CN107737583B - Dual-mode identification chromatography medium and preparation method thereof - Google Patents

Dual-mode identification chromatography medium and preparation method thereof Download PDF

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CN107737583B
CN107737583B CN201710805339.2A CN201710805339A CN107737583B CN 107737583 B CN107737583 B CN 107737583B CN 201710805339 A CN201710805339 A CN 201710805339A CN 107737583 B CN107737583 B CN 107737583B
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施伟
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Taizhou University
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    • 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
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/268—Polymers created by use of a template, e.g. molecularly imprinted polymers
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    • B01J20/28083—Pore diameter being in the range 2-50 nm, i.e. mesopores
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Abstract

The invention discloses a dual-mode identification chromatography medium with a mixed mode and a molecular imprinting mode and a preparation method thereof. Selecting mixed mode medium and template protein for full adsorption; polymerizing tetramethoxysilane and phenyltriethoxysilane on the surface of the medium to form a imprinting layer; and washing the imprinting medium with hydrochloric acid solution, sodium hydroxide solution and deionized water in sequence to remove the template protein, thereby obtaining the dual-mode chromatography medium. The novel chromatography medium developed by the invention has two recognition capabilities of a mixed mode and a molecular imprinting mode, greatly improves the removal capability of the mixed mode medium on macromolecular impurities, improves the chromatography selectivity, and retains the good adsorption performance of the mixed mode, pH dependence and salt-tolerant adsorption capability by utilizing the steric hindrance of a molecular imprinting polymer on macromolecular substances.

