CN104725559B - Thiophilic chromatography material and its preparation method and application - Google Patents
Thiophilic chromatography material and its preparation method and application Download PDFInfo
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- 239000003960 organic solvent Substances 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 21
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- 238000007142 ring opening reaction Methods 0.000 claims abstract description 17
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical class [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 3
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Abstract
The invention discloses a kind of thiophilic chromatography material and its preparation method and application, the preparation method includes：A, in the presence of initiator, GMA and methylene-bisacrylamide are carried out in pore-foaming agent copolyreaction copolymer poly (GMA co MBAA) are obtained；B, in the presence of the first organic solvent, copolymer poly (GMA co MBAA) and sulfanilamide (SN) are carried out into ring-opening reaction copolymer poly (SFA (GMA co MBAA)) are obtained.The sulfone sulfide group stabilization for having high mechanical strength, permeability good by thiophilic chromatography material obtained in the method and modifying, what the thiophilic chromatography material was capable of antagonist carries out selective enrichment and separation.
Description
Technical field
The present invention relates to chromatographic material, in particular it relates to a kind of thiophilic chromatography material and its preparation method and application.
Background technology
With the fast development of molecular biology, immune engineering and genetic engineering, antibody disease treatment, bio-pharmaceuticals and
Biotechnology field serves more and more extensive effect.In domestic and international market, the demand of antibody is improved year by year, according to
The latest survey of Business Communication companies shows that since nineteen ninety-three, the demand of antibody continues with 4.4%
Growth rate is increased, wherein, only the market share of monoclonal antibody has just reached hundreds of hundred million dollars [referring to Nature
Biotech.,2010,28,917-924].However, the use of antibody, especially vivo applications, have strict requirements to purity,
The antibody purification technology and method for developing low-cost high-efficiency can not only promote the progress of scientific research, and contain huge
Business opportunity.
Thiophilic chromatography method also known as close sulphur chromatography [referring to Analytica Chimica Acta.2010,658,18-31],
It is a kind of method being enriched with for antibody with separate.So far, domestic and foreign scholars are devoted to various thiophilic chromatography materials always
Preparation and be applied to the enrichment of antibody with purifying.Early in the eighties in last century, Porath etc. [referring to FEBS Lett,
1985,185,306-310] divinyl sulfone activated agarose is just used, T2gel gels is obtained, obtain earliest thiophilic chromatography point
From material.Hereafter, Yannick etc. [referring to J.Chromatogr.B, 2004,808,51-56] is in hollow fiber film polyethylene diethyl
Thiophilic reagent is modified on enol, the separation to IgG in complex system is realized.2010, Lakhiar etc. [referring to
Chromatogr., 2010,72, the 205-213] ethylenediamine on the surface bond of glucan, the electricity for being used to neutralize glucan is born
Property, sulfone-sulfide group is then re-introduced into, successfully the antibody in mouse ascites is enriched with and has been separated.China Foochow is big
Fu Rong etc. [referring to Natural Science, 2000,28 (6), 110-114] isolates and purifies ovum using the T2gel of commercialization
Yellow immunoglobulin (IgY), activity recovery may be up to 70%.Huaqiao University the great grade of money [referring to Biotech.Progr.,
2009,25,376-383] the correlative study work of thiophilic magnetic microsphere is engaged in, they carry out the polymer microballoon of superparamagnetism
It is fully hydrolyzed, then by Michael addition reactions, thiophilic group, the thiophilic magnetic microsphere is coupled on the surface of magnetic microsphere
Can be used for the purifying of Detection of Antibody in Serum and excellent effect.
However, agarose, gel belong to soft matrix, it is impossible to bear high pressure；And the rear modification on magnetic microsphere is easily sent out
The loss of raw functionalization group.This kind of mechanical strength is small, high cost, and storage is difficult, and cannot repeatedly use, and meets
Not high-throughout antibody purification.
The content of the invention
It is an object of the invention to provide a kind of thiophilic chromatography material and its preparation method and application, by obtained in the method
The sulfone that thiophilic chromatography material has high mechanical strength, permeability good and modifies-sulfide group stabilization, the thiophilic chromatography material can
Antagonist carries out selective enrichment and separation.
