CN116262222A - Preparation method of diethylaminoethyl natural polysaccharide chromatographic medium - Google Patents
Preparation method of diethylaminoethyl natural polysaccharide chromatographic medium Download PDFInfo
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- CN116262222A CN116262222A CN202310030203.4A CN202310030203A CN116262222A CN 116262222 A CN116262222 A CN 116262222A CN 202310030203 A CN202310030203 A CN 202310030203A CN 116262222 A CN116262222 A CN 116262222A
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 69
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 69
- 150000004676 glycans Chemical class 0.000 title claims abstract description 61
- 239000012501 chromatography medium Substances 0.000 title claims abstract description 32
- MZVQCMJNVPIDEA-UHFFFAOYSA-N [CH2]CN(CC)CC Chemical group [CH2]CN(CC)CC MZVQCMJNVPIDEA-UHFFFAOYSA-N 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 239000002609 medium Substances 0.000 claims abstract description 73
- 238000006243 chemical reaction Methods 0.000 claims abstract description 37
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 37
- 239000011159 matrix material Substances 0.000 claims abstract description 33
- 125000003700 epoxy group Chemical group 0.000 claims abstract description 30
- 150000001875 compounds Chemical class 0.000 claims abstract description 26
- STMDPCBYJCIZOD-UHFFFAOYSA-N 2-(2,4-dinitroanilino)-4-methylpentanoic acid Chemical compound CC(C)CC(C(O)=O)NC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O STMDPCBYJCIZOD-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000005576 amination reaction Methods 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 82
- 239000000243 solution Substances 0.000 claims description 41
- 238000005937 allylation reaction Methods 0.000 claims description 31
- 230000031709 bromination Effects 0.000 claims description 29
- 238000005893 bromination reaction Methods 0.000 claims description 29
- 239000004005 microsphere Substances 0.000 claims description 28
- 238000005406 washing Methods 0.000 claims description 28
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 claims description 16
- JGJLWPGRMCADHB-UHFFFAOYSA-N hypobromite Inorganic materials Br[O-] JGJLWPGRMCADHB-UHFFFAOYSA-N 0.000 claims description 15
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 12
- 229920000936 Agarose Polymers 0.000 claims description 11
- -1 hydroxyl polysaccharide Chemical class 0.000 claims description 11
- MECNWXGGNCJFQJ-UHFFFAOYSA-N 3-piperidin-1-ylpropane-1,2-diol Chemical compound OCC(O)CN1CCCCC1 MECNWXGGNCJFQJ-UHFFFAOYSA-N 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 239000002245 particle Substances 0.000 claims description 8
- 229920000647 polyepoxide Polymers 0.000 claims description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 229920002307 Dextran Polymers 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 5
- 229920002678 cellulose Polymers 0.000 claims description 5
- 239000001913 cellulose Substances 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 4
- PLDLPVSQYMQDBL-UHFFFAOYSA-N 2-[[3-(oxiran-2-ylmethoxy)-2,2-bis(oxiran-2-ylmethoxymethyl)propoxy]methyl]oxirane Chemical compound C1OC1COCC(COCC1OC1)(COCC1OC1)COCC1CO1 PLDLPVSQYMQDBL-UHFFFAOYSA-N 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 230000003301 hydrolyzing effect Effects 0.000 claims 1
- 238000005342 ion exchange Methods 0.000 abstract description 28
- 102000004169 proteins and genes Human genes 0.000 abstract description 19
- 108090000623 proteins and genes Proteins 0.000 abstract description 19
- 239000002994 raw material Substances 0.000 abstract description 14
- 230000000694 effects Effects 0.000 abstract description 11
- 238000000746 purification Methods 0.000 abstract description 9
- 230000004048 modification Effects 0.000 abstract description 7
- 238000012986 modification Methods 0.000 abstract description 7
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 abstract description 6
- 125000003277 amino group Chemical group 0.000 abstract description 5
- 238000001179 sorption measurement Methods 0.000 abstract description 5
- 229910052751 metal Inorganic materials 0.000 abstract description 4
- 238000005571 anion exchange chromatography Methods 0.000 abstract description 2
- 239000002184 metal Substances 0.000 abstract description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 40
- 239000008367 deionised water Substances 0.000 description 29
- 229910021641 deionized water Inorganic materials 0.000 description 29
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 27
- 239000003446 ligand Substances 0.000 description 13
- 238000011068 loading method Methods 0.000 description 10
- 239000011543 agarose gel Substances 0.000 description 9
- 239000012086 standard solution Substances 0.000 description 9
- 238000005349 anion exchange Methods 0.000 description 8
- 238000004140 cleaning Methods 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 8
- 239000011734 sodium Substances 0.000 description 8
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 7
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 7
