CN114534310B - Composite anion chromatography medium and preparation method and application thereof - Google Patents
Composite anion chromatography medium and preparation method and application thereof Download PDFInfo
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- CN114534310B CN114534310B CN202210194759.2A CN202210194759A CN114534310B CN 114534310 B CN114534310 B CN 114534310B CN 202210194759 A CN202210194759 A CN 202210194759A CN 114534310 B CN114534310 B CN 114534310B
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- 239000012501 chromatography medium Substances 0.000 title claims abstract description 58
- 239000002131 composite material Substances 0.000 title claims abstract description 25
- 238000005571 anion exchange chromatography Methods 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title abstract description 17
- -1 amine salt Chemical class 0.000 claims abstract description 36
- 239000003446 ligand Substances 0.000 claims abstract description 27
- 239000004593 Epoxy Substances 0.000 claims abstract description 23
- 239000011159 matrix material Substances 0.000 claims abstract description 18
- 238000000926 separation method Methods 0.000 claims abstract description 16
- 239000012190 activator Substances 0.000 claims abstract description 15
- 238000004132 cross linking Methods 0.000 claims abstract description 15
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims abstract description 11
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims abstract description 11
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims abstract description 11
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 8
- 238000000746 purification Methods 0.000 claims description 17
- 102000004169 proteins and genes Human genes 0.000 claims description 16
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 13
- 150000001450 anions Chemical class 0.000 claims description 12
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 claims description 11
- 238000007142 ring opening reaction Methods 0.000 claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 229910052736 halogen Inorganic materials 0.000 claims description 6
- 150000002367 halogens Chemical class 0.000 claims description 6
- STMDPCBYJCIZOD-UHFFFAOYSA-N 2-(2,4-dinitroanilino)-4-methylpentanoic acid Chemical group CC(C)CC(C(O)=O)NC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O STMDPCBYJCIZOD-UHFFFAOYSA-N 0.000 claims description 5
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 claims description 5
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- 230000005526 G1 to G0 transition Effects 0.000 claims description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
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- 150000001412 amines Chemical group 0.000 claims description 3
- 125000003277 amino group Chemical group 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
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- 125000000524 functional group Chemical group 0.000 claims description 3
- 150000004676 glycans Chemical class 0.000 claims description 3
- 239000003999 initiator Substances 0.000 claims description 3
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- 239000000377 silicon dioxide Substances 0.000 claims description 3
- 125000001424 substituent group Chemical group 0.000 claims description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
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- 230000000379 polymerizing effect Effects 0.000 claims description 2
- 238000011068 loading method Methods 0.000 abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 16
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 10
- 238000001742 protein purification Methods 0.000 abstract description 4
- 150000002500 ions Chemical class 0.000 abstract description 3
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- 239000000872 buffer Substances 0.000 description 16
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- 108091003079 Bovine Serum Albumin Proteins 0.000 description 10
- 229940098773 bovine serum albumin Drugs 0.000 description 10
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 102000008192 Lactoglobulins Human genes 0.000 description 5
- 108010060630 Lactoglobulins Proteins 0.000 description 5
- 238000005349 anion exchange Methods 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 4
- 108060003951 Immunoglobulin Proteins 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000011049 filling Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 102000018358 immunoglobulin Human genes 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 125000000129 anionic group Chemical group 0.000 description 3
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- 238000010828 elution Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 239000012500 ion exchange media Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 238000003825 pressing Methods 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000005341 cation exchange Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
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- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 2
- 229960003506 piperazine hexahydrate Drugs 0.000 description 2