To achieve these goals, the invention provides a kind of preparation method of thiophilic chromatography material, the preparation method
Including：
A, in the presence of initiator, by GMA and methylene-bisacrylamide in pore-foaming agent
Carry out copolyreaction and copolymer poly (GMA-co-MBAA) is obtained；
B, in the presence of the first organic solvent, copolymer poly (GMA-co-MBAA) and sulfanilamide (SN) are carried out into ring-opening reaction
Copolymer poly (SFA- (GMA-co-MBAA)) is obtained.
Present invention provides a kind of thiophilic chromatography material, the thiophilic chromatography material is by above-mentioned method preparation
.
Present invention also offers a kind of above-mentioned thiophilic chromatography material antibody enrichment with separate in application.
By above-mentioned technical proposal, the present invention first in the presence of initiator, by GMA and
Methylene-bisacrylamide carries out copolyreaction and copolymer poly (GMA-co-MBAA) is obtained；Then by sulfanilamide (SN) and copolymer
Poly (GMA-co-MBAA) carries out ring-opening reaction, so as to sulfone-sulfide group modification is arrived into copolymer poly (GMA-co-MBAA)
On be made copolymer poly (SFA- (GMA-co-MBAA)), i.e. thiophilic chromatography material.The thiophilic chromatography material is the netted of crosslinking
Structure, mechanical strength is big and permeability is good.In the presence of high salt concentration, the thiophilic chromatography material can be appropriate with antibody surface
There is specific adsorption in site, in the presence of low concentration of salt, antibody can be eluted out from thiophilic chromatography material.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Brief description of the drawings
Accompanying drawing is, for providing a further understanding of the present invention, and to constitute the part of specification, with following tool
Body implementation method is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is the reaction principle schematic diagram that copolymer poly (SFA- (GMA-co-MBAA)) is prepared in embodiment 1；
Fig. 2 is the Fourier-infrared absorpting light spectra for detecting the thiophilic chromatography materials A 1 in example 1；
Fig. 3 is to detect scanning electron microscope (SEM) photograph of the thiophilic chromatography materials A 2 in the case where amplifying 1300 times in example 2；
Fig. 4 is to detect scanning electron microscope (SEM) photograph of the thiophilic chromatography materials A 2 in the case where amplifying 5000 times in example 2；
Fig. 5 is MALDI-TOF MS figure of the thiophilic chromatography materials A 2 to IgY eluents in application examples 3；
Fig. 6 is electrophoretogram of the thiophilic chromatography materials A 2 to IgY eluents in application examples 4.
Specific embodiment
Specific embodiment of the invention is described in detail below.It should be appreciated that described herein specific
Implementation method is merely to illustrate and explain the present invention, and is not intended to limit the invention.
The invention provides a kind of preparation method of thiophilic chromatography material, the preparation method includes：
A, in the presence of initiator, by GMA (GMA) and methylene-bisacrylamide
(MBAA) copolyreaction is carried out in pore-foaming agent copolymer poly (GMA-co-MBAA) is obtained；
B, in the presence of the first organic solvent, copolymer poly (GMA-co-MBAA) and sulfanilamide (SN) (SFA) are carried out into open loop
Reaction is obtained copolymer poly (SFA- (GMA-co-MBAA)).
In the present invention, initiator is any conventional initiator in this area, can be that organic peroxide draws
Hair agent, inorganic peroxide initiator, azo-initiator or redox initiator, it is contemplated that the mildness of reaction temperature,
Preferably, the initiator is azo-initiator, in order to further improve efficiency of initiation, it is highly preferred that the initiator is selected
One kind or many from azodiisobutyronitrile (AIBN), AMBN (AMBN), azo-bis-iso-dimethyl (AIBME)
Kind.
In the present invention, the specific species of pore-foaming agent can be selected in scope wide, but in order to improve copolyreaction
Yield, it is preferable that the pore-foaming agent be selected from dimethyl sulfoxide, lauryl alcohol, acetone, PEG-200 (trade mark for PEG-200 poly- second
Glycol), in the PEG-300 polyethylene glycol of PEG-300 (trade mark for) and PEG-10000 (trade mark is the polyethylene glycol of PEG-1000)
One or more.