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 7
- 229910052794 bromium Inorganic materials 0.000 description 7
- 239000012535 impurity Substances 0.000 description 7
- 239000001632 sodium acetate Substances 0.000 description 7
- 235000017281 sodium acetate Nutrition 0.000 description 7
- 239000000499 gel Substances 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 239000004593 Epoxy Substances 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 229920001503 Glucan Polymers 0.000 description 3
- 238000010560 atom transfer radical polymerization reaction Methods 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 3
- 235000019345 sodium thiosulphate Nutrition 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- RAGSWDIQBBZLLL-UHFFFAOYSA-N 2-chloroethyl(diethyl)azanium;chloride Chemical compound Cl.CCN(CC)CCCl RAGSWDIQBBZLLL-UHFFFAOYSA-N 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- 229920002271 DEAE-Sepharose Polymers 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- RTRZIWZZYGDMFE-UHFFFAOYSA-N 1-chloro-n,n-diethylethanamine;hydron;chloride Chemical compound Cl.CCN(CC)C(C)Cl RTRZIWZZYGDMFE-UHFFFAOYSA-N 0.000 description 1
- JVIPLYCGEZUBIO-UHFFFAOYSA-N 2-(4-fluorophenyl)-1,3-dioxoisoindole-5-carboxylic acid Chemical compound O=C1C2=CC(C(=O)O)=CC=C2C(=O)N1C1=CC=C(F)C=C1 JVIPLYCGEZUBIO-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- PTHCMJGKKRQCBF-UHFFFAOYSA-N Cellulose, microcrystalline Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC)C(CO)O1 PTHCMJGKKRQCBF-UHFFFAOYSA-N 0.000 description 1
- 229920001425 Diethylaminoethyl cellulose Polymers 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 101710188314 Protein V Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- WKXGVFOESMDKRF-UHFFFAOYSA-L dipotassium;bromate;bromide Chemical compound [K+].[K+].[Br-].[O-]Br(=O)=O WKXGVFOESMDKRF-UHFFFAOYSA-L 0.000 description 1
- 239000006167 equilibration buffer Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- STZCRXQWRGQSJD-GEEYTBSJSA-M methyl orange Chemical compound [Na+].C1=CC(N(C)C)=CC=C1\N=N\C1=CC=C(S([O-])(=O)=O)C=C1 STZCRXQWRGQSJD-GEEYTBSJSA-M 0.000 description 1
- 229940012189 methyl orange Drugs 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/3085—Chemical treatments not covered by groups B01J20/3007 - B01J20/3078
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F251/00—Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
- B01J2220/48—Sorbents characterised by the starting material used for their preparation
- B01J2220/4812—Sorbents characterised by the starting material used for their preparation the starting material being of organic character
- B01J2220/4825—Polysaccharides or cellulose materials, e.g. starch, chitin, sawdust, wood, straw, cotton
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention relates to the technical field of biology, and discloses a preparation method of diethylaminoethyl natural polysaccharide chromatographic medium. The invention is characterized in that a polysaccharide matrix is firstly activated by a polyepoxy compound to introduce a plurality of active hydroxyl reaction sites, then allyl glycidyl ether is used for modifying the surface of the polysaccharide matrix, allyl is then brominated, and then the polysaccharide-based anion exchange chromatography medium is obtained through amination. In the preparation process of the invention, no metal element which is easy to denature protein is adopted; meanwhile, epoxy groups which do not react with hydroxyl groups are fully hydrolyzed into hydroxyl groups, so that subsequent amination of the epoxy groups can be avoided, and therefore, the residual epoxy groups and amine groups on proteins cannot be subjected to nonspecific adsorption, and further the purification effect and the service life of a medium are affected. In addition, the method has the advantages of easily available raw materials, high allyl modification and amination efficiency, and high ion exchange capacity of the obtained diethylaminoethyl natural polysaccharide chromatographic medium.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of diethylaminoethyl natural polysaccharide chromatographic medium.
Background
With the vigorous development of the biomedical field, the market demand for stable and efficient protein separation materials is also urgent. Natural macromolecular materials such as cellulose, agarose and dextran have been widely used as chromatographic media in the field of separation and purification, mainly because of their abundant sources, low production cost, easy modification, non-toxicity and good biocompatibility. For example, diethylaminoethyl sepharose is a weak anion exchange type gel filler, which has been widely used for separation and purification of proteins, but the common preparation raw material diethylaminoethyl chloride hydrochloride is incorporated into the second class of supervision products published in 2018 and implemented in 2019, 1 st, and < regulatory rules of monitoring chemicals of the people's republic of China > implementation rules, so raw materials are not easily available to be an important problem for production thereof.
