CN111420644A - Preparation method of composite mixed-mode polymer chromatography medium - Google Patents
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Abstract
The invention discloses a preparation method of a composite mixed mode polymer chromatographic medium, which comprises the following steps: mixing core-shell polymer microspheres with amine compounds with charges and hydrophobic groups, adding a solvent capable of dissolving amine ligands, keeping a certain reaction temperature and a certain rotation speed for reaction, and performing suction filtration and washing after the reaction to remove unreacted residues to obtain polymer microspheres mixed with the ligands; and (3) mixing a neutral high molecular compound with the molecular weight of 1000000-10000000 with the polymer microsphere of the mixed mode ligand, and keeping a certain reaction temperature and a certain rotation speed for reaction to enable the surface of the polymer microsphere to be electrically neutral, so that the composite mixed mode polymer chromatographic medium is prepared. The invention not only has the volume exclusion function, the anion exchange function and the hydrophobic function at the same time, but also has the advantages of multiple functions, wide application range, good separation effect, simple preparation process, few operation steps, low preparation cost, safety and environmental protection.
Description
Technical Field
The invention relates to the field of modification and modification of polymer chromatographic packing, in particular to a preparation method of a composite mixed-mode polymer chromatographic medium.
Background
The mixed mode ligand is a compound with double or multiple functions, can simultaneously play multiple functions to selectively adsorb feed liquid, thereby realizing high-efficiency purification effect, and can be widely applied to the fields of complex systems such as vaccine development, virus purification, chromatographic separation and the like.
At present, the existing chromatographic medium usually has only one function, such as size exclusion function, ion exchange function, hydrophobic function, affinity function, etc., and in order to achieve the purity requirement in the whole purification process, multi-step separation is required by using the chromatographic medium with multiple functions, and the increase of separation steps inevitably leads to the reduction of the recovery rate of the target product, the reduction of the activity and the increase of the operation cost. At present, most of chromatographic media with two or more functions adopt coupled mixed mode ligands, and the mixed mode ligands need to have functional groups with multiple functions on one molecular chain, so that the effect of capturing a target object can be improved only by ensuring the synergistic effect between the functional groups, and the selection of the functional groups is greatly limited. The existing commonly used mixed mode ligand is combined with an ion exchange function and a hydrophobic function, and cannot be combined with the mixed mode ligand because the volume exclusion function requires no charge on the surface of a medium, but the volume exclusion function has the advantages of good biocompatibility, low non-specific adsorption and the like, and can play a key role in the purification of a complex system. The affinity function is often applied to capture specific protein and is not suitable for removing impurities from various substances in a complex system. Therefore, a mixed-mode chromatography medium with wide application range, low preparation cost, multiple functional effects and good separation effect is urgently needed.
Disclosure of Invention
The invention provides a preparation method of a composite mixed-mode polymer chromatographic medium, aiming at solving the technical problems of the existing chromatographic medium in the purification of a complex system, the prepared composite mixed-mode polymer chromatographic medium has the advantages of volume exclusion function, anion exchange function and hydrophobic function, multiple functions, wide application range, good separation effect, simple preparation process, less operation steps, low preparation cost, safety and environmental protection, and solves the problems of complex operation steps, high cost, low efficiency, small medium application range and the like of moderate and fine purification of the chromatographic medium applied to the purification of complex systems such as viruses and the like.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a composite mixed mode polymer chromatography medium comprises the following steps:
step 2, surface neutralization of the core-shell microspheres: and (2) mixing a neutral high molecular compound with the molecular weight of 1000000-10000000 with the polymer microsphere of the mixed-mode ligand prepared in the step (1), and reacting at a certain reaction temperature and a certain rotation speed to prepare the composite mixed-mode polymer chromatographic medium.
Preferably, in step 1, the amine compound is an amine compound which can react with an epoxy group and has a hydrophobic group; the dosage of the amine compound is 0.5-5 times of the total mass of the core-shell polymer microsphere.