In step a of the invention, the specific consumption of each raw material can be selected in scope wide, but in order to improve altogether
The speed and yield of poly- reaction, it is preferable that relative to the GMA of 1 weight portion, the di-2-ethylhexylphosphine oxide third
The consumption of acrylamide is 1-5 weight portions, and the consumption of the initiator is 0.1-5% weight portions, and the consumption of the pore-foaming agent is 1-
10 weight portions.
In step a of the invention, the specific reaction condition of copolyreaction can be selected in scope wide, but in order to
Improve the speed and yield of copolyreaction, it is preferable that the copolyreaction meets following condition：Reaction temperature is 40-90 DEG C, instead
It is 2-24h between seasonable.
In step b of the invention, the first organic solvent can be that any one in this area for ring-opening reaction has
Machine solvent, but in order to improve the speed and yield of ring-opening reaction, it is preferable that first organic solvent be selected from acetonitrile, acetone,
One or more in methyl alcohol and ethanol.
In step b of the invention, the specific consumption of each material can be selected in scope wide, but in order to improve out
The speed and yield of ring reaction, it is preferable that relative to the copolymer poly (GMA-co-MBAA) of 1 weight portion, the use of the sulfanilamide (SN)
It is 0.5-2 weight portions to measure, and the consumption of first organic solvent is 10-50 weight portions.
In step b of the invention, the specific reaction condition of ring-opening reaction can be selected in scope wide, but in order to
Improve the speed and yield of ring-opening reaction, it is preferable that the ring-opening reaction meets following condition：Reaction temperature is 65-75 DEG C, instead
It is 10-14h between seasonable.
In the present invention, in order to remove the unreacted small-molecule substance adhered on copolymer poly (GMA-co-MBAA),
So as to improve the purity of copolymer poly obtained in ring-opening reaction (SFA- (GMA-co-MBAA)), it is preferable that before step b,
Methods described also includes：Second organic solvent is extracted or drip washing to copolymer poly (GMA-co-MBAA).Wherein,
Extraction can be carried out by apparatus,Soxhlet's, and drip washing can be carried out by way of column chromatography.
In the present invention, in order to remove unreacted small point adhered on copolymer poly (SFA- (GMA-co-MBAA))
Sub- material, further improves the purity of copolymer poly (SFA- (GMA-co-MBAA)), it is preferable that described after step b
Method also includes：3rd organic solvent is extracted or drip washing to copolymer poly (SFA- (GMA-co-MBAA)).Its
In, extraction again may be by apparatus,Soxhlet's to be carried out, and the mode that drip washing again may be by column chromatography is carried out.
In the step of above-mentioned extraction or drip washing, the second organic solvent is conventional in the art with the 3rd organic solvent
Organic solvent, can select in scope wide, but in order to improve the removal effect of small-molecule substance, it is preferable that second has
Machine solvent is each independently selected from one or more in methyl alcohol, acetonitrile and ethanol with the 3rd organic solvent.
In the present invention, the reaction vessel of copolyreaction and ring-opening reaction can be glass container conventional in the art,
Such as flask, test tube and chromatographic column, but in order to obtain the thiophilic chromatography material of given shape, it is preferable that the copolyreaction and
Ring-opening reaction is carried out in open glass container or the capillary of sealing.Thiophilic color so in open glass container
Spectrum material is bulk, and then by grinding to form the thiophilic chromatography material of powdery, the powdery thiophilic chromatography material can be irrigated to color
In spectrum post, so that antagonist is enriched with and is separated.Except of course that the thiophilic chromatography material of bulk, by the capillary in sealing
In carry out copolyreaction and ring-opening reaction, so obtained thiophilic chromatography material is just fixed in capillary, such thiophilic color
Spectrum material can may be repeated in capillary and use.
In the above-described embodiment, in order that obtained thiophilic chromatography material can be more stably fixed in capillary,
Preferably, before step a, methods described also includes：Capillary is cleaned, then by the capillary after cleaning the 4th
In the presence of organic solvent haptoreaction is carried out with 3- (methacryloxypropyl) propyl trimethoxy silicane (γ-MAPS).So
Just can be as shown in figure 1, the silane group of so γ-MAPS just can be carried out with the hydroxyl on the silica of the inner surface of capillary
Reaction, then the carbon-carbon double bond of γ-MAPS can be reacted with the double bond on copolymer poly (GMA-co-MBAA), and then
So that copolymer poly (GMA-co-MBAA) is fixed in capillary, then by SFA and copolymer poly (GMA-co-MBAA)
Ring-opening reaction is carried out, so that obtained copolymer poly (SFA- (GMA-co-MBAA)) is tightly fixed in capillary.