Description

Dual-mode identification chromatography medium and preparation method thereof
Technical Field
The invention relates to a dual-mode identification chromatography medium with a mixed mode and a molecular imprinting mode and a preparation method thereof, belonging to the protein chromatography separation technology in the field of biochemical engineering.
Background
In recent years, the development of biomacromolecule drugs represented by protein drugs is more and more rapid, the application of biomacromolecule drugs in the field of treatment and diagnosis is continuously expanded, and the proportion of the biomacromolecule drugs in the whole medical field is continuously increased. Compared with chemical drugs, biological drugs not only require high purity, but also maintain biological activity, so the separation and purification difficulty is high. For the whole production process of the biological medicine, the cost of the separation and purification steps is as high as 50-80%, and the method becomes a bottleneck for preventing the further industrialization of the biological medicine. How to further improve the separation and purification efficiency of biological drugs becomes an urgent problem to be solved.
Chromatography is the most important technology in the field of separation and purification, and with the development of biotechnology, novel media and methods are continuously introduced. Mixed mode chromatography is an emerging chromatography technique with great potential in recent years. The medium of the technology has multiple action modes, including electrostatic action force, hydrophobic action force, hydrogen bond action force and the like. Therefore, the mixed mode chromatography has the advantages of higher selectivity, larger adsorption capacity, salt-tolerant adsorption, lower cost and the like. Are currently used as an alternative or supplement to many traditional modes of chromatography. The selectivity of mixed mode chromatography is still unsatisfactory. One important reason is that mixed mode ligands have a small recognition region for proteins, a single recognition site, and are easily confused with impurities, such as mixed mode ligand 4-mercaptoethylpyridine, and antibody recognition sites of Y319 and L309(Acta Chim. Sinica,2010, 68: 1597) on the Fc fragment; 5-aminobenzimidazole then interacts mainly with His 435, Met 252 and Ile 253 on the Fc-fragment (J.Sep.Sci.,2015, 38: 2387). Therefore, on the basis of keeping the original advantages of the mixed mode, the method is an effective way by adding an additional recognition mode.
Molecular imprinting is an excellent molecular recognition technique, proposed by Wulff et al, Germany in the 80 th century (Tetrahedron Lett.,1973: 4329). It realizes recognition by matching with a target object on molecular size, shape and functional sites, and is widely applied to the field of separation analysis. Recently, the number of reports of molecular imprinting applied to biological macromolecules is increasing, but molecular imprinting in the field of macromolecules still presents a great challenge compared to small molecules. The reason for this is that the complex structure and huge size of the macromolecular substance hinder the performance and mass transfer efficiency of the imprinting process. In general, most research efforts have been directed to eliminating such steric hindrance and improving mass transfer efficiency. However, from another point of view, the molecular imprinting polymer reasonably utilizes the steric hindrance effect of macromolecular substances, and can improve the resolution capability of the medium on macromolecular impurities. The invention selects the surface molecular imprinting technology for mixed-mode medium surface polymerization, and guarantees the mass transfer efficiency of the target protein while preventing macromolecular impurities from entering the medium. The medium developed finally fully keeps the excellent adsorption capacity of the mixed-mode medium, the pH dependence and the salt-tolerant adsorption performance are realized, and meanwhile, the selectivity of the chromatography medium is greatly improved, so that the method is suitable for separation and purification of macromolecular substances with impurities.
Disclosure of Invention
The invention aims to provide a dual-mode identification chromatography medium with a mixed mode and a molecular imprinting mode and a preparation method thereof.
The dual-mode identification chromatography medium with the mixed mode and the molecular imprinting mode comprises a chromatography matrix, a mixed mode ligand and an imprinting layer, wherein the chromatography matrix is hydrophilic porous microspheres with hydroxyl groups, the mixed mode ligand is tryptamine, and imprinting polymerization monomers are tetramethoxysilane and phenyltriethoxysilane.
The structure of the surface of the chromatography medium is shown in FIG. 1.
The chromatography matrix is hydrophilic microspheres with a porous structure and surface hydroxyl groups. The chromatography matrix is agarose gel or cellulose microspheres.
The surface of the chromatography medium is provided with a mixed mode ligand and a molecular imprinting layer at the same time.
The preparation method of the dual-mode identification chromatography medium with the mixed mode and the molecular imprinting mode comprises the following steps:
1) preparing a mixed-mode chromatography medium from hydrophilic porous microspheres, draining, adding 1-5 times of saturated adsorption capacity protein, adsorbing in 20mM buffer solution at pH 4-8, shaking at 25 deg.C and 120rpm for 12 hr.
2) Adding 1:3 tetramethoxysilane and phenyltriethoxysilane into the adsorbent system, adjusting pH to 9.3 with ammonia water, and performing polymerization reaction for 1-16 hr.
3) And (3) sequentially using 0.1M hydrochloric acid solution, 0.1M sodium hydroxide solution and deionized water to clean the medium, destroying hydrophobic acting force between the protein and the ligand, desorbing the template protein, and obtaining the dual-mode identification chromatography medium.
The dual-mode identification chromatography medium with the mixed mode and the molecular imprinting mode, which is developed by the invention, can be used for chromatographic separation of macromolecular substances with impurities, and has the following advantages: (1) the target protein has large adsorption capacity and strong processing capacity; (2) the adsorption process can be regulated by pH, and by regulating the pH to be near 4, the full desorption can be realized by electrostatic repulsion between the mixed-mode ligand and the protein, so that the damage of strong acid and strong base elution conditions to the protein activity is avoided; (3) the medium retains excellent salt-tolerant adsorption capacity, and the adsorption capacity is reduced less in a higher salinity range, so that the feed liquid does not need to be diluted or salted, and the operation efficiency is improved; (4) in a dynamic adsorption mode, the selectivity of the medium on macromolecular protein impurities is greatly improved, the competitive adsorption of the impurity proteins and the target protein is effectively prevented, and the purity of a chromatography product is improved; (5) the imprinting layer further improves the stability of the medium, and the obtained dual-mode identification medium can be recycled for multiple times without the reduction of adsorption performance. The novel dual-mode identification medium developed by the invention takes tryptamine as a mixed-mode ligand, and uses tetramethoxysilane and phenyltriethoxysilane to polymerize and form an imprinting layer, so that the specific adsorption performance of mixed-mode chromatography is retained, and meanwhile, the selectivity of macromolecular impurities is greatly improved, and the novel dual-mode identification medium can be used for primary separation of protein purification.
Drawings