Preferably, in step 1, the solvent is at least one of methanol, ethanol, dimethyl sulfoxide and isopropanol; the dosage of the solvent is 2-5 times of the total volume of the core-shell polymer microsphere.
Preferably, in the step 1, the reaction temperature is 30-60 ℃, the rotation speed is 80-140 r/min, and the reaction time is 12-48 h.
Preferably, in step 2, the neutral polymer compound is one or a mixture of two of aldehyde agarose, aldehyde dextran, aldehyde cellulose and aldehyde polyvinyl alcohol; the dosage of the neutral high molecular compound is 0.1-5 times of the total mass of the polymer microspheres of the mixed mode ligand prepared in the step 1.
Preferably, in the step 2, the reaction temperature is 5-25 ℃, the rotation speed is 80-140 r/min, and the reaction time is 24-48 h.
Preferably, the composite mixed-mode polymer chromatographic medium is a porous polymer microsphere with a core-shell structure, the pore diameter of a shell layer is 50-300 nm, the surface of the shell layer is neutral and hydrophilic, the pore diameter of a core is 200-500 nm, the shell layer and the core are mutually communicated, and the composite mixed-mode polymer chromatographic medium has a mixed chromatographic function of anion and hydrophobic interaction.
Preferably, the protein binding capacity of the composite mixed mode polymer chromatography medium is kept at 73-50 mg/m L under the condition that the salt concentration is 0-1 mol/L.
Preferably, the ion exchange capacity of the composite mixed mode polymer chromatography medium is 0.19-0.23 mmol/m L.
According to the technical scheme provided by the invention, the mixed-mode ligand with the anion exchange function and the hydrophobic function is coupled to the core-shell polymer microsphere, and the coupled ligand density is controlled by selecting compounds with different ionic strengths and hydrophobic strengths and regulating and controlling the reaction time, the reaction temperature and the ratio of the microsphere to the ligand, so that the inner surface and the outer surface of the microsphere have the anion exchange function and the hydrophobic function. Then, neutral hydrophilic macromolecules with high molecular weight are used for modifying the surface of a shell layer of the microsphere, so that the surface of the microsphere is a neutral hydrophilic layer, the aim of reducing the nonspecific adsorption of protein is fulfilled, and meanwhile, the pore diameter of the shell layer of the microsphere is small, so that molecules such as large-size virus particles and the like can be effectively prevented from entering the interior of the microsphere. The preparation method has the advantages of simple operation steps, easy control of reaction, ingenious design idea, low cost of used reagents, environmental protection, wide application range of the prepared composite mixed-mode polymer chromatographic medium, high separation and purification efficiency, and strong-reactivity hydroxyl on the surface of the shell layer, and can be used for continuously modifying other ligands to expand other functions.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a scanning electron micrograph of a composite mixed mode polymer chromatography medium according to example 1 of the present invention.
FIG. 2 is a SEM photograph of composite mixed mode polymer chromatographic medium of example 2 of the present invention.
FIG. 3 is a SEM photograph of composite mixed mode polymer chromatographic medium of example 3 of the present invention.
FIG. 4 is a SEM photograph of composite mixed mode polymer chromatographic medium of example 4 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
The preparation method of the composite mixed mode polymer chromatography medium provided by the invention is described in detail below. Details which are not described in detail in the embodiments of the invention belong to the prior art which is known to the person skilled in the art.
A preparation method of a composite mixed mode polymer chromatographic medium can comprise the following steps:
Step 2, surface neutralization of the core-shell microspheres: and (2) uniformly mixing a neutral high molecular compound with the molecular weight of 1000000-10000000 with the polymer microsphere of the mixed-mode ligand prepared in the step (1), keeping the reaction temperature at 5-25 ℃ and the rotating speed at 80-140 r/min for reaction for 24-48 h to enable the surface of the polymer microsphere to be electrically neutral, and after the reaction is finished, performing suction filtration and washing to remove unreacted residues, so that the composite polymer microsphere with the neutral surface and the mixed-mode group inside is prepared, namely the composite mixed-mode polymer chromatography medium.