In the present invention, the 4th organic dissolution can be any one in this area, but in order that obtain copolymer poly
(SFA- (GMA-co-MBAA)) can more stably be located at capillary in, it is preferable that the 4th organic solvent be selected from methyl alcohol,
One or more in acetonitrile and ethanol.
When processing capillary, the consumption of each material can be selected in scope wide, but in order to improve altogether
The stability of polymers poly (SFA- (GMA-co-MBAA)), it is preferable that relative to the 3- (methacryloxypropyl) third of 1 weight portion
Base trimethoxy silane, the consumption of the 4th organic solvent is 0.5-10 weight portions.
Catalytic actual conditions can be selected in scope wide in the present invention, but in order that SFA can
Reacted with the hydroxyl on the silica of the inner surface of capillary as much as possible, it is preferable that the haptoreaction meet with
Lower condition：Reaction temperature is 40-60 DEG C, and the reaction time is 8-12h.
In addition, the cleaning of capillary can be cleaning way conventional in the art, such as pickling, alkali cleaning, distilled water and have
Machine solvent clean, but in order that capillary can clean it is cleaner, it is preferable that capillary sequentially passes through alkali cleaning, water
Wash, pickling, washing and the cleaning of organic solvent.
The invention provides a kind of thiophilic chromatography material, the thiophilic chromatography material is prepared by above-mentioned method.
Present invention also offers a kind of above-mentioned thiophilic chromatography material antibody enrichment with separate in application.
Below will the present invention will be described in detail by embodiment.In following examples, infared spectrum parameter passes through Fu
In leaf-infrared spectrometer (IR-21, Japanese Shimadzu Corporation) measure, ESEM collection of illustrative plates parameter and X-ray energy spectrum parameter pass through
Field emission scanning electron microscope (S-4800, HIT Hitachi) is measured, mass spectrometry parameters pass through Matrix Assisted Laser Desorption
Ionization time of flight mass spectrometry (4800TOF/TOF, American AB Sciex company) is measured, and electrophoretic parameters pass through HPCE
(P/ACEtmMDQ, U.S. Bake Man) measure.
GMA, methylene-bisacrylamide, 3- (methacryloxypropyl) propyl trimethoxy silicon
Alkane, azodiisobutyronitrile are bought in Aladdin and are the commercially available product of Shanghai Jing Chun biochemical technologies Co., Ltd.
For the ability of enrichment and the separation of the antagonist of thiophilic chromatography material, with yolk immune globulin in following detection example
(IgY) is detected to thiophilic chromatography material in vain.
Embodiment 1
1) DMSO the and 1.74g lauryl alcohols of MBA, 2g of GMA, 0.6g of 0.3g are placed in centrifuge tube, shaken through being vortexed,
Ultrasound degassing, be well mixed, add the AIBN of initiator 10mg, be vortexed again for, ultrasound, after obtaining the solution of homogeneous clarification,
Constant temperature copolyreaction 12h obtains bulk material M1 in 75 DEG C of water-bath；
2) take 3g bulk materials M1 to be cut into small pieces, be put into apparatus,Soxhlet's, add 50mL methyl alcohol, extracted at 110 DEG C
Obtain bulk material M2 within 24 hours.
3) bulk material M2 is placed in vacuum drying chamber, is dried 12 hours at 100 DEG C, you can obtain copolymer poly
(GMA-co-MBAA)。
4) sulfanilamide (SN) of 5g is dissolved in the solution of 10ml acetonitriles, then with 3g copolymers poly (GMA-co-MBAA) 70
Ring-opening reaction 12h at a temperature of DEG C, obtains bulk material M3.
5) and then by 3g bulk materials M3 it is put into extractor, adds 50mL methyl alcohol, 24 is extracted at a temperature of 110 DEG C small
When, obtain copolymer poly (SFA- (GMA-co-MBAA)), i.e. thiophilic chromatography materials A 1.