Chinese patent application CN105727911a proposes a method of Atom Transfer Radical Polymerization (ATRP) using glycidyl methacrylate grafted onto agarose gel media to mount active epoxy groups, then diethylamine modification to obtain diethylaminoethyl agarose chromatography media. However, the method disclosed in this patent has the following drawbacks: firstly, after the ATRP reaction is finished, although EDTA solution is used for cleaning to remove copper catalyst, partial copper is still difficult to avoid to remain in a medium, and denaturation and inactivation of protein are easy to cause in later use; secondly, the amination reaction of the epoxy groups is incomplete, so that the residual epoxy groups and amine groups on the protein are easy to generate nonspecific adsorption, and the purification effect and the service life of the medium are influenced. In addition, the weak hydroxyl activity of the polysaccharide matrix surface of the commonly used ion exchange medium affects ligand density, resulting in lower ion exchange capacity, which is one of the reasons for poor protein separation and purification effects.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of diethylaminoethyl natural polysaccharide chromatographic medium. The invention is characterized in that a polysaccharide matrix is firstly activated by a polyepoxy compound to introduce a plurality of active hydroxyl reaction sites, then allyl glycidyl ether is used for modifying the surface of the polysaccharide matrix, allyl is then brominated, and then the polysaccharide-based anion exchange chromatography medium is obtained through amination. In the preparation process of the invention, no metal element which is easy to denature protein is adopted; according to the invention, epoxy groups which do not react with hydroxyl groups in the polyepoxy compound are fully hydrolyzed into hydroxyl groups, so that the number of active hydroxyl groups can be increased, and amination of the epoxy groups can be avoided, so that nonspecific adsorption of residual epoxy groups and amine groups on proteins can not be caused, and further the purification effect and the service life of a medium are influenced. In addition, the method has the advantages of easily available raw materials, high allyl modification and amination efficiency, and high ion exchange capacity of the obtained diethylaminoethyl natural polysaccharide chromatographic medium.
The specific technical scheme of the invention is as follows:
a preparation method of diethylaminoethyl natural polysaccharide chromatography medium comprises the following steps:
s1: the method comprises the steps of firstly activating a polysaccharide matrix by using a polyepoxy compound to introduce a plurality of active hydroxyl reaction sites to obtain a poly-active hydroxyl polysaccharide matrix, and then introducing inert groups on the surface of the poly-active hydroxyl polysaccharide matrix by using allyl glycidyl ether to obtain an allylation medium.
S2: brominating unsaturated bonds on the surface of the allylation medium obtained in the step S1 to obtain a bromination medium.
S3: and aminating the bromination medium obtained by S2 by using diethylamine to obtain the diethylaminoethyl natural polysaccharide chromatography medium.
The preparation method of the invention has the following advantages: firstly, the invention does not adopt metal elements which are easy to denature protein; secondly, aiming at the problem that the hydroxyl activity of the surface of the polysaccharide matrix is weaker, a plurality of active hydroxyl reaction sites are introduced into the surface of the polysaccharide matrix; in addition, the method of the invention fully hydrolyzes epoxy groups which do not react with hydroxyl groups in the polyepoxy compound into hydroxyl groups, thereby not only increasing the number of active hydroxyl groups, but also avoiding amination of epoxy groups, so that the residual epoxy groups and amine groups on protein do not generate nonspecific adsorption, and further the purification effect and the service life of the medium are affected. The diethylaminoethyl natural polysaccharide chromatographic medium prepared by the method has high exchange capacity which can reach 150-280 mu mol/mL.
Preferably, in S1, the step of activating the polysaccharide matrix with the polyepoxide compound to introduce a plurality of reactive hydroxyl reactive sites specifically comprises: and (3) oscillating polysaccharide matrix, dimethyl sulfoxide and NaOH solution at 35-45 ℃ for reaction, then adding polyepoxy compound for reaction, adding NaOH solution to fully hydrolyze unreacted epoxy groups, and washing off unreacted polyepoxy compound to obtain the poly-active hydroxyl polysaccharide matrix.
Polysaccharide matrix surfaces are rich in hydroxyl groups, but we have found during the course of the study that these hydroxyl groups are less reactive. In order to increase ligand density of diethylaminoethyl natural polysaccharide chromatographic medium and to increase ion exchange capacity, epoxy group of polyepoxy compound is reacted with hydroxyl group on polysaccharide substrate surface, then unreacted epoxy group is hydrolyzed to convert into multiple active hydroxyl reaction sites, and the subsequent reaction can be carried out by utilizing the active hydroxyl reaction sites to increase ligand density of finally obtained ion exchange medium and increase ion exchange capacity.
Preferably, in S1, the polyepoxy compound is selected from trimethylolpropane triglycidyl ether, glycerol tripropyloxy triglycidyl ether or pentaerythritol glycidyl ether.