- AVRVZRUEXIEGMP-UHFFFAOYSA-N piperazine;hexahydrate Chemical compound O.O.O.O.O.O.C1CNCCN1 AVRVZRUEXIEGMP-UHFFFAOYSA-N 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000005086 pumping Methods 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 235000011152 sodium sulphate Nutrition 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
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- 230000007613 environmental effect Effects 0.000 description 1
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- 238000001914 filtration Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
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- 239000003960 organic solvent Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 238000000164 protein isolation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- PUVAFTRIIUSGLK-UHFFFAOYSA-M trimethyl(oxiran-2-ylmethyl)azanium;chloride Chemical compound [Cl-].C[N+](C)(C)CC1CO1 PUVAFTRIIUSGLK-UHFFFAOYSA-M 0.000 description 1
- 238000011514 vinification Methods 0.000 description 1
- 239000010888 waste organic solvent Substances 0.000 description 1
- 239000011240 wet gel Substances 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
- B01D15/361—Ion-exchange
-
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Peptides Or Proteins (AREA)
Abstract
The application relates to the technical field of chromatographic chromatography, in particular to a composite anion chromatography medium, a preparation method thereof and application thereof in antibody protein purification. The composite anion chromatography medium comprises a matrix and a functional ligand, wherein the functional ligand is epoxy amine salt and polyvinylpyrrolidone which are covalently combined by a crosslinking activator, and the epoxy amine salt is bonded to the surface of the matrix. The application adopts the chromatographic medium compounded by the epoxy amine salt and the polyvinylpyrrolidone, has high dynamic combination loading capacity and low eluting ion strength besides the separation function of high selectivity and high efficiency. The preparation method of the composite anion chromatography medium provided by the application has the advantages that the preparation process is simple and convenient, the toxicity of the used chemical reagent is low, and only the water phase is used, so that the preparation method is environment-friendly.
Description
Technical Field
The application relates to the technical field of chromatographic chromatography, in particular to a composite anion chromatography medium, a preparation method thereof and application thereof in antibody protein purification.
Background
With the rapid development of the biomedical market, the antibody medicament has excellent curative effect and safety on diseases, has gradually and prominently important effects in the treatment process of tumors and cardiovascular diseases, in particular autoimmune diseases, and is the medicament with the highest growth rate in the current biological medicaments. Among them, monoclonal antibodies (MAbs) have been a research hotspot by virtue of their excellent properties. Because of the continuous innovation of MAb large-scale efficient culture technology, the expression concentration of antibody protein (also called immunoglobulin) is greatly improved, which brings great pressure to antibody protein purification. In order to obtain highly purified antibody proteins in a simpler and more efficient manner, a large number of researchers have studied the methods of antibody isolation and purification.
Traditional methods of antibody protein isolation are based on selective reversible precipitation of antibody protein-containing fragments while removing other proteins from the solution. The reagents used are typically ethanol, polyethylene glycol, ammonium sulfate, potassium phosphate or octanoic acid, etc. The conventional separation method produces products with low purity and is time-consuming and labor-consuming, and the reagents used in addition cause serious environmental problems. Thus, the currently common method of antibody protein purification is typically chromatography. The method utilizes two mutually immiscible phases (stationary phase and mobile phase), and in a separation system, samples to be purified in the mobile phase and the stationary phase perform a series of interactions, and the purpose of separation is achieved by utilizing the difference of physical or chemical properties of the samples. In order to improve the efficiency of separation and purification, researchers are struggling to develop the most excellent chromatographic medium (stationary phase). The chromatographic medium is generally composed of ligands containing functional groups and a solid phase carrier, and separation and purification are carried out according to different interaction principles of each chromatographic medium. A classical three-step purification strategy is generally employed: crude purification-intermediate purification-fine purification. The main purpose of crude purification is to capture, concentrate and stabilize a sample, and in order to remove various potential impurities to meet the requirements of drug safety, intermediate purification and fine purification are required after crude purification to remove Host Cell Proteins (HCPs), host DNA, antibody aggregates, variants, etc., and common chromatographic techniques can be classified into affinity purification media, ion exchange media, hydrophobic media, gel filtration media, etc.