The preparation method of the composite mixed mode polymer chromatographic medium can comprise the following embodiments:
(1) in step 1, the core-shell polymer microsphere can be a porous polymer microsphere with a core-shell structure described in chinese patent CN 201810615178.5.
(2) In the step 1, the amine compound can react with an epoxy group and has a hydrophobic group; for example: the amine compound may be pentylamine, hexylamine, octylamine, aniline, or the like. The dosage of the amine compound is 0.5-5 times of the total mass of the core-shell polymer microsphere.
(3) In the step 1, the solvent capable of dissolving the amine ligand is at least one of methanol, ethanol, dimethyl sulfoxide and isopropanol; the dosage of the solvent is 2-5 times of the total volume of the core-shell polymer microsphere.
(4) In the step 2, the neutral polymer compound is one or a mixture of two of aldehyde agarose, aldehyde dextran, aldehyde cellulose and aldehyde polyvinyl alcohol; the dosage of the neutral high molecular compound is 0.1-5 times of the total mass of the polymer microspheres of the mixed mode ligand prepared in the step 1.
(5) The composite mixed-mode polymer chromatographic medium is a porous polymer microsphere with a core-shell structure, the particle size range of the porous polymer microsphere is 50-100 mu m, the pore diameter of a shell layer is 50-300 nm, the surface of the shell layer is neutral and hydrophilic, the pore diameter of a core is 200-500 nm and is communicated with each other, the composite mixed-mode polymer chromatographic medium has a mixed chromatographic function of anion and hydrophobic interaction, and can be applied to virus purification.
(6) The ion exchange capacity of the composite mixed mode polymer chromatography medium is 0.19-0.23 mmol/m L.
(7) The salt tolerance of the composite mixed mode polymer chromatographic medium is remarkably improved, the protein binding capacity is kept at 73-50 mg/m L under the condition that the salt concentration is 0-1 mol/L. the mixed mode ligand coupled in the composite mixed mode polymer chromatographic medium has stronger salt tolerance, and the composite mixed mode polymer chromatographic medium can be widely applied to complex systems sensitive to the environment, such as moderate and fine purification of proteins and viruses with larger molecular weights.
Specifically, on the basis of preparing the polymer microsphere with the core-shell structure by using a suspension polymerization method, mixed-mode ligands are introduced to the inner surface and the outer surface of the microsphere to play a role in anion exchange and a role in hydrophobicity, and then the surface of a shell layer is neutralized to play a role in similar size exclusion, so that the composite mixed-mode polymer chromatographic medium with the size exclusion function, the anion exchange function and the hydrophobic function is prepared. The preparation method has the advantages of simple process, few operation steps, lower cost, safety, environmental protection and unique design method, the surface of the composite mixed-mode polymer chromatographic medium does not generate adsorption, and the composite mixed-mode polymer chromatographic medium can adsorb proteins with negative charges or hydrophobicity inside the medium, so that the medium and fine purification method can be widely applied to medium and fine purification of complex systems containing different kinds of hybrid proteins, such as virus fermentation liquor and the like.
In conclusion, the composite mixed-mode polymer chromatographic medium prepared by the embodiment of the invention has the advantages of volume exclusion function, anion exchange function and hydrophobic function, multiple functional effects, wide application range, good separation effect, simple preparation process, few operation steps, low preparation cost, safety and environmental protection, and solves the problems of complex operation steps, high cost, low efficiency, small medium application range and the like when the chromatographic medium is applied to the purification of complex systems such as viruses and the like.
In order to more clearly show the technical scheme and the technical effects provided by the present invention, the following detailed description is provided with specific examples of the preparation method of the composite mixed mode polymer chromatographic medium provided by the present invention.