It should be noted that in the present invention, it was found in experiments that the number of epoxy groups on the polyepoxide compound has a certain effect on the hydroxyl grafting effect on the polysaccharide substrate surface. Wherein, if the epoxy groups of the multi-epoxy compound are too small, the final active hydroxyl groups are less; conversely, if the number of epoxy groups in the polyepoxide is too large, too many branches are formed on the same polyepoxide molecule, which is likely to cause steric hindrance and affect the reactivity of the epoxy groups. Finally, the comparison shows that the same polyepoxide compound has better effect of containing 3-4 symmetrically distributed epoxy groups.
Preferably, in S1, the mass ratio of the polyepoxy compound to the polysaccharide matrix is 0.8-3:1.
Preferably, in S1, the natural polysaccharide gel microspheres are selected from agarose microspheres, cellulose microspheres or dextran microspheres.
Preferably, in S1, the particle size of the natural polysaccharide gel microsphere is 45-165 μm, and the content of the natural polysaccharide is 2-12%.
Preferably, in S1, the volume ratio of the allyl glycidyl ether to the natural polysaccharide matrix is 0.5-2:1.
Preferably, in S2, bromine water is adopted for bromination, the volume ratio of the bromine water to the allylation medium is 0.1-0.4:1, and the concentration of the bromine water is 1.5-3 wt%.
Preferably, in S2, the reaction temperature is 40-70 ℃ and the reaction time is 1-3 h.
Preferably, in S3, the amination is carried out by means of an aqueous solution of 20 to 40% by weight of diethylamine.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention firstly uses epoxy group of the polyepoxy compound to react with hydroxyl group on the surface of polysaccharide matrix, then hydrolyzes unreacted epoxy group to convert into a plurality of active hydroxyl reaction sites, and the subsequent reaction can be carried out by utilizing the active hydroxyl reaction sites, so that the ligand density of the finally obtained ion exchange medium can be improved, and the ion exchange capacity can be improved. The diethylaminoethyl natural polysaccharide chromatographic medium prepared by the invention can realize higher ion exchange capacity (150-280 mu mol/mL), so that the loading capacity of the protein in the medium can be improved, and the purification efficiency can be improved.
(2) The preparation method does not adopt metal elements which are easy to denature protein; the method of the invention fully hydrolyzes epoxy groups which do not react with hydroxyl groups in the polyepoxy compound into hydroxyl groups, and can avoid amination of the epoxy groups, so that the residual epoxy groups and amine groups on protein can not generate nonspecific adsorption, thereby influencing the purification effect and the service life of the medium.
(3) The preparation method has simple raw material sources, and the raw material diethylamino chloroethane hydrochloride which is not easy to obtain and controlled is changed into the diethylamine which is easy to obtain and safe.
Detailed Description
The invention is further described below with reference to examples.
General examples
A preparation method of diethylaminoethyl natural polysaccharide chromatographic medium comprises the following steps:
s1: polysaccharide matrix (preferably agarose microsphere, cellulose microsphere or dextran microsphere, particle diameter is 45-165 μm, natural polysaccharide content is 2-12%), dimethyl sulfoxide and NaOH solution are shake reacted at 35-45 deg.C, then poly epoxy compound (selected from trimethylolpropane triglycidyl ether, glycerol tripropoxy triglycidyl ether or pentaerythritol glycidyl ether, mass ratio of poly epoxy compound to polysaccharide matrix is 0.8-3:1) is added to react, naOH solution is added to fully hydrolyze unreacted epoxy group, and unreacted poly epoxy compound is washed out, so as to obtain poly active hydroxyl polysaccharide matrix.
S2: then activating the multi-active hydroxyl polysaccharide matrix with allyl glycidyl ether according to the volume ratio of 0.5-2:1, and introducing inert groups on the surface of the polysaccharide matrix to prepare the allylation medium.
S3: brominating unsaturated bonds on the surface of the allylation medium obtained by S1 with bromine water according to the volume of 0.1-0.4:1, wherein the concentration of the bromine water is 1.5-3 wt%, the reaction temperature is 40-70 ℃, and the reaction time is 1-3 h, thus obtaining the bromination medium.
S4: amination of the bromination medium obtained by S2 with a diethylamine aqueous solution having a concentration of 20-40 wt% yields a diethylaminoethyl natural polysaccharide chromatography medium.
