.Known by the variation of protein concentration before BSA solution absorption under more different pH and after absorption, this medium has certain adsorption capacity to the BSA with different electric charges.
Claims (4)
1. the chromatographic media with compound aglucon, this medium can be divided into kernel and shell two parts, and kernel and shell are modified with two kinds of different aglucons respectively, and the preparation method is as follows:
(1) modification of kernel: by the agarose gel microsphere prepared, with anion aglucon (as the diethylin ethyl), be modified into anionic exchange medium.
(2) modification of shell: the solid-to-liquid ratio by 0.2-0.8 joins kernel in the solution that agarose concentration is 5%-8%, at rotating speed, be 800-1200 rpm, oil-water ratio is 5: 1-8: 1, utilize suspension method to be wrapped up the kernel of having modified the anion aglucon, after crosslinked, modify the shell Ago-Gel with cation aglucon (as sulfonic group), so just being prepared into a kind of shell is the ball-type chromatographic media with compound aglucon that cation aglucon and kernel are the anion aglucon.
2. chromatographic media as described in claim 1, according to different requirements, two kinds of aglucons are interchangeable, and available anion aglucon is modified shell, with the cation aglucon, modifies kernel.
3. chromatographic media as described in claim 1, is characterized in that having on same medium the modification of inside and outside two kinds of different aglucons, and present shell core formula and distribute.
4. chromatographic media as described in claim 1, the modification density of two kinds of aglucons can be different.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108815879A (en) * | 2018-06-13 | 2018-11-16 | 浙江月旭材料科技有限公司 | A kind of compounded mix and preparation method thereof |
| CN109647361A (en) * | 2019-02-22 | 2019-04-19 | 北京石油化工学院 | A kind of preparation method of composite polymer chromatography media |
| CN109806916A (en) * | 2019-03-15 | 2019-05-28 | 中科森辉微球技术（苏州）有限公司 | High performance anion exchange media and preparation method thereof |
| CN113750571A (en) * | 2021-09-10 | 2021-12-07 | 杭州先端生物科技有限公司 | Method for prolonging service life of ion exchange filler |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1457919A (en) * | 2003-05-16 | 2003-11-26 | 天津大学 | Method for preparing high density core material coated with thin shell medium of agarose gel |
| WO2011102790A1 (en) * | 2010-02-19 | 2011-08-25 | Ge Healthcare Bio-Sciences Ab | Method for production of chromatography media |
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- 2012-05-15 CN CN2012101481781A patent/CN103418358A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1457919A (en) * | 2003-05-16 | 2003-11-26 | 天津大学 | Method for preparing high density core material coated with thin shell medium of agarose gel |
| WO2011102790A1 (en) * | 2010-02-19 | 2011-08-25 | Ge Healthcare Bio-Sciences Ab | Method for production of chromatography media |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108815879A (en) * | 2018-06-13 | 2018-11-16 | 浙江月旭材料科技有限公司 | A kind of compounded mix and preparation method thereof |
| CN109647361A (en) * | 2019-02-22 | 2019-04-19 | 北京石油化工学院 | A kind of preparation method of composite polymer chromatography media |
| CN109806916A (en) * | 2019-03-15 | 2019-05-28 | 中科森辉微球技术（苏州）有限公司 | High performance anion exchange media and preparation method thereof |
| CN109806916B (en) * | 2019-03-15 | 2021-12-21 | 中科森辉微球技术（苏州）有限公司 | High-performance anion exchange medium and preparation method thereof |
| CN113750571A (en) * | 2021-09-10 | 2021-12-07 | 杭州先端生物科技有限公司 | Method for prolonging service life of ion exchange filler |
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Application publication date: 20131204 |