The chromatography medium of the composite mixed-mode polymer is a composite polymer microsphere with multiple functions, the surface of the chromatography medium plays a role in intercepting molecular weight in a fixed range, and the interior of the microsphere plays a role in anion exchange and hydrophobic functions.A scanning electron microscope is used for representing the shell structure of the outer surface and the pore structure of the interior of the chromatography medium of the composite mixed-mode polymer, an acid-base titration method is used for measuring the coupling amount of ion exchange capacity representing mixed-mode ligand, a static adsorption method is used for measuring the static binding capacity under different salt concentrations (0-1 mol/L NaCl), different adsorption temperatures (10-25 ℃) and different adsorption times (2-8 h) to represent the capacity of capturing protein, and meanwhile, hydroxyl with strong reaction activity still exists on the surface of the microsphere, so that the chromatography medium lays a foundation for subsequent function expansion and has good development prospects in the fields of separation and purification of protein and virus, biopharmaceutical and the like.
Example 1
A preparation method of a composite mixed mode polymer chromatographic medium can comprise the following steps:
step 1A, coupling mixed mode ligand: mixing core-shell polymer microspheres (PGMA-EDMA) with amine compounds (pentylamine) which have charges and hydrophobic groups, adding a solvent (methanol) capable of dissolving amine ligands, stirring uniformly, wherein the amount of the methanol is 2 times of the total volume of the core-shell polymer microspheres, then putting the mixture on a shaking bed, keeping the reaction temperature at 30 ℃ and the rotation speed at 80r/min for reaction, reacting for 12h, and performing suction filtration and washing to remove unreacted residues after the reaction is finished to obtain the ligand polymer microspheres with the mixed mode.
Step 2A, surface neutralization of the core-shell microspheres: mixing a neutral high molecular compound (aldehyde agarose with the molecular weight of 1000000) with the polymer microspheres with the mixed-mode ligands prepared in the step 1A, wherein the dosage of the aldehyde agarose is 0.1 time of the total mass of the polymer microspheres with the mixed-mode ligands prepared in the step 1A, then placing the mixture on a shaking bed, keeping the reaction temperature at 5 ℃ and the rotating speed at 80r/min for reaction, keeping the reaction time at 24h, and enabling the surfaces of the polymer microspheres to be electrically neutral, and after the reaction is finished, performing suction filtration and washing by using deionized water and ethanol to remove unreacted residues, thereby preparing the composite polymer microspheres with neutral surfaces and mixed-mode groups inside, namely the composite mixed-mode polymer chromatography medium.
Specifically, the following tests were performed on the chromatography medium of the composite mixed-mode polymer prepared in example 1 of the present invention:
(1) after drying the composite polymer microspheres prepared in the embodiment 1 of the invention in a 70 ℃ oven, observing the shell structure of the outer surface and the internal pore structure of the composite polymer microspheres by using a scanning electron microscope, thereby obtaining a scanning electron microscope photograph of the composite mixed-mode polymer chromatographic medium shown in figure 1; fig. 1a is a scanning electron microscope photograph of a shell structure of the composite polymer microsphere prepared in example 1 of the present invention, and fig. 1b is a scanning electron microscope photograph of an internal pore structure of the composite polymer microsphere prepared in example 1 of the present invention. As can be seen from fig. 1 a: the composite polymer microsphere prepared in the embodiment 1 of the invention has an obvious shell structure on the surface, and the aperture is very small and ranges from 100 nm to 300 nm. As can be seen from fig. 1 b: the composite polymer microsphere prepared in the embodiment 1 of the invention has mutually communicated inner pores and the pore diameter range of 200-500 nm.
(2) For the composite mixed-mode polymer chromatographic medium prepared in the embodiment 1 of the invention, an acid-base titration method is adopted to measure ion exchange capacity to represent the coupling amount of the mixed-mode ligand, and a static adsorption method is adopted to adsorb for 2 hours at 10 ℃ to respectively measure the static binding capacity under the salt concentration of 0 mol/L and 0.4 mol/L NaCl to represent the capacity of capturing protein, so that the results shown in the following table 1 are obtained:
TABLE 1
Note: BSA in Table 1 above is bovine serum albumin
As can be seen from the above table 1, the average value of the ion exchange capacity of the composite mixed mode polymer chromatography medium prepared in example 1 of the present invention measured by three parallel experiments is 0.22 + -0.1 mmol/ml, which proves that the ligand is successfully connected to the microspheres and the coupling amount is very high, and the average value of the BSA static loading of the composite mixed mode polymer chromatography medium prepared in example 1 of the present invention measured by adsorption for 2h at 10 ℃ is 69 + -4 mg/ml (0 mol/L NaCl) and 65 + -1 mg/ml (0.4 mol/L NaCl), which proves that the mixed mode ligand has very strong protein capturing capability and very small variation of the loading amount at high salt concentration, and has certain salt tolerance compared with the common mixed mode polymer chromatography medium.