Embodiment 2
1) capillary is rinsed into 30min with NaOH (0.1M), water, HCl (0.1M), water and methyl alcohol successively.Then will rinse
The inwall nitrogen stream of capillary afterwards blows 10h dryings.At 50 DEG C, dried capillary is placed in γ-MAPS and methyl alcohol
Mixed solution (volume ratio of γ-MAPS and methyl alcohol be 1:1, mass ratio is 1.3:1) haptoreaction 10h in, after reaction terminates
Capillary is rinsed with methyl alcohol, nitrogen drying obtains the capillary of cleaning.
2) GMA (30mg), MBAA (60mg), DMSO (200mg), lauryl alcohol (174mg) and AIBN (1mg) are placed in
In the centrifuge tube of 1.5mL, vortex, ultrasound obtain the solution of homogeneous clarification.Then the solution is injected into rapidly above-mentioned cleaning
In capillary, silicon rubber sealing two ends are used, in 75 DEG C of water-bath constant temperature copolyreaction 12h.
3) above-mentioned capillary is taken out, is connected on high pressure liquid chromatography pump, rinsing capillary as mobile phase with methyl alcohol is obtained altogether
Polymers poly (GMA-co-MBAA).
4) sulfanilamide (SN) of 25.0mg is dissolved in the solution of 1ml acetonitriles, at a temperature of 70 DEG C, is continuously injected into high-pressure pump
In stating the capillary containing copolymer poly (GMA-co-MBAA), ring-opening reaction 12h.After reaction terminates, capillary is taken out, connect
On high pressure liquid chromatography pump, integral post is rinsed with acetonitrile and methyl alcohol successively, obtain copolymer poly (SFA- (GMA-co-
MBAA)), i.e. thiophilic chromatography materials A 2.
Detection example 1
Thiophilic chromatography materials A 1 is detected by Fourier-infrared spectrometer (IR-21, Japanese Shimadzu Corporation), is examined
Survey result and see Fig. 2, as known in the figure sulfuryl (1020.3cm-1), phenyl ring (621.1,657.7,952.9cm-1) etc. characteristic group
Absworption peak, and then show that successfully sulfone-sulfide group has been gone up in modification in thiophilic chromatography materials A 1.
Detection example 2
By field emission scanning electron microscope (S-4800, HIT Hitachi) to thiophilic chromatography materials A 2 and
Copolymer poly (GMA-co-MBAA) carries out X-ray elementary analysis detection in embodiment 2, and testing result is shown in Table 1, can by the table
Know, 4 the step of by embodiment 2), sulfanilamide (SN) group successfully modification on copolymer poly (GMA-co-MBAA).
Table 1
Detection example 3
Thiophilic chromatography materials A 2 is examined by field emission scanning electron microscope (S-4800, HIT Hitachi)
Survey, testing result is shown in Fig. 3 and Fig. 4, it can be seen that thiophilic chromatography materials A 2 is highly cross-linked porous material, the material is
The material of the 3D skeleton structures of a diameter of 30-70nm.
Preparation example 1
The preparation of the IgY standard liquids of 1mg/mL：At 25 DEG C, 2mg IgY are dissolved in the 0.5M aqueous sodium sulfates of 2ml
The IgY standard liquids of 1mg/mL are obtained in liquid.
Preparation example 2
The preparation of the citric acid solution of 1M：At 25 DEG C, 2.1014g citric acids are dissolved in appropriate distilled water, and be settled to
10mL is configured to the aqueous citric acid solution of 1M.
Preparation example 3
The preparation of sample solution (CPB)：At 25 DEG C, 0.7163g disodium hydrogen phosphates and 3.5510g sodium sulphate are dissolved in 45mL
Distilled water in, then with the aqueous citric acid solution of 1M adjust to pH be 6.2, be then settled to 50mL and obtain sample solution.
Preparation example 4
The preparation of eluent (PB)：At 25 DEG C, by the distilled water of 0.7163g disodium hydrogen phosphates 45mL, 1M is then used
Aqueous citric acid solution adjust to pH be 6.2, be then settled to 50mL and obtain eluent.