Ion exchange media are commonly used to separate immunoglobulins and are largely divided into two categories, anion exchange media and cation exchange media. In anion exchange chromatography, the immunoglobulin negative charge amino acid terminal chain will interact with the positively charged ligand of the chromatographic medium. In contrast, cation exchange chromatography, i.e. the positively charged amino acid end chains of immunoglobulins, will interact with negatively charged ligands of the chromatographic medium. In order to widen the application range of ion exchange media, patent WO9729825 (Amersham Pharmacia Biotech AB) discloses a mixed anion exchange medium which can provide double interactions of positive charges and hydrogen bonds as early as 1997. In addition, patent WO9965607 (Amersham Pharmacia Biotech AB) discloses a mixed cation exchange medium that can provide both negative charge and hydrogen bonding interactions. However, the chromatographic medium-binding substances mentioned in the above two patents are relatively strong, and require a high eluting ionic strength when eluting the bound substances, which may cause denaturation of the macromolecular substances.
To solve the above problems, patent WO0138228 (Belew et al) discloses a mixed high salt ligand, each comprising a positively charged nitrogen and a thioether bond at a distance of 1 to 7 atoms from the positively charged nitrogen, by which negatively charged species in the sample can be removed by means of the anion exchange medium. And which can adsorb the target substance at a high salt concentration (e.g., 0.25M NaCl). Because of the property of high salt concentration resistance, the sample does not need to be diluted in the process of separation and purification, thus greatly reducing the total volume of the required sample, reducing the cost and simplifying the work. However, the preparation process of the mixed anion chromatography medium is uncontrollable, which can cause different ligand products, seriously affect the separation and purification effects, and possibly lead to lower separation and purification efficiency than the common medium.
Whereas patent US6702943 (Johansson et al) discloses a mixed anion chromatography medium containing anion exchange groups and a hydrophobic structural ligand. In addition, some commercial mixed anion chromatography media, such as capto sphere from Cytiva, are also a mixed anion chromatography media containing anion exchange groups and hydrophobic structural ligands. The ligand composition of the mixed anion chromatography medium in the preparation process is easy to control, and the mixed anion chromatography medium has good separation and purification effects. However, the preparation process of the chromatographic medium is often complex, and various chemical reagents with strong toxicity, such as various substances containing benzene ring structures, are involved. Meanwhile, various exchange and mixing problems of the water phase and the organic solvent are involved in the preparation process, so that more time and labor are wasted, and meanwhile, the treatment of the waste organic solvent is also a great difficulty.
Disclosure of Invention
In one aspect, the application provides a composite anion chromatography medium and application thereof in separation and purification of antibody proteins, wherein the chromatography medium has high selectivity and high-efficiency separation effect, higher dynamic binding capacity and lower eluting ion strength, and the adopted technical scheme is as follows: a composite anion chromatography medium comprises a matrix and a functional ligand, wherein the functional ligand is epoxy amine salt and polyvinylpyrrolidone which are covalently combined by a crosslinking activator, and the epoxy amine salt is bonded to the surface of the matrix.
In some embodiments, the matrix is a polysaccharide selected from dextran, agarose, and cellulose.
In some embodiments, the matrix is a synthetic polymer selected from the group consisting of polystyrene, polyethylene, and silica.
In some embodiments, the epoxy amine salt is an amine or amine salt comprising a primary, secondary, tertiary, or quaternary amine group, and has a substituted or unsubstituted C 1-20 Linear or branched alkanes or alkenes.
In some embodiments, the substituents on the carbon chain of the epoxide amine salt are selected from amino, hydroxyl, and halogen.
In some embodiments, the crosslinking activator is a difunctional or polyfunctional substituted compound comprising an allyl group selected from the group consisting of halogen, epoxy, and carboxyl.
In some embodiments, the crosslinking activator is allyl glycidyl ether.