Example 2
A preparation method of a composite mixed mode polymer chromatographic medium can comprise the following steps:
step 1B, coupling mixed mode ligand: mixing core-shell polymer microspheres (PGMA-EDMA) with amine compounds (hexylamine) which have charges and hydrophobic groups, wherein the dosage of the hexylamine is 1 time of the total mass of the core-shell polymer microspheres, adding a solvent (ethanol) capable of dissolving amine ligands, uniformly stirring, the dosage of the ethanol is 3 times of the total volume of the core-shell polymer microspheres, then placing the mixture on a shaking bed to react at 40 ℃ and at the rotating speed of 100r/min for 24h, and after the reaction is finished, performing suction filtration and washing to remove unreacted residues to obtain the ligand polymer microspheres with the mixed mode.
Step 2B, surface neutralization of the core-shell microspheres: mixing a neutral high molecular compound (aldehyde dextran with the molecular weight of 4000000) with the polymer microsphere with the mixed mode ligand prepared in the step 1B, wherein the dosage of the aldehyde dextran is 2 times of the total mass of the polymer microsphere with the mixed mode ligand prepared in the step 1B, then placing the mixture on a shaking bed, keeping the reaction temperature at 10 ℃ and the rotating speed at 100r/min for reaction, keeping the reaction time at 30h, and enabling the surface of the polymer microsphere to be electrically neutral, and after the reaction is finished, performing suction filtration and washing by using deionized water and ethanol to remove unreacted residues, so as to prepare the composite polymer microsphere with the neutral surface and the mixed mode group inside, namely the composite mixed mode polymer chromatography medium.
Specifically, the following tests were performed on the chromatography medium of the composite mixed-mode polymer prepared in example 2 of the present invention:
(1) after drying the composite polymer microspheres prepared in the embodiment 2 of the invention in a 70 ℃ oven, observing the shell structure of the outer surface and the internal pore structure by using a scanning electron microscope, thereby obtaining a scanning electron microscope picture of the composite mixed mode polymer chromatographic medium shown in figure 2; fig. 2a is a scanning electron microscope photograph of a shell structure of the composite polymer microsphere prepared in example 2 of the present invention, and fig. 2b is a scanning electron microscope photograph of an internal pore structure of the composite polymer microsphere prepared in example 2 of the present invention. As can be seen from fig. 2 a: the composite polymer microsphere prepared in the embodiment 2 of the invention has an obvious shell structure on the surface, and the aperture is very small and ranges from 100 nm to 200 nm. As can be seen from fig. 2 b: the composite polymer microsphere prepared in the embodiment 2 of the invention has mutually communicated inner pores and the aperture range of 200-300 nm.
(2) For the composite mixed-mode polymer chromatographic medium prepared in the embodiment 2 of the invention, an acid-base titration method is adopted to measure ion exchange capacity to represent the coupling amount of the mixed-mode ligand, and a static adsorption method is adopted to adsorb for 4 hours at 15 ℃ to respectively measure the static binding capacity under the salt concentration of 0 mol/L and 0.6 mol/L NaCl to represent the capacity of capturing protein, so that the results shown in the following table 2 are obtained:
TABLE 2
Note: BSA in Table 2 above is bovine serum albumin
As can be seen from the above table 2, the average value of the ion exchange capacity of the composite mixed mode polymer chromatography medium prepared in example 2 of the present invention measured by three parallel experiments is 0.23 + -0.2 mmol/ml, which proves that the ligand is successfully connected to the microspheres and the coupling amount is very high, and the average value of the BSA static loading of the composite mixed mode polymer chromatography medium prepared in example 2 of the present invention measured by adsorption for 4h at 15 ℃ is 70 + -2 mg/ml (0 mol/L NaCl) and 64 + -1 mg/ml (0.6 mol/L NaCl), which proves that the mixed mode ligand has very strong protein capturing capability and very small variation of the loading amount at high salt concentration, and has certain salt tolerance compared with the common mixed mode polymer chromatography medium.