Preparation example 5
The preparation of MALDI-TOF MS Matrix Solutions：At 25 DEG C, by 100mg gallic acids (SA), 3mL acetonitriles and 7mL
It is prepared after 0.1% trifluoroacetic acid mixing.
Preparation example 6
The preparation of cushioning liquid is separated by electrophoresis：By the disodium hydrogen phosphate of 1.9501g, 0.7305g sodium chloride, 7.2095g ten
Sodium dialkyl sulfate is dissolved in 240mL distilled water, and it is 6.5 to adjust solution to pH with 1M sodium hydrate aqueous solutions, is settled to
250mL is obtained electrophoretic separation cushioning liquid.
Application examples 1
1) 200min will be balanced in the sample solution injection thiophilic chromatography materials A 2 of 1mL, is then poured into the 1mg/ml's of 50 μ L
The immobilized 60min of IgY standard liquids；
2) after IgY is immobilized, is injected with the sample solution of 20 μ L and be rinsed in thiophilic chromatography materials A 2, after obtaining IgY enrichments
The Liquid Residue not being eluted；
3) IgY is eluted into prepared IgY eluents by the eluent injection thiophilic chromatography materials A 2 of 6 μ L；
4) the IgY eluents of 0.5 μ L are mixed with the MALDI-TOF MS Matrix Solutions of 0.5 μ L, is then dropped to
On MALDI-TOF MS target plates, then dry, cleaned with frozen water to remove salt solution, finally ionized with Matrix Assisted Laser Desorption and flown
Row time mass spectrum (4800TOF/TOF, American AB Sciex company) carries out mass spectral characteristi.Characterization result is shown in Fig. 5, and a is bent in the figure
Line is the blank control group (Na of 0.5M2SO4Solution) mass spectrogram, b curves for 1mg/mL IgY standard liquids mass spectrogram, c
Curve is the mass spectrogram of the Liquid Residue not being eluted after IgY is enriched with, and d curves are the mass spectrogram of IgY eluents, it can be seen that
Thiophilic chromatography materials A 2 can carry out effective enrichment in the presence of high salt concentration to IgY, right in the presence of low concentration of salt
IgY is effectively eluted.
Can be obtained by Fig. 5, using MALDI-TOF MS methods, can learn that thiophilic chromatography materials A 1 being capable of depositing in high salt concentration
Effective enrichment is carried out to IgY lower, and IgY can be eluted out in the presence of low concentration of salt.
Application examples 2
1) 200min will be balanced in the sample solution injection thiophilic chromatography materials A 2 of 1mL, is then poured into the 1mg/ml's of 50 μ L
The immobilized 60min of IgY standard liquids；
2) after IgY is immobilized, is injected with the sample solution of 20 μ L and be rinsed in thiophilic chromatography materials A 2, after obtaining IgY enrichments
The Liquid Residue not being eluted；
3) IgY is eluted into prepared IgY eluents by the eluent injection thiophilic chromatography materials A 2 of 6 μ L；
4) by HPCE (P/ACEtmMDQ, U.S. Bake Man) electrophoresis detection is carried out to IgY eluents,
Specifically testing conditions are：In the vitreous silica capillary that internal diameter is 75 μm (total length is 56.5cm, and effective length is 50cm)
In carry out；Infrared detection wavelength is 214nm, and sample size is 5s × 0.5psi.Electrophoresis detection result is shown in Fig. 6, and a curves are in the figure
Blank control group (the Na of 0.5M2SO4Solution) electrophoretogram, b curves for 1mg/mL IgY standard liquids electrophoretogram, c curves
The electrophoretogram of the Liquid Residue not being eluted after being enriched with for IgY, d curves are the electrophoretogram of IgY eluents, it can be seen that thiophilic
Chromatographic material A2 can carry out effective enrichment in the presence of high salt concentration to IgY, and IgY is entered in the presence of low concentration of salt
The effective wash-out of row.
Using the method, can equally learn that thiophilic chromatography materials A 1 can be carried out in the presence of high salt concentration to IgY
, can be eluted out for IgY in the presence of low concentration of salt by effective enrichment.
