A chromatographic medium based on zirconium dioxide and its preparation method

Abstract

This invention provides a zirconium dioxide-based chromatography medium and its preparation method. The zirconium dioxide-based chromatography medium uses zirconium dioxide as the matrix, and the surface of the matrix is ​​coated with polyacrylate and cross-linked aminocellulose. The cross-linked aminocellulose is activated and then linked to ligands. The zirconium dioxide-based chromatography medium of this invention exhibits good hydrophilicity, good biocompatibility, and high separation efficiency.

Description

Technical Field

[0001] This invention relates to chromatographic separation technology, and in particular to a chromatographic medium based on zirconium dioxide and its preparation method. Background Technology

[0002] Currently, the affinity chromatography media used for the purification and separation of protein drugs are mainly agarose products. However, these products have low mechanical strength, and their low pressure tolerance during industrial production limits the improvement of separation efficiency and also restricts the service life of such chromatography media.

[0003] To address the aforementioned issues, zirconium dioxide, with its high mechanical strength, can be used as a matrix material instead of agarose. However, zirconium dioxide has poor hydrophilicity and biocompatibility, resulting in low separation efficiency of the chromatographic medium. Summary of the Invention

[0004] The purpose of this invention is to provide a chromatographic medium based on zirconium dioxide and its preparation method, so as to solve the problem of low separation efficiency of chromatographic media based on zirconium dioxide in the prior art.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0006] A chromatographic medium based on zirconium dioxide, wherein the surface of the zirconium dioxide matrix is ​​coated with polyacrylate and cross-linked aminocellulose; the cross-linked aminocellulose is activated and then linked to a ligand.

[0007] Preferably, the zirconium dioxide has a particle size of 30-80 nm.

[0008] Preferably, the ligand is ProA.

[0009] This invention also provides a method for preparing a chromatographic medium based on zirconium dioxide, comprising the following steps:

[0010] (1) Take zirconium dioxide, polyacrylate and aminocellulose, mix them and react to obtain a modified matrix;

[0011] (2) Activate the modified matrix obtained in step (1) to obtain the activated modified matrix;

[0012] (3) Add a ligand to the activated modified matrix obtained in step (2) and after the reaction, a chromatography medium with zirconium dioxide as the matrix is ​​obtained.

[0013] Preferably, in step (1), the mass ratio of zirconium dioxide, polyacrylate, and aminocellulose is 1:(1-2):1.5.

[0014] Preferably, step (1) specifically includes: taking zirconium dioxide, polyacrylate, and aminocellulose, adding them to an ethanol solution with a volume fraction of 60-70%, mixing them evenly, stirring and reacting at 60-80℃ for 3-5 hours, filtering, and obtaining the modified matrix.

[0015] Preferably, in step (2), the modified matrix is ​​activated by bonding epichlorohydrin.

[0016] Preferably, step (2) specifically includes: taking the modified matrix obtained in step (1), adding propylene oxide, acetone and NaOH, stirring and reacting at 30-40℃ for 1-3 hours, filtering, and obtaining the activated modified matrix.

[0017] Preferably, in step (3), the mass ratio of the ligand to the activated modified matrix is ​​1:(1.5-2.0).

[0018] Preferably, step (3) specifically includes: taking the activated modified matrix obtained in step (2), adding ligand, ethanol, and NaOH, stirring and reacting at 40-45℃ for 20-30h, filtering, and obtaining a chromatography medium with zirconium dioxide as the matrix.

[0019] The above-described solution of the present invention has at least the following beneficial effects:

[0020] The present invention relates to a zirconium dioxide-based chromatography medium. The zirconium dioxide matrix is ​​coated with polyacrylate and cross-linked aminocellulose. The cross-linked aminocellulose, after activation, connects to ligands. This zirconium dioxide-based chromatography medium significantly improves the mechanical properties of the chromatography medium. The use of polyacrylate and cross-linked aminocellulose to coat the zirconium dioxide improves its hydrophilicity and biocompatibility. The combined coating of polyacrylate and aminocellulose on the zirconium dioxide surface forms a more stable cross-linked structure, resulting in a more stable chromatography medium with higher separation efficiency. Detailed Implementation

[0021] Unless otherwise specified in the embodiments of this invention, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products; different manufacturers and models of raw materials do not affect the implementation of the technical solution or the achievement of the technical effect of this invention.