Example 3
A preparation method of a composite mixed mode polymer chromatographic medium can comprise the following steps:
step 1C, coupling mixed mode ligands: mixing core-shell polymer microspheres (PGMA-EDMA) with amine compounds (octylamine) which have charges and hydrophobic groups, wherein the dosage of the octylamine is 1.5 times of the total mass of the core-shell polymer microspheres, adding a solvent (dimethyl sulfoxide) capable of dissolving amine ligands, stirring uniformly, the dosage of the dimethyl sulfoxide is 4 times of the total volume of the core-shell polymer microspheres, then placing the core-shell polymer microspheres on a shaking bed to react at 50 ℃ and at a rotating speed of 120r/min for 36 hours, and after the reaction is finished, performing suction filtration and washing to remove unreacted residues to obtain the polymer microspheres with mixed ligands.
Step 2C, surface neutralization of the core-shell microspheres: mixing a neutral high molecular compound (aldehyde cellulose with the molecular weight of 6000000) with the polymer microsphere with the mixed-mode ligand prepared in the step 1C, wherein the dosage of the aldehyde cellulose is 3 times of the total mass of the polymer microsphere with the mixed-mode ligand prepared in the step 1C, placing the mixture on a shaking bed, keeping the reaction temperature at 15 ℃ and the rotating speed at 120r/min for reaction for 40 hours to ensure that the surface of the polymer microsphere is electrically neutral, and after the reaction is finished, performing suction filtration and washing by using deionized water and ethanol to remove unreacted residues to prepare the composite polymer microsphere with the neutral surface and the mixed-mode group inside, namely the composite mixed-mode polymer chromatography medium.
Specifically, the following tests were performed on the chromatography medium of the composite mixed-mode polymer prepared in example 3 of the present invention:
(1) after drying the composite polymer microspheres prepared in the embodiment 3 of the invention in a 70 ℃ oven, observing the shell structure of the outer surface and the internal pore structure by using a scanning electron microscope, thereby obtaining a scanning electron microscope picture of the composite mixed mode polymer chromatographic medium shown in figure 3; fig. 3a is a scanning electron microscope photograph of a shell structure of the composite polymer microsphere prepared in example 3 of the present invention, and fig. 3b is a scanning electron microscope photograph of an internal pore structure of the composite polymer microsphere prepared in example 3 of the present invention. As can be seen from fig. 3 a: the composite polymer microsphere prepared in the embodiment 3 of the invention has an obvious shell structure on the surface, and the aperture is very small and is 50-150 nm. As can be seen from fig. 3 b: the composite polymer microsphere prepared in the embodiment 3 of the invention has interconnected inner pores and the pore diameter is 100-400 nm.
(2) For the composite mixed-mode polymer chromatographic medium prepared in the embodiment 3 of the invention, an acid-base titration method is adopted to measure ion exchange capacity to represent the coupling amount of the mixed-mode ligand, and a static adsorption method is adopted to adsorb for 6 hours at 20 ℃ to respectively measure the static binding capacity under the salt concentration of 0 mol/L and 0.8 mol/L NaCl to represent the capacity of capturing protein, so that the results shown in the following table 3 are obtained:
TABLE 3
Note: BSA in Table 3 above is bovine serum albumin
As can be seen from the above Table 3, the average value of the ion exchange capacity of the composite mixed mode polymer chromatography medium prepared in example 3 of the present invention measured by three parallel experiments is 0.19 + -0.2 mmol/ml, which proves that the ligand is successfully connected to the microspheres and the coupling amount is very high, and the average value of the BSA static loading of the composite mixed mode polymer chromatography medium prepared in example 3 of the present invention measured by adsorption for 6h at 20 ℃ is 64 + -2 mg/ml (0 mol/L NaCl) and 56 + -1 mg/ml (0.8 mol/L NaCl), which proves that the mixed mode ligand has very strong protein capturing capability and very small variation of the loading amount at high salt concentration, and has a certain salt tolerance compared with the general ion exchange ligand.