The preferred embodiment of the present invention described in detail above, but, the present invention is not limited in above-mentioned implementation method
Detail, in range of the technology design of the invention, various simple variants can be carried out to technical scheme, this
A little simple variants belong to protection scope of the present invention.
It is further to note that each particular technique feature described in above-mentioned specific embodiment, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy is no longer separately illustrated.
Additionally, can also be combined between a variety of implementation methods of the invention, as long as it is without prejudice to originally
The thought of invention, it should equally be considered as content disclosed in this invention.
Claims (16)
1. a kind of preparation method of thiophilic chromatography material, it is characterised in that the preparation method includes：
A, in the presence of initiator, GMA and methylene-bisacrylamide are carried out in pore-foaming agent
Copolyreaction is obtained copolymer poly (GMA-co-MBAA)；
B, in the presence of the first organic solvent, copolymer poly (GMA-co-MBAA) is opened with P-aminobenzene-sulfonamide
Ring reaction is obtained copolymer poly (SFA- (GMA-co-MBAA))；
Wherein, relative to the GMA of 1 weight portion, the consumption of the methylene-bisacrylamide is 1-5
Weight portion, the consumption of the initiator is 0.1-5% weight portions, and the consumption of the pore-foaming agent is 1-10 weight portions；Relative to 1 weight
The copolymer poly (GMA-co-MBAA) of part is measured, the consumption of the P-aminobenzene-sulfonamide is 0.5-2 weight portions, described first
The consumption of organic solvent is 10-50 weight portions.
2. preparation method according to claim 1, wherein, the initiator is selected from azodiisobutyronitrile, azo diisoamyl
One or more in nitrile, azo-bis-iso-dimethyl.
3. preparation method according to claim 1, wherein, the pore-foaming agent is selected from dimethyl sulfoxide, lauryl alcohol, acetone, board
One kind or many in the polyethylene glycol of the polyethylene glycol, the polyethylene glycol of trade mark PEG-300 and trade mark PEG-1000 of number PEG-200
Kind.
4. the preparation method according to any one in claim 1-3, wherein, the copolyreaction meets following condition：
Reaction temperature is 40-90 DEG C, and the reaction time is 2-24h.
5. preparation method according to claim 4, wherein, first organic solvent is selected from acetonitrile, methyl alcohol and ethanol
One or more.
6. the preparation method according to any one in claim 1-3, wherein, the ring-opening reaction meets following condition：
Reaction temperature is 65-75 DEG C, and the reaction time is 10-14h.
7. the preparation method according to any one in claim 1-3, wherein, before step b, methods described is also wrapped
Include：Second organic solvent is extracted or drip washing to copolymer poly (GMA-co-MBAA).
8. the preparation method according to any one in claim 1-3, wherein, after step b, methods described is also wrapped
Include：3rd organic solvent is extracted or drip washing to copolymer poly (SFA- (GMA-co-MBAA)).
9. the preparation method according to any one in claim 1-3, wherein, second organic solvent and the described 3rd
Organic solvent is each independently selected from one or more in methyl alcohol, acetonitrile and ethanol.
10. the preparation method according to any one in claim 1-3, wherein, the copolyreaction and ring-opening reaction exist
Carried out in open glass container or the capillary of sealing.
11. preparation methods according to claim 10, wherein, before step a, methods described also includes：By capillary
Cleaned, then by the capillary after cleaning in the presence of the 4th organic solvent with 3- (methacryloxypropyl) propyl group front three
TMOS carries out haptoreaction.
12. preparation methods according to claim 11, wherein, the 4th organic solvent be selected from acetonitrile, acetone, methyl alcohol and
One or more in ethanol.
13. preparation method according to claim 11 or 12, wherein, relative to the 3- (methacryloxypropyl) of 1 weight portion
Propyl trimethoxy silicane, the consumption of the 4th organic solvent is 0.5-10 weight portions.
14. preparation methods according to claim 13, wherein, the haptoreaction meets following condition：Reaction temperature is
40-60 DEG C, the reaction time is 8-12h.
15. a kind of thiophilic chromatography materials, it is characterised in that the thiophilic chromatography material is by any one in claim 1-14
Method described in is prepared.
A kind of 16. thiophilic chromatography materials according to claim 15 antibody enrichment with separate in application.
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