Example 1
S1, shaking and washing 10g of agarose gel microspheres (agarose content is 6%, average particle size is 90 μm), 6mL of dimethyl sulfoxide and 6mL of 20wt% NaOH solution, and shaking and reacting for 10min at 40 ℃. Then, after the reaction was continued for 3 hours by adding 8g of trimethylolpropane triglycidyl ether, 3mL of 20wt% NaOH solution was added and the reaction was continued for 1 hour to hydrolyze the unreacted epoxy groups sufficiently. And washing unreacted trimethylolpropane triglycidyl ether with pure water to obtain the multi-active hydroxyl agarose gel microspheres. The reaction scheme for S1 is shown below:
s2, 10g of the washed and evenly shaken multi-active hydroxyl agarose gel microspheres obtained by S1, 6mL of 30wt% NaOH solution and 0.02g of NaBH 4 ，1g Na 2 SO 4 4mL of allyl glycidyl ether was added to a 100mL Erlenmeyer flask and reacted with shaking at 40℃for 16h. After completion, the sample was washed with ethanol and deionized water to remove unreacted starting materials and impurities to give an allylated medium having an allylated ligand density of 180. Mu. Mol/mL.
S3, sequentially adding 10mL of allylation medium obtained in the step S1, 20mL of deionized water, 1g of sodium acetate, 2mL of 1.5wt% bromine water and 40 ℃ into a 100mL three-necked flask, and washing with a large amount of deionized water after the completion of the reaction to obtain a bromination medium.
S4, vibrating and reacting 10mL of the bromination medium obtained in the step S3 in 100mL of 20wt% diethylamine water solution at 30 ℃ for 12h to enable bromine on the bromination medium to fully react, and washing with ethanol and deionized water to obtain the anion exchange medium with the ion exchange capacity of 156 mu mol/mL.
The reaction scheme for S2-S4 is shown below:
example 2
S1, the same as in the embodiment 1.
S2, sequentially shaking and cleaning 10g of the multi-active hydroxyl agarose gel microspheres, 6mL of 30wt% NaOH solution and 0.02g of NaBH 4 ，1g Na 2 SO 4 8mL of allyl glycidyl ether is added into a 100mL conical flask, shaking reaction is carried out for 16h at 40 ℃, after completion, the sample is washed by ethanol and deionized water, unreacted raw materials and impurities are removed, and an allylation medium is obtained, wherein the allylation ligand density is 248 mu mol/mL.
S3, sequentially adding 10mL of allylation medium obtained in the step S1, 20mL of deionized water, 1g of sodium acetate, 2mL of 1.5wt% bromine water and the like into a 100mL three-necked flask, reacting for 1h at 40 ℃, and washing with a large amount of deionized water after completion to obtain a bromination medium.
S4, vibrating and reacting 10mL of the bromination medium obtained in the S1 in 100mL of 20wt% diethylamine water solution at 30 ℃ for 12h to enable bromine on the bromination medium to fully react, and washing with ethanol and deionized water to obtain the anion exchange medium with the ion exchange capacity of 205 mu mol/mL.
Example 3
S1, the same as in the embodiment 1.
S2, sequentially shaking and cleaning 10g of the multi-active hydroxyl agarose gel microspheres, 6mL of 30wt% NaOH solution and 0.02g of NaBH 4 ，1g Na 2 SO 4 15mL of allyl glycidyl ether is added into a 100mL conical flask, shaking reaction is carried out for 16h at 40 ℃, after completion, the sample is washed by ethanol and deionized water, unreacted raw materials and impurities are removed, and an allylation medium is obtained, and the allylation ligand density is 276 mu mol/mL.
S3, sequentially adding 10mL of allylation medium obtained in the example 1, 20mL of deionized water, 1g of sodium acetate, 3mL of 2wt% bromine water and 40 ℃ into a 100mL three-necked flask, reacting for 1h, and washing with a large amount of deionized water after completion to obtain a bromination medium.
S4, vibrating and reacting 10mL of the bromination medium obtained in the step S3 in 100mL of 30wt% diethylamine water solution at 30 ℃ for 12h to enable bromine on the bromination medium to fully react, and washing with de-ethanol and ionized water to obtain an anion exchange medium with the ion exchange capacity of 230 mu mol/mL.
Example 4
S1, the same as in the embodiment 1.
S2, sequentially shaking and cleaning 10g of the multi-active hydroxyl agarose gel microspheres, 6mL of 30wt% NaOH solution and 0.02g of NaBH 4 ，1g Na 2 SO 4 20mL of allyl glycidyl ether is added into a 100mL conical flask, shaking reaction is carried out for 16h at 40 ℃, after completion, the sample is washed by ethanol and deionized water, unreacted raw materials and impurities are removed, and an allylation medium is obtained, wherein the allylation ligand density is 302 mu mol/mL.
S3, sequentially adding 10mL of allylation medium obtained in the step S1, 20mL of deionized water, 1g of sodium acetate and 4mL of 2wt% bromine water into a 100mL three-necked flask, reacting at room temperature for 40min, and washing with a large amount of deionized water after completion to obtain a bromination medium.