Example 4
A preparation method of a composite mixed mode polymer chromatographic medium can comprise the following steps:
step 1D, coupling mixed mode ligand: mixing core-shell polymer microspheres (PGMA-EDMA) and amine compounds (aniline) with charges and hydrophobic groups, wherein the dosage of the aniline is 2 times of the total mass of the core-shell polymer microspheres, adding a solvent (isopropanol) capable of dissolving amine ligands, uniformly stirring, and the dosage of the isopropanol is 5 times of the total volume of the core-shell polymer microspheres, then putting the mixture on a shaking bed, keeping the reaction temperature at 60 ℃ and the rotating speed at 140r/min for reaction for 48h, and after the reaction is finished, carrying out suction filtration and washing to remove unreacted residues, thus obtaining the polymer microspheres with mixed-mode ligands.
Step 2D, surface neutralization of the core-shell microspheres: mixing a neutral high molecular compound (aldehyde polyvinyl alcohol with the molecular weight of 10000000) with the polymer microspheres with the mixed-mode ligands prepared in the step 1D, wherein the dosage of the aldehyde polyvinyl alcohol is 5 times of the total mass of the polymer microspheres with the mixed-mode ligands prepared in the step 1D, then placing the mixture on a shaking bed, keeping the reaction temperature at 25 ℃ and the rotating speed at 140r/min for reaction, keeping the reaction time at 48h, enabling the surfaces of the polymer microspheres to be electrically neutral, and after the reaction is finished, performing suction filtration and washing by using deionized water and ethanol to remove unreacted residues, so as to prepare the composite polymer microspheres with neutral surfaces and mixed-mode groups inside, namely the composite mixed-mode polymer chromatography medium.
Specifically, the following tests were performed on the chromatography medium of the composite mixed-mode polymer prepared in example 4 of the present invention:
(1) after drying the composite polymer microspheres prepared in the embodiment 4 of the invention in a 70 ℃ oven, observing the shell structure of the outer surface and the internal pore structure by using a scanning electron microscope, thereby obtaining a scanning electron microscope picture of the composite mixed mode polymer chromatographic medium shown in figure 4; fig. 4a is a scanning electron microscope photograph of a shell structure of the composite polymer microsphere prepared in example 4 of the present invention, and fig. 4b is a scanning electron microscope photograph of an internal pore structure of the composite polymer microsphere prepared in example 4 of the present invention. As can be seen from fig. 4 a: the composite polymer microsphere prepared in the embodiment 4 of the invention has an obvious shell structure on the surface, and the aperture is very small and is 50-200 nm. As can be seen from fig. 4 b: the composite polymer microsphere prepared in the embodiment 4 of the invention has mutually communicated inner pores and the aperture range of 100-300 nm.
(2) For the composite mixed-mode polymer chromatographic medium prepared in the embodiment 4 of the invention, an acid-base titration method is adopted to measure ion exchange capacity to represent the coupling amount of the ligand in the mixed mode, and a static adsorption method is adopted to adsorb for 8 hours at 25 ℃ to respectively measure the static binding capacity under the salt concentration of 0 mol/L and 1 mol/L NaCl to represent the capacity of capturing protein, so that the results shown in the following table 4 are obtained:
TABLE 4
Note: BSA in Table 4 above is bovine serum albumin
As can be seen from Table 4 above, the average value of the ion exchange capacity of the composite mixed mode polymer chromatography medium prepared in example 4 of the present invention measured by three parallel experiments is 0.22 + -0.2 mmol/ml, which proves that the ligand is successfully connected to the microspheres and the coupling amount is very high, and the average value of the BSA static loading of the composite mixed mode polymer chromatography medium prepared in example 4 of the present invention measured by adsorption for 8h at 25 ℃ is 61 + -2 mg/ml (0 mol/L NaCl) and 50 + -1 mg/ml (1 mol/L NaCl), which proves that the mixed mode ligand has very strong protein capturing ability and very small variation of the loading amount at high salt concentration, and has a certain salt tolerance compared with the general ion exchange ligand.