On the other hand, the application provides a method for preparing the composite anion chromatography medium, which has simple preparation process, low toxicity of the used chemical reagent and water phase only, and forms an environment-friendly preparation method, and the adopted technical scheme is as follows: the method comprises the following steps: adding an epoxy amine salt with a proper proportion into an alkaline matrix at a ring opening temperature for full reaction so as to enable the epoxy amine salt to be grafted onto the matrix; adding a proper amount of crosslinking activator into the reaction product to react with the hydroxyl after the ring opening of the epoxy amine salt so as to activate the amine salt ligand; and adding vinyl pyrrolidone into the activated amine salt ligand, and polymerizing the vinyl pyrrolidone under the action of an initiator and grafting the vinyl pyrrolidone onto a crosslinking activator, thereby obtaining the composite anion chromatography medium.
In some embodiments, the crosslinking activator is allyl glycidyl ether, the epoxy groups contained therein react with hydroxyl groups after ring opening of the epoxide amine salt at ring opening temperature, and the allyl groups contained therein react with polyvinylpyrrolidone.
The technical scheme adopted by the application has at least the following beneficial effects:
n-vinyl pyrrolidone (NVP) is a precursor of polyvinyl pyrrolidone (PVP), PVP is a homopolymer with excellent solubility, low toxicity, biocompatibility, chemical stability and biological inertia, and is widely applied to the fields of medical and health, daily chemical industry, food, beverage, wine making, textile printing and dyeing and the like, and an amide bond exists on the N-vinyl pyrrolidone and can form a hydrogen bond with an amide bond on polypeptide, so that the purification purpose is achieved; the epoxy amine salt can be combined with polypeptide with negative charge to play the role of separation and purification; the application adopts the chromatographic medium compounded by the epoxy amine salt and the polyvinylpyrrolidone, has high dynamic combination loading capacity and low eluting ion strength besides the separation function of high selectivity and high efficiency.
2. The composite anion chromatographic medium provided by the application is used for filling a chromatographic column, and the obtained chromatographic column can be used for purifying proteins, in particular antibodies, antibody fragments or fusion proteins containing antibodies.
3. The preparation method of the composite anion chromatography medium provided by the application has the advantages that the preparation process is simple and convenient, the toxicity of the used chemical reagent is low, and only the water phase is used, so that the preparation method is environment-friendly.
Drawings
For a clearer description of the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a synthetic route diagram of a composite anion chromatographic medium according to example 2 of the application;
FIG. 2 is a functional spectrum of a composite anion chromatography medium according to example 3 of the present application;
FIG. 3 is a functional spectrum of a Q Beatarose FF chromatographic medium according to example 3 of the present application;
FIG. 4 is a graph showing a 10% dynamic loading of the composite anion chromatographic medium according to example 4 of the present application;
FIG. 5 is a graph of a 10% dynamic loading assay of a Q Beatarose FF chromatographic medium according to example 5 of the present application.
Detailed Description
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the teachings of the present application, and such equivalents are intended to fall within the scope of the application as defined in the appended claims. The various reagents commonly used in the examples are all commercially available products.
Example 1
Based on the amide bond capable of bonding with polypeptide and the positive charge carried by epoxy amine salt and capable of interacting with the amino acid end chain of negative charge of antibody protein, the inventor designs a composite anion chromatography medium comprising a matrix and a functional ligand, wherein the functional ligand is epoxy amine salt and polyvinylpyrrolidone which are covalently bonded by a crosslinking activator, and the epoxy amine salt is bonded to the surface of the matrix.
In some preferred embodiments, the matrix is a natural polymer or a synthetic polymer, wherein the natural polymer is a polysaccharide selected from dextran, agarose and cellulose; the synthetic polymer is selected from polystyrene, polyethylene and silica.
In other preferred embodiments, the epoxide amine salt is an amine or amine salt comprising a primary, secondary, tertiary or quaternary amine group and having a substituted or unsubstituted C 1-20 Linear or branched alkanes or alkenes, wherein the substituents on the amine epoxide carbon chain are selected from amino, hydroxyl and halogen.