S4, vibrating and reacting 10mL of the bromination medium obtained in the step S3 in 100mL of 40wt% diethylamine water solution at 30 ℃ for 12h to enable bromine on the bromination medium to fully react, and washing with ethanol and deionized water to obtain the anion exchange medium with the ion exchange capacity of 252 mu mol/mL.
Example 5
S1, shaking and cleaning 10g of cellulose gel microspheres, wherein the cellulose content is 6%, the average particle size is 95 mu m,6mL of dimethyl sulfoxide and 6mL of 20wt% NaOH solution, and vibrating and reacting for 10min at 40 ℃. Then, after the reaction was continued for 3 hours by adding 8g of trimethylolpropane triglycidyl ether, 3mL of 20wt% NaOH solution was added and the reaction was continued for 1 hour to hydrolyze the unreacted epoxy groups sufficiently. Washing unreacted trimethylolpropane triglycidyl ether with pure water to obtain the multi-active hydroxy cellulose gel microsphere.
S2, sequentially shaking and cleaning 10g of the multi-active hydroxy cellulose gel microspheres, 6mL of 30wt% NaOH solution and 0.02g of NaBH 4 ，1g Na 2 SO 4 15mL of allyl glycidyl ether is added into a 100mL conical flask, shaking reaction is carried out for 16h at 40 ℃, after completion, the sample is washed by ethanol and deionized water, unreacted raw materials and impurities are removed, and the allylation medium is obtained, wherein the allylation ligand density is 310 mu mol/mL.
S3, sequentially adding 10mL of allylation medium obtained in the step S1, 20mL of deionized water, 1g of sodium acetate and 4mL of 2wt% bromine water into a 100mL three-necked flask, reacting at room temperature for 40min, and washing with a large amount of deionized water after completion to obtain a bromination medium.
S4, vibrating and reacting 10mL of the bromination medium obtained in the step S3 in 100mL of 40wt% diethylamine water solution at 30 ℃ for 12h to enable bromine on the bromination medium to fully react, and washing with ethanol and deionized water to obtain the anion exchange medium with the ion exchange capacity of 240 mu mol/mL.
Example 6
S1, shaking and cleaning 10g of dextran gel microspheres, wherein the dextran content is 6%, the average particle size is 93 mu m,6mL of dimethyl sulfoxide and 6mL of 20wt% NaOH solution, and vibrating and reacting for 10min at 40 ℃. Then, after the reaction was continued for 3 hours by adding 8g of trimethylolpropane triglycidyl ether, 3mL of 20wt% NaOH solution was added and the reaction was continued for 1 hour to hydrolyze the unreacted epoxy groups sufficiently. Washing unreacted trimethylolpropane triglycidyl ether with pure water to obtain the multi-active hydroxyl glucan gel microsphere.
S2, sequentially shaking and cleaning 10g of the multi-active hydroxyl glucan gel microspheres, 6mL of 30wt% NaOH solution and 0.02g of NaBH 4 ，1g Na 2 SO 4 15mL of allyl glycidyl ether is added into a 100 mL-conical flask, shaking reaction is carried out for 16h at 40 ℃, after completion, the sample is washed by ethanol and deionized water, unreacted raw materials and impurities are removed, and an allylation medium is obtained, wherein the allylation ligand density is 289 mu mol/mL.
S3, sequentially adding 10mL of allylation medium obtained in the step S1, 20mL of deionized water, 1g of sodium acetate and 4mL of 2wt% bromine water into a 100mL three-necked flask, reacting at room temperature for 40min, and washing with a large amount of deionized water after completion to obtain a bromination medium.
S4, vibrating and reacting 10mL of the bromination medium obtained in the step S3 in 100mL of 40wt% diethylamine water solution at 30 ℃ for 12h to enable bromine on the bromination medium to fully react, and washing with ethanol and deionized water to obtain the anion exchange medium with the ion exchange capacity of 276 mu mol/mL.
Comparative example 1
The comparative example is a diethylaminoethyl chloride hydrochloride modified agarose chromatography medium comprising the steps of:
s1, sequentially adding 10mL of shaking-cleaned agarose gel microspheres with agarose content of 6%, average particle size of 90 mu m,4mL of 30wt% NaOH solution and 10mL of 5M diethyl chloroethane hydrochloride solution into a 100mL conical flask, and carrying out shaking reaction for 8 hours at 50 ℃.
S2, after the reaction is finished, washing the solution to be neutral, then washing the solution with 20mL of 1M HCl solution, and finally washing the solution to be neutral by deionized water to obtain the diethylamine ethyl agarose chromatography medium, wherein the ion exchange capacity is 153 mu mol/mL.