In conclusion, the composite mixed-mode polymer chromatographic medium prepared by the embodiment of the invention has the advantages of volume exclusion function, anion exchange function and hydrophobic function, multiple functional effects, wide application range, good separation effect, simple preparation process, few operation steps, low preparation cost, safety and environmental protection, and solves the problems of complex operation steps, high cost, low efficiency, small medium application range and the like when the chromatographic medium is applied to the purification of complex systems such as viruses and the like.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (9)
1. A preparation method of a composite mixed mode polymer chromatographic medium is characterized by comprising the following steps:
step 1, coupling mixed mode ligand: mixing core-shell polymer microspheres with amine compounds with charges and hydrophobic groups, adding a solvent capable of dissolving amine ligands, keeping a certain reaction temperature and a certain rotation speed for reaction, and performing suction filtration and washing after the reaction to remove unreacted residues to obtain polymer microspheres mixed with the ligands;
step 2, surface neutralization of the core-shell microspheres: and (2) mixing a neutral high molecular compound with the molecular weight of 1000000-10000000 with the polymer microsphere of the mixed-mode ligand prepared in the step (1), and reacting at a certain reaction temperature and a certain rotation speed to prepare the composite mixed-mode polymer chromatographic medium.
2. The method for preparing the chromatography medium of the composite mixed mode polymer according to claim 1, wherein in the step 1, the amine compound is an amine compound which can react with an epoxy group and has a hydrophobic group; the dosage of the amine compound is 0.5-5 times of the total mass of the core-shell polymer microsphere.
3. The method for preparing the chromatography medium of the composite mixed mode polymer according to claim 1 or 2, wherein in the step 1, the solvent is at least one of methanol, ethanol, dimethyl sulfoxide and isopropanol; the dosage of the solvent is 2-5 times of the total volume of the core-shell polymer microsphere.
4. The preparation method of the composite mixed mode polymer chromatographic medium according to claim 1 or 2, wherein in the step 1, the reaction temperature is 30-60 ℃, the rotation speed is 80-140 r/min, and the reaction time is 12-48 h.
5. The method for preparing the chromatography medium of the composite mixed-mode polymer according to claim 1 or 2, wherein in the step 2, the neutral polymer compound is one or a mixture of two of aldehyde agarose, aldehyde dextran, aldehyde cellulose and aldehyde polyvinyl alcohol; the dosage of the neutral high molecular compound is 0.1-5 times of the total mass of the polymer microspheres of the mixed mode ligand prepared in the step 1.
6. The preparation method of the composite mixed mode polymer chromatographic medium according to claim 1 or 2, wherein in the step 2, the reaction temperature is 5-25 ℃, the rotation speed is 80-140 r/min, and the reaction time is 24-48 h.
7. The preparation method of the composite mixed mode polymer chromatographic medium according to claim 1 or 2, wherein the composite mixed mode polymer chromatographic medium is a porous polymer microsphere with a core-shell structure, the pore diameter of the shell layer is 50-300 nm, the surface of the shell layer is neutral and hydrophilic, the pore diameter of the core is 200-500 nm, the shell layer and the core are mutually communicated, and the composite mixed mode polymer chromatographic medium has an anion and hydrophobic interaction mixed chromatographic function.
8. The preparation method of the composite mixed mode polymer chromatographic medium according to claim 1 or 2, wherein the protein binding capacity of the composite mixed mode polymer chromatographic medium is maintained at 73-50 mg/m L at a salt concentration of 0-1 mol/L.
9. The preparation method of the composite mixed mode polymer chromatographic medium according to claim 1 or 2, wherein the ion exchange capacity of the composite mixed mode polymer chromatographic medium is 0.19-0.23 mmol/m L.
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