[0022] Example 1

[0023] In this embodiment, the chromatography medium is based on zirconium dioxide. The surface of the matrix is ​​coated with polyacrylate and cross-linked aminocellulose. After activation, the cross-linked aminocellulose is linked to the ligand.

[0024] The zirconium dioxide has a particle size of 30 nm; the ligand is ProA, i.e., protein A.

[0025] The method for preparing the zirconium dioxide-based chromatography medium in this embodiment includes the following steps:

[0026] (1) Take zirconium dioxide, polyacrylate and aminocellulose, add them to an ethanol solution with a volume fraction of 60%, mix them evenly, stir and react at 80°C for 3 hours, filter, and obtain the modified matrix.

[0027] The mass ratio of zirconium dioxide, polyacrylate, and aminocellulose is 1:1.5:1.5. In this embodiment, the mass of zirconium dioxide is 50g; the volume of the ethanol solution is 500ml.

[0028] (2) Take the modified matrix obtained in step (1), add propylene oxide, acetone and NaOH, stir and react at 30°C for 2 hours, filter, and obtain the activated modified matrix.

[0029] The amount of propylene oxide is 30 ml, the amount of acetone is 80 ml, and the amount of NaOH is 12 g.

[0030] (3) Take the activated modified matrix obtained in step (2), add ligand, ethanol and NaOH, stir and react at 45°C for 20 h, filter, and obtain the chromatography medium with zirconium dioxide as matrix.

[0031] The mass ratio of the ligand to the activated modified matrix is ​​1:2; the ethanol is anhydrous ethanol, and the amount used is 100 ml; the NaOH is 10 g.

[0032] Example 2

[0033] In this embodiment, the chromatography medium is based on zirconium dioxide. The surface of the matrix is ​​coated with polyacrylate and cross-linked aminocellulose. After activation, the cross-linked aminocellulose is linked to the ligand.

[0034] The zirconium dioxide has a particle size of 80 nm; the ligand is ProA, i.e., protein A.

[0035] The method for preparing the zirconium dioxide-based chromatography medium in this embodiment includes the following steps:

[0036] (1) Take zirconium dioxide, polyacrylate and aminocellulose, add them to an ethanol solution with a volume fraction of 65%, mix them evenly, stir and react at 70°C for 4 hours, filter, and obtain the modified matrix.

[0037] The mass ratio of zirconium dioxide, polyacrylate, and aminocellulose is 1:2:1.5. In this embodiment, the mass of zirconium dioxide is 50g; the volume of the ethanol solution is 500ml.

[0038] (2) Take the modified matrix obtained in step (1), add propylene oxide, acetone and NaOH, stir and react at 35°C for 3 hours, filter, and obtain the activated modified matrix.

[0039] The amount of propylene oxide is 30 ml, the amount of acetone is 80 ml, and the amount of NaOH is 12 g.

[0040] (3) Take the activated modified matrix obtained in step (2), add ligand, ethanol and NaOH, stir and react at 45°C for 25 h, filter, and obtain the chromatography medium with zirconium dioxide as matrix.

[0041] The mass ratio of the ligand to the activated modified matrix is ​​1:1.5; the ethanol is anhydrous ethanol, and the amount used is 100 ml; the NaOH is 10 g.

[0042] Example 3

[0043] In this embodiment, the chromatography medium is based on zirconium dioxide. The surface of the matrix is ​​coated with polyacrylate and cross-linked aminocellulose. After activation, the cross-linked aminocellulose is linked to the ligand.

[0044] The zirconium dioxide has a particle size of 55 nm; the ligand is ProA, i.e., protein A.

[0045] The method for preparing the zirconium dioxide-based chromatography medium in this embodiment includes the following steps:

[0046] (1) Take zirconium dioxide, polyacrylate and aminocellulose, add them to an ethanol solution with a volume fraction of 70%, mix them evenly, stir and react at 60°C for 5 hours, filter, and obtain the modified matrix.

[0047] The mass ratio of zirconium dioxide, polyacrylate, and aminocellulose is 1:1:1.5. In this embodiment, the mass of zirconium dioxide is 50g; the volume of the ethanol solution is 500ml.