FIG. 1 is a schematic view showing the structural composition of the surface of the bimodal identification chromatography medium in example 1. The mixed mode recognition and the molecular imprinting recognition exist on the surface of the medium at the same time.
FIG. 2 is an IR spectrum of 740cm of the bimodal identification chromatography medium preparation process of example 1-1Is an N-H characteristic peak which indicates the successful coupling of tryptamine ligand and is 1200-950 cm-1And 696cm-1The imprinting polymerization is successful as indicated by characteristic peaks formed by Si-O-Si and C-H carried by tetramethoxysilane and phenyltriethoxysilane.
Fig. 3 is a protein permeation adsorption experiment of the dual-mode identification chromatography medium and the mixed-mode medium obtained in example 1 on bovine serum albumin, which is a target protein, and an impurity protein bovine antibody, wherein the dual-mode identification chromatography medium has better adsorption capacity on the target protein, and simultaneously effectively reduces the adsorption on the impurity protein.
FIG. 4 is a high performance liquid chromatography analysis chart of bovine serum albumin separated from bovine serum by the dual-mode identification chromatography medium and the mixed-mode medium obtained in example 1, and an elution component comparison chart shows that the dual-mode identification chromatography effectively improves the product purity.
Detailed Description
The invention is further described by way of examples below:
example 1
10g of agarose gel microspheres are taken, and tryptamine is taken as a ligand to prepare a mixed-mode chromatography medium. The medium was drained, protein was added in 3 times the saturated adsorption capacity, and adsorption was carried out in 20mM buffer, pH 5, temperature 25 ℃, shaker at 120rpm, 12 hours. Adding 1:3 tetramethoxysilane and phenyltriethoxysilane into the adsorbent system, adjusting pH to 9.3 with ammonia water, and performing polymerization reaction for 1 hr. And (3) sequentially using 0.1M hydrochloric acid solution, 0.1M sodium hydroxide solution and deionized water to clean the medium, destroying hydrophobic acting force between the protein and the ligand, and desorbing the template protein to obtain the dual-mode identification chromatography medium. The medium has an average pore size of 33.3nm as determined by nitrogen adsorption.
Example 2
10g of agarose gel microspheres are taken, and tryptamine is taken as a ligand to prepare a mixed-mode chromatography medium. The medium was drained, protein was added in 3 times the saturated adsorption capacity, and adsorption was carried out in 20mM buffer, pH 5, temperature 25 ℃, shaker at 120rpm, 12 hours. Adding 1:3 tetramethoxysilane and phenyltriethoxysilane into the adsorbent system, adjusting pH to 9.3 with ammonia water, and performing polymerization reaction for 2 hr. And (3) sequentially using 0.1M hydrochloric acid solution, 0.1M sodium hydroxide solution and deionized water to clean the medium, destroying hydrophobic acting force between the protein and the ligand, and desorbing the template protein to obtain the dual-mode identification chromatography medium. The medium has an average pore size of 27.7nm as determined by nitrogen adsorption.
Example 3
10g of agarose gel microspheres are taken, and tryptamine is taken as a ligand to prepare a mixed-mode chromatography medium. The medium was drained, protein was added in 3 times the saturated adsorption capacity, and adsorption was carried out in 20mM buffer, pH 5, temperature 25 ℃, shaker at 120rpm, 12 hours. Adding 1:3 tetramethoxysilane and phenyltriethoxysilane into the adsorbent system, adjusting pH to 9.3 with ammonia water, and performing polymerization reaction for 4 hr. And (3) sequentially using 0.1M hydrochloric acid solution, 0.1M sodium hydroxide solution and deionized water to clean the medium, destroying hydrophobic acting force between the protein and the ligand, and desorbing the template protein to obtain the dual-mode identification chromatography medium. The average pore size of the medium was 26.7nm as determined by nitrogen adsorption.
Example 4
And (3) carrying out a static adsorption performance test on the dual-mode identification chromatography medium, and comparing with a mixed-mode medium. Firstly, fully cleaning a medium by using deionized water, balancing the medium by using a phosphate buffer solution with the pH value of 5.0 for about 30min, carrying out suction filtration, weighing 0.04g of the medium, adding the medium into a 2mL centrifuge tube, and adding 1.5mL of buffer solutions with different bovine serum albumin concentrations; placing the centrifuge tube in a constant temperature mixing instrument, adsorbing at 25 deg.C and 1200rpm for 3h, and taking supernatant to determine bovine serum albumin concentration; and (3) calculating the adsorption capacity of the medium according to the material balance, drawing an adsorption isotherm, and fitting by using a Langmuir equation to obtain the adsorption capacity and the dissociation constant. The dual-mode identification medium prepared in the embodiment 1 of the invention has the saturated adsorption capacity of 70.53mg/mL resin and the dissociation constant of 0.16 mg/mL; while the saturated adsorption capacity of the mixed mode media was 72.49mg/mLresin with a dissociation constant of 0.46 mg/mL. The results show that the dual-mode recognition medium maintains the adsorption capacity of the mixed-mode medium on the target protein.
Example 5
And (3) testing the salt-tolerant adsorption performance of the dual-mode identification chromatography medium. Firstly, fully cleaning a medium by using deionized water, balancing the medium by using a phosphate buffer solution with the pH value of 5.0 for about 30min, carrying out suction filtration, weighing 0.04g of the medium, adding the medium into a 2mL centrifuge tube, and adding 1.5mL of buffer solutions (the NaCl concentration is 0.4 and 0.8M) with different bovine serum albumin concentrations; placing the centrifuge tube in a constant temperature mixing instrument, adsorbing at 25 deg.C and 1200rpm for 3h, and taking supernatant to determine bovine serum albumin concentration; and (3) calculating the adsorption capacity of the medium according to the material balance, drawing an adsorption isotherm, and fitting by using a Langmuir equation to obtain the adsorption capacity and the dissociation constant. The dual-mode identification medium prepared in the embodiment 1 of the invention has a saturated adsorption capacity of 51.15mg/mL resin and a dissociation constant of 0.31mg/mL under the condition of 0.4M NaCl concentration; the saturated adsorption capacity under the condition of 0.8M NaCl concentration was 48.93mg/mLresin, and the dissociation constant was 0.43 mg/mL. The result shows that the dual-mode identification medium maintains the good salt-resistant adsorption capacity of the mixed-mode medium.
Example 6
And (3) testing the dynamic adsorption performance of the dual-mode identification chromatography medium, and comparing the dynamic adsorption performance with the mixed-mode medium. A2 mg/mL bovine serum albumin and bovine antibody solution was prepared using a pH 5.0 phosphate buffer. Taking 3mL mediumThe material was packed into a chromatography column (diameter 1cm), equilibrated with phosphate buffer pH 5.0, linear flow rate 76cm/h, loaded, and the column outlet protein concentration change was monitored on-line with a UV detector (280 nm). Recording the sample loading volume at 10% penetration, and calculating the dynamic adsorption capacity Q of the medium to bovine serum albumin and bovine antibody at 10% penetration according to the material balance10％. The dual-mode recognition medium prepared in the embodiment 1 of the invention maintains the dynamic adsorption capacity similar to that of a mixed-mode medium on the target protein, but obviously reduces the adsorption of impurity proteins, and effectively prevents competitive adsorption of the impurity proteins.