In a further preferred embodiment, the crosslinking activator is a difunctional or polyfunctional allyl-containing substituted compound, the functional groups being selected from the group consisting of halogen, epoxy and carboxyl groups, preferably allyl glycidyl ether.
Example 2: referring to FIG. 2, preparation of a composite anion chromatography medium
1) Incorporation of 2, 3-epoxypropyl trimethyl ammonium chloride (GTA) onto a high rigidity agarose matrix
Adding 100g of high-rigidity agarose (Bestalose 6 FF) into a round-bottom flask, adding 1kg of purified water, stirring thoroughly to swell, adding 110g of Na 2 SO 4 After stirring at 30℃for 1 hour, 15ml of 1M sodium hydroxide solution was added to the round-bottomed flask, and after stirring for dissolution, the temperature was raised to 55℃and 50g of GTA was added for reaction for 8 hours. The product was washed 3 times with absolute ethanol, washed once with purified water, and suction filtered to obtain agarose-GTA gel.
2) Allyl glyceryl ether activation
100g of the agarose-GTA prepared in the step (1) was taken and put into a round-bottomed flask, 1kg of purified water was added, 70g of sodium hydroxide was added, allyl glycerol ether was added after stirring and dissolution, and the reaction was carried out at 55℃for 12 hours. After 12 hours, the resulting activated gel was washed with 10 volumes of purified water, ethanol, and purified water in this order.
3) Grafted Vinyl Pyrrolidone (VP)
100mL of the gel was filtered, washed with 5L of purified water several times, washed with 3X 150mL of 1.0M sodium sulfate, and filtered and drained after each wash. The gel described above and 1.0M sodium sulfate solution (total weight 244.7 g) were added to a round bottom flask followed by 26.3g of vinylpyrrolidone and 0.25g of the radical initiator Azobisisobutyronitrile (AIBN). Nitrogen was vented at 200rpm for 25 minutes and the round bottom flask was placed in Gan Youyu (45 ℃) and reacted until a polymer suspension (approximately 2mm in diameter) was formed. After the reaction was continued overnight, 200g of purified water was added to dilute the reaction solution. After filtration, it was washed with 20L of purified water and 1L of 20% ethanol in this order.
Example 3: complex anionic ligand chromatographic medium and Q Beatarose FF chromatographic medium function assay
beta-Lactoglobulin (beta-Lactoglobulin A and beta-Lactoglobulin B) and KatG are used to detect the function of chromatographic medium, linearly increasing salt concentration gradient is used to elute protein, and the function of medium is evaluated based on the retention volume of protein.
(1) Experimental sample and reagent
Buffer a:20mM piperazine hexahydrate (pH 6.20.+ -. 0.05); buffer B:20mM piperazine hexahydrate, 0.3M NaCl (pH 6.20.+ -. 0.05); 1% acetone; beta-Lactoglobulin; kat G.
(2) Experimental method
a) And (3) column loading: 6g of wet gel medium was weighed, an equal amount of water was added to make a 50% suspension, the suspension was poured into an EzsScreen 4.4mL column tube and the column was packed at a flow rate of 1.5mL/min for 10min.
b) And (3) measuring the column effect: the packed column was connected to an Exploer100 system and the column was equilibrated with purified water at a flow rate of 0.4mL/min until the conductance was stable. 1% acetone was injected into 100. Mu.l of the loading ring under Load. In the reject state, the column effect was measured at a flow rate of 0.4ml/min, the UV peak was completely out and the procedure was ended at the same time as the equilibrium. The ultraviolet peak is integrated by UNICON software, and the theoretical plate number N and the asymmetry factor As (N is more than or equal to 3000, and As is in the range of 0.8-1.5) are calculated by software.
c) Chromatography column balancing: the column was connected to an Exploer100 system and equilibrated with equilibration buffer at a flow rate of 1ml/min until baseline was stationary.
d) Loading: 22mg of beta-Lactoglobulin was weighed, dissolved in 1ml of Kat G, and then filtered with a 0.45 μm filter. The above samples were injected into 1000 μl loading ring and the Exploer100 system program was run.
(3) Analysis of results: the retention volume of the sample was calculated from the peak time of the sample.
FIGS. 2 and 3 are functional assays of mixed anion ligand chromatography media and Q Beatarose FF chromatography media, respectively. From the comparison of fig. 2 and fig. 3, it is obvious that the three peaks obtained in fig. 2 have no obvious tailing phenomenon and symmetrical peak shapes, and compared with the conventional Q Beatarose FF chromatographic medium, the mixed anion ligand chromatographic medium has the separation effect of higher selectivity and higher efficiency.
Example 4: 10% dynamic load determination of composite anionic ligand chromatographic medium
Bovine Serum Albumin (BSA) at a concentration of 4.0mg/ml was used as a protein sample and bound to the mixed anionic ligand chromatography medium at high pH, and medium binding capacity was calculated when 10% protein was running through.
(1) Experimental sample and reagent
Column loading solution: 20% ethanol, 0.2M NaCl; buffer a:50mM Tris (pH 8.0.+ -. 0.05); buffer B:50mM Tris 1M NaCl (pH 8.0.+ -. 0.05); 1% acetone.
Sample: a proper amount of buffer A was added to a beaker of 0.64g BSA, and the absorbance at 280nm was measured by a spectrophotometer to give a final concentration of 4.0.+ -. 0.1mg/ml.
(2) Experimental method
a) And (3) column loading: 10ml of medium was weighed, washed 3 times with column-packed solution, and then drained. 7g of medium was transferred to a beaker and 10ml of column-packed solution was added to mix well. 5ml of the suspension is poured into a BHR5/100 column tube provided with a column filling device, and the upper layer of the column filling device is filled with the column filling solution. The column is pressed for 4min at the flow rate of 25ml/min, the column loading device is disassembled, and the height of the rubber surface is adjusted to be 10cm plus or minus 0.2cm. And (5) mounting the column head, continuing to press the column for 2min, scribing at the rubber surface, stopping the flow rate, screwing off the column head, and pressing the column head to the scribing position.
b) And (3) measuring the column effect: the packed column was connected to AKTA and equilibrated with 20% ethanol at a flow rate of 0.33ml/min until the conductance equilibrated. Ensure that 1% acetone and 20% ethanol solution are injected into 20 mu l of loading ring under Load state, change the loading valve into the project state, measure the column effect of the chromatographic column at the flow rate of 0.2ml/min, and end the procedure after the ultraviolet peak is completely out. The ultraviolet peak is integrated by UNICON software to calculate the theoretical plate number N and the asymmetry factor As. (As should be in the range of 0.8-1.4)
c) Chromatography column balancing: after washing the pump with buffer a, the column was connected to the AKTA system and equilibrated with buffer a at a flow rate of 2.0ml/min until baseline was stationary.
d) Sample UV assay: the ultraviolet is zeroed, the sample passes through an ultraviolet detector at a flow rate of 2.0ml/min through a Bypass, and the ultraviolet absorption value of the sample is recorded after the sample is stabilized. The sample introduction was stopped and the sample in the system line was rinsed off with buffer a.
e) Loading: pumping the sample into the chromatographic column at a flow rate of 2.0ml/min while collecting the flow-through, stopping the sample feeding when the UV value is the sum of the base flow-through and the 10% sample UV value, and recording the collected flow-through volume V FT 。
f) Washing and eluting: the column was washed with buffer A at a flow rate of 2.0ml/min until baseline was stationary, and then eluted with buffer B at a flow rate of 2.0ml/min, and the elution peak was collected.
(3) Analysis of results
C BSA Concentration of BSA sample; v (V) FT A flow-through volume for collection; v (V) c Is the volume of the chromatographic column.
The results were averaged over the results of the two column assays.
Example 5: 10% dynamic load assay of Q Beatarose FF chromatographic medium
Protein samples were prepared using Bovine Serum Albumin (BSA) at a concentration of 4.0mg/ml and bound to Q Beatarose FF in Tris buffer pH 8.0, and medium binding capacity was calculated when 10% protein was running through.
(1) Experimental sample and reagent
Buffer a:50mM Tris (pH 8.0.+ -. 0.05); buffer B:0.1M acetic acid pH 3.0.+ -. 0.05;4M NaOH;5M HCl;1% acetone;
sample: 0.4g BSA was weighed into a beaker, 100ml buffer A was added, and the absorbance at 280nm was measured with a spectrophotometer to give a final sample concentration of 4.0.+ -. 0.1mg/ml.
(2) Experimental method
a) And (3) column loading: about 10g of medium is weighed, 20ml of water is added, the mixture is stirred uniformly and then settled for 1 to 2 minutes, and the mixture is pumped out and repeated for 4 times. Repeating the above washing steps by using 20% ethanol solution, adding 3ml of 20% ethanol into 3g of medium, stirring uniformly to prepare 50% suspension, pouring the suspension into an HR5/100 column tube, pressing the column at a flow rate of 1ml/min until the interface is clear and stable, removing the HR5/100 column loader, mounting the column head, mounting the column at a flow rate of 13ml/min for 5min, blocking the lower end after the column pressing is finished, and adjusting the column head to be just contacted with the rubber surface, wherein the column height is 10.2+/-0.2 cm.
b) And (3) measuring the column effect: the packed column was connected to an AKTA system, the column was equilibrated with purified water at a flow rate of 1.0ml/min until baseline was stationary, and 1% acetone was injected into 20. Mu.l of loading ring under Load. In the project state, the column effect was measured at a flow rate of 0.1ml/min, the salt peak was completely out and the procedure was ended when the conductivity was equilibrated. The salt peaks were integrated by UNICORN software and the theoretical plate number N and asymmetry factor As (N > 3000, as should be in the range of 0.8-1.5) were calculated by software.
c) Chromatography column balancing: the BSA samples were directly injected into the detector with a syringe at 23±2 ℃ and UV values were recorded. The column was connected to the AKTA system and equilibrated with buffer a at a flow rate of 1.0ml/min until baseline plateau.
d) Loading: pumping the sample into the chromatographic column at a flow rate of 2.0ml/min while collecting the flow-through, stopping the sample feeding when the UV value is the sum of the base flow-through and the 10% sample UV value, and recording the collected flow-through volume V FT 。
e) Washing and eluting: the column was washed with buffer A at a flow rate of 2.0ml/min until baseline plateau. The column was eluted with buffer B at a flow rate of 2.0ml/min, and the elution peak was collected.
(3) Analysis of results
Wherein A is 280 Absorbance after 4-fold dilution of BSA samples; 4 is dilution multiple; v (V) FT Is a flow-through volume; 0.67 is the extinction coefficient; v is the bed volume.
The results were averaged over the results of the two column assays.
FIGS. 4 and 5 are 10% dynamic loading determinations for mixed anion ligand chromatography media and Q Beatarose FF chromatography media, respectively. As is evident from the comparison of FIG. 4 and FIG. 5, the dynamic loading of FIG. 4 is 5-6 times that of the Q Beatarose FF chromatographic medium, and generally the higher the protein binding capacity is, the more difficult the later elution is, and the eluting peak height and peak shape are basically consistent compared with those of FIG. 4 and FIG. 5, and the volume of the used eluent is not much, so the mixed anion ligand chromatographic medium has high dynamic loading and simultaneously has no problem of eluting difficulty, which indicates that the chromatographic medium of the application is easier to elute, is beneficial to repeated use and saves cost.
The foregoing examples illustrate only a few embodiments of the application and are described in detail herein without thereby limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of the application should be assessed as that of the appended claims.
Claims (8)
1. The composite anion chromatography medium is characterized by comprising a matrix and a functional ligand, wherein the functional ligand is epoxy amine salt and polyvinylpyrrolidone which are covalently combined by a crosslinking activator, and the epoxy amine salt is bonded to the surface of the matrix; wherein,,
the crosslinking activator is a compound substituted by a difunctional group or a polyfunctional group containing allyl, and the functional group is selected from halogen, epoxy and carboxyl; and
the epoxy amine salt is an amine or amine salt containing a primary, secondary, tertiary or quaternary amine group and having a substituted or unsubstituted C 1-20 Linear or branched alkanes or alkenes.
2. The composite anion chromatographic medium of claim 1, wherein the matrix is a polysaccharide selected from the group consisting of dextran, agarose and cellulose.
3. The composite anion chromatographic medium of claim 1, wherein the matrix is a synthetic polymer selected from the group consisting of polystyrene, polyethylene and silica.
4. The composite anion chromatographic medium of claim 1, wherein the substituents on the carbon chain of the amine epoxide salt are selected from the group consisting of amino, hydroxyl and halogen.
5. The composite anion chromatographic medium of claim 1, wherein the crosslinking activator is allyl glycidyl ether.
6. Use of a complex anion chromatography medium according to any one of claims 1-5 as a stationary phase packed in a chromatography column for the separation and purification of antibody proteins.
7. A method of preparing a composite anion chromatographic medium of any of claims 1-5, comprising the steps of:
adding an epoxy amine salt with a proper proportion into an alkaline matrix at a ring opening temperature for full reaction so as to enable the epoxy amine salt to be grafted onto the matrix;
adding a proper amount of crosslinking activator into the reaction product to react with the hydroxyl after the ring opening of the epoxy amine salt so as to activate the amine salt ligand;
and adding vinyl pyrrolidone into the activated amine salt ligand, and polymerizing the vinyl pyrrolidone under the action of an initiator and grafting the vinyl pyrrolidone onto a crosslinking activator, thereby obtaining the composite anion chromatography medium.
8. The method according to claim 7, wherein the crosslinking activator is allyl glycidyl ether, the epoxy group contained therein reacts with the hydroxyl group after the ring opening of the amine epoxide salt at the ring opening temperature, and the allyl group contained therein reacts with polyvinylpyrrolidone.
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| CN105396628A (en) * | 2015-12-06 | 2016-03-16 | 杭州飞山浩科技有限公司 | Preparation method of polyethylene polyamine graft-modified polystyrene-divinyl benzene ion chromatographic packing |
| CN111001443A (en) * | 2019-12-20 | 2020-04-14 | 浙江工业大学 | Preparation method of poly (amine-epichlorohydrin) agglomeration graft type anion chromatographic packing |
| CN111818980A (en) * | 2018-03-08 | 2020-10-23 | 生物辐射实验室股份有限公司 | Anion exchange-hydrophobic mixed mode chromatography resin |
| WO2021099522A1 (en) * | 2019-11-21 | 2021-05-27 | Merck Millipore Ltd. | Methods for coupling a ligand to a composite material |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105396628A (en) * | 2015-12-06 | 2016-03-16 | 杭州飞山浩科技有限公司 | Preparation method of polyethylene polyamine graft-modified polystyrene-divinyl benzene ion chromatographic packing |
| CN111818980A (en) * | 2018-03-08 | 2020-10-23 | 生物辐射实验室股份有限公司 | Anion exchange-hydrophobic mixed mode chromatography resin |
| WO2021099522A1 (en) * | 2019-11-21 | 2021-05-27 | Merck Millipore Ltd. | Methods for coupling a ligand to a composite material |
| CN111001443A (en) * | 2019-12-20 | 2020-04-14 | 浙江工业大学 | Preparation method of poly (amine-epichlorohydrin) agglomeration graft type anion chromatographic packing |
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