[0048] (2) Take the modified matrix obtained in step (1), add propylene oxide, acetone and NaOH, stir and react at 40°C for 1 h, filter, and obtain the activated modified matrix.

[0049] The amount of propylene oxide is 30 ml, the amount of acetone is 80 ml, and the amount of NaOH is 12 g.

[0050] (3) Take the activated modified matrix obtained in step (2), add ligand, ethanol and NaOH, stir and react at 40°C for 30 h, filter, and obtain the chromatography medium with zirconium dioxide as matrix.

[0051] The mass ratio of the ligand to the activated modified matrix is ​​1:1.8; the ethanol is anhydrous ethanol, and the amount used is 100 ml; the NaOH is 10 g.

[0052] Example 4

[0053] In this embodiment, the chromatography medium is based on zirconium dioxide. The surface of the matrix is ​​coated with polyacrylate and cross-linked aminocellulose. After activation, the cross-linked aminocellulose is linked to the ligand.

[0054] The zirconium dioxide has a particle size of 60 nm; the ligand is ProA, i.e., protein A.

[0055] The method for preparing the zirconium dioxide-based chromatography medium in this embodiment includes the following steps:

[0056] (1) Take zirconium dioxide, polyacrylate and aminocellulose, add them to an ethanol solution with a volume fraction of 60-70%, mix them evenly, stir and react at 65℃ for 3h, filter, and obtain the modified matrix.

[0057] The mass ratio of zirconium dioxide, polyacrylate, and aminocellulose is 1:1.6:1.5. In this embodiment, the mass of zirconium dioxide is 50g; the volume of the ethanol solution is 500ml.

[0058] (2) Take the modified matrix obtained in step (1), add propylene oxide, acetone and NaOH, stir and react at 35°C for 3 hours, filter, and obtain the activated modified matrix.

[0059] The amount of propylene oxide is 30 ml, the amount of acetone is 80 ml, and the amount of NaOH is 12 g.

[0060] (3) Take the activated modified matrix obtained in step (2), add ligand, ethanol and NaOH, stir and react at 40°C for 20 h, filter, and obtain the chromatography medium with zirconium dioxide as matrix.

[0061] The mass ratio of the ligand to the activated modified matrix is ​​1:1.6; the ethanol is anhydrous ethanol, and the amount used is 100 ml; the NaOH is 10 g.

[0062] Effect Experiment Example

[0063] To verify the technical effectiveness of the zirconium dioxide-based chromatography medium described in this invention, the following experiments were conducted:

[0064] Using the zirconium dioxide-based chromatography media obtained in Examples 1-4, and IgG antibody as the target protein, each affinity chromatography medium was packed into columns for loading tests. The chromatography experiments were repeated three times with the same concentration of target protein. After each chromatography experiment, the column was washed with a mixed solution of 0.5 mol / L sodium hydroxide and 0.15 mol / L sodium chloride for 2 column volumes, and then equilibrated with 5 column volumes of equilibration buffer. The loading of the third chromatography experiment was recorded.

[0065] The results of the experiment are as follows:

[0066]

[0067] Based on the above experimental results, it can be seen that the present invention uses zirconium dioxide as a matrix. Due to the high mechanical strength of zirconium dioxide, its use as a matrix for chromatography can improve the pressure resistance during column packing and greatly extend the service life of the chromatography medium. More importantly, by coating its surface with polyacrylate and cross-linked aminocellulose, and activating the cross-linked aminocellulose to connect with ligands, the problems of poor hydrophilicity and poor biocompatibility of zirconium dioxide can be improved, resulting in a chromatography medium with good stability and high separation efficiency.

[0068] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. A method for the preparation of a chromatography medium based on zirconium dioxide, characterized in that, It comprises the following steps: (1) Take zirconium dioxide, polyacrylate, amino cellulose, mix, and then react to obtain a modified matrix; specifically, take zirconium dioxide, polyacrylate, and amino cellulose, add to an ethanol solution with a volume fraction of 60-70%, mix uniformly, stir and react at 60-80°C for 3-5h, filter, and obtain the modified matrix; The mass ratio of the zirconium dioxide, polyacrylate, and amino cellulose is 1: (1-2): 1.5; (2) Activate the modified matrix obtained in step (1) to obtain an activated modified matrix; the activation is performed by bonding epichlorohydrin to the modified matrix; (3) Add a ligand