Comparative example 2 (differing from example 4 in that no multi-reactive hydroxylation was carried out)
S1, 10g of shaking and washing agarose gel microspheres (agarose content is 6%, average particle size is 90 μm), 6mL of 30wt% NaOH solution, 0.02g of NaBH 4 ，1g Na 2 SO 4 20mL of allyl glycidyl ether is added into a 100mL conical flask, shaking reaction is carried out for 16h at 40 ℃, after completion, the sample is washed by ethanol and deionized water, unreacted raw materials and impurities are removed, and an allylation medium is obtained, wherein the allylation ligand density is 216 mu mol/mL.
S2, sequentially adding 10mL of allylation medium obtained in the step S1, 20mL of deionized water, 1g of sodium acetate and 4mL of 2wt% bromine water into a 100mL three-necked flask, reacting at room temperature for 40min, and washing with a large amount of deionized water after completion to obtain a bromination medium.
S3, vibrating and reacting 10mL of the bromination medium obtained in the S3 in 100mL of 40wt% diethylamine water solution at 30 ℃ for 12h to fully react bromine on the bromination medium, and washing with ethanol and deionized water to obtain the anion exchange medium with the ion exchange capacity of 158 mu mol/mL.
Performance testing
(one) allyl ligand density testing method:
s1, placing 1mL of allylation medium into a 250mL conical flask, adding 30mL of 0.1M potassium bromide-potassium bromate solution, adding 10mL of 6M HCl, shaking uniformly, rapidly covering a cover, sealing a bottle opening, and standing in a dark place for 20min.
S2, rapidly adding 10ml of 20% KI solution, and placing in a dark place for 5min.
S3, titrating the standard solution of 0.1M sodium thiosulfate to light yellow, adding 1-2 mL of 1wt% starch indicator, continuously titrating until the solution is colorless as an end point, and recording the volume V of consumed sodium thiosulfate 1 Unmodified natural polysaccharide chromatographic media are blank. Ligand density was calculated according to the following formula:
in the formula, MNA 2 S 2 O 3 Is the standard of sodium thiosulfateConcentration of solution (M), V 0 Volume of sodium thiosulfate consumed (mL), V for allylation medium g Is allylation medium volume (mL).
(II) ion exchange capacity test method:
s1, taking 1mL of diethylaminoethyl natural polysaccharide chromatographic medium in a measuring cylinder, standing overnight, and determining the volume of the medium;
s2, transferring to a gravity column, adding 5mL of 0.1M HCl standard solution for washing, adding deionized water for washing to neutrality, and adding 5mL of 0.1M NaOH standard solution for washing to neutrality.
S3, adding 5mL of 0.1M NaOH standard solution, and collecting effluent.
S4, washing the microspheres to be neutral, collecting effluent liquid into the pre-effluent liquid, and dripping 3 drops of 2% methyl orange serving as an indicator.
S5, titrating with 0.1M HCl standard solution, and obtaining the consumed HCl volume V when the solution turns orange to be the titration end point HCl The ion exchange capacity was calculated according to the following formula:
ion exchange capacity= (V HCl M HCl -V NaOH M NaOH )/V g (μmol/mL)
Wherein V is NaOH Is the volume (mL) of NaOH standard solution, M NaOH Is the molar concentration (M), V of NaOH standard solution HCI Volume of HCl standard solution (mL), M HCl The molar concentration (M) of HCl standard solution; v (V) g Is the volume (mL) of the diethylaminoethyl natural polysaccharide chromatography medium.
TABLE 1 ion exchange capacities of different natural polysaccharide chromatography media
The results show that examples 2-7 all have higher ion exchange capacities, and the ion exchange capacities are all 200 mu mol.mL -1 The ion exchange capacity of example 6 was even up to 276. Mu. Mol. AbovemL -1 . Comparative examples 1 and 2 and commercial DEAE Sepharose FF, which were not activated by the polyepoxy compound, each had ion exchange capacities of 150. Mu. Mol.mL -1 Left and right. It is noted that the ion exchange capacities of example 4 using the activated polysaccharide matrix and comparative example 2 using the conventional polysaccharide matrix differ by 94. Mu. Mol.mL under the same experimental conditions -1 This suggests that the surface active hydroxyl groups of the polysaccharide matrix after polyepoxide activation are significantly increased, increasing the reaction sites for allylation, thereby increasing the ion exchange capacity.
(III) dynamic load testing method:
diethylaminoethyl natural polysaccharide chromatography media (10 mL) was loaded onto an AKTA protein purification system, equilibration buffer 40mmol/LTris-HCl (pH 8.3) solution. Bovine serum albumin was passed through the system and the baseline was leveled to give UV 280nm absorbance A, and 10% A was used as termination signal to record protein V consumed 0 . The flow rate was 0.5mL/min, the sample loading was stopped after the flow-through reached the stop signal, elution was performed with 40mmol/L Tris-HCl (pH 8.3) solution, and the elution peaks were collected and the effluent V and the eluent protein concentration were determined, respectively. The media loading is calculated according to the following formula:
DBC＝C×(V 0 -V)/V g
wherein DBC is medium loading (mg), C is eluent protein concentration (mg/mL), V 0 Protein volume (mL) for baseline consumption, V for eluent volume (mL), V g Is the volume (mL) of the natural polysaccharide medium.