Claims (5)

1. A dual-mode recognition chromatography medium with a mixed mode and a molecular imprinting mode is characterized by comprising a chromatography matrix and a recognition mode, wherein the chromatography matrix is a hydrophilic porous microsphere with hydroxyl, mixed mode recognition is provided by tryptamine ligand, and a molecular imprinting layer is obtained by polymerizing tetramethoxysilane and phenyltriethoxysilane;
the structure of the surface of the chromatography medium is as follows:
Figure FDA0002403546160000011
2. the dual mode recognition chromatography medium with mixed mode and molecularly imprinted mode as claimed in claim 1, wherein the chromatography matrix is a hydrophilic microsphere with porous structure and surface hydroxyl group.
3. The dual mode recognition chromatography medium with mixed mode and molecular imprinting mode of claim 1 or 2, wherein the chromatography matrix is agarose gel or cellulose microspheres.
4. The dual-mode recognition chromatography medium with mixed mode and molecular imprinting mode as claimed in claim 1, wherein the chromatography medium surface has both mixed mode ligand and molecular imprinting layer.
5. A method for preparing a dual-mode recognition chromatography medium having a mixed mode and a molecularly imprinted mode according to claim 1, comprising the steps of:
1) preparing a mixed-mode chromatography medium by using hydrophilic porous microspheres, draining, adding 1-5 times of saturated adsorption capacity protein, adsorbing in a 20mM buffer solution, controlling the pH to be 4-8, carrying out shaking table at 25 ℃ and 120rpm for 12 hours;
2) adding 1:3 tetramethoxysilane and phenyltriethoxysilane into the adsorption system, adjusting pH to 9.3 with ammonia water, and performing polymerization reaction for 1-16 hr;
3) and (3) sequentially using 0.1M hydrochloric acid solution, 0.1M sodium hydroxide solution and deionized water to clean the medium, destroying hydrophobic acting force between the protein and the ligand, and desorbing the template protein to obtain the dual-mode identification chromatography medium.
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