TABLE 2 dynamic loading of different natural polysaccharide chromatography media on bovine serum albumin
| Name of the name | load/mg.mL -1 |
| Example 1 | 80 |
| Example 2 | 96 |
| Example 3 | 104 |
| Example 4 | 136 |
| Example 5 | 130 |
| Example 6 | 128 |
| Comparative example 1 | 59 |
| Comparative example 2 | 60 |
| Commercialization DEAE Sepharose FF | 57 |
The results show that the media obtained in examples 1-6 all present higher dynamic loadings, i.e., up to 80 mg.mL for protein -1 The above. Wherein the dynamic loading of the diethylaminoethyl glucan chromatography medium of example 6 on bovine serum albumin can be as high as 128 mg.mL -1 . Likewise, the dynamic loading of bovine serum albumin by the diethylaminoethyl cellulose chromatography medium of example 5 and the diethylaminoethyl agarose chromatography medium of example 4 were both 130 mg.mL -1 136 mg/mL -1 The above. Comparative examples 1 and 2 using polysaccharide matrices not activated by polyepoxide and commercial medium DEA under the same experimental conditionsThe E Sepharose FF loadings were 59mg mL, respectively -1 、60mg·mL -1 And 57 mg.mL -1 . This demonstrates that diethylaminoethyl natural polysaccharide chromatographic media greatly increases protein loading after suitable polyepoxy, allyl, and diethylamine modifications.
The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.
The raw materials and equipment used in the invention are common raw materials and equipment in the field unless specified otherwise; the methods used in the present invention are conventional in the art unless otherwise specified.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification, variation and equivalent transformation of the above embodiment according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.
Claims (10)
1. A preparation method of diethylaminoethyl natural polysaccharide chromatographic medium is characterized in that: the method comprises the following steps:
s1: firstly, activating a polysaccharide matrix by using a polyepoxy compound to introduce a plurality of active hydroxyl reaction sites to obtain a poly-active hydroxyl polysaccharide matrix, and then introducing inert groups on the surface of the poly-active hydroxyl polysaccharide matrix by using allyl glycidyl ether to prepare an allylation medium;
s2: brominating unsaturated bonds on the surface of the allylation medium obtained in the step S1 to obtain a bromination medium;
s3: and aminating the bromination medium obtained by S2 by using diethylamine to obtain the diethylaminoethyl natural polysaccharide chromatography medium.
2. A method of preparation as claimed in claim 3, wherein: in S1, the step of activating the polysaccharide matrix with the polyepoxide compound to introduce a plurality of reactive hydroxyl reactive sites specifically includes: and (3) oscillating polysaccharide matrix, dimethyl sulfoxide and NaOH solution at 35-45 ℃ for reaction, then adding polyepoxy compound for reaction, adding NaOH solution for fully hydrolyzing unreacted epoxy groups, and washing off unreacted polyepoxy compound to obtain the poly-active hydroxyl polysaccharide matrix.
3. The preparation method according to claim 1 or 2, characterized in that: in S1, the polyepoxy compound is selected from trimethylolpropane triglycidyl ether, glycerol tripropyloxy triglycidyl ether or pentaerythritol glycidyl ether.
4. The method of manufacturing as claimed in claim 2, wherein: in S1, the mass ratio of the polyepoxy compound to the polysaccharide matrix is 0.8-3:1.
5. The preparation method according to claim 1 or 2, characterized in that: in S1, the natural polysaccharide gel microsphere is selected from agarose microsphere, cellulose microsphere or dextran microsphere.
6. The method of manufacturing according to claim 5, wherein: in the S1, the particle size of the natural polysaccharide gel microsphere is 45-165 mu m, and the content of the natural polysaccharide is 2-12%.
7. The preparation method according to claim 1 or 2, characterized in that: in S1, the volume ratio of the allyl glycidyl ether to the natural polysaccharide matrix is 0.5-2:1.
8. The preparation method according to claim 1 or 2, characterized in that: and S2, bromination is carried out by adopting bromine water, the volume ratio of the bromine water to the allylation medium is 0.1-0.4:1, and the concentration of the bromine water is 1.5-3 wt%.
9. The method of preparing as claimed in claim 8, wherein: in S2, the reaction temperature is 40-70 o And C, reacting for 1-3 h.
10. The preparation method according to claim 1 or 2, characterized in that: and S3, amination is carried out through a diethylamine aqueous solution with the weight percentage of 20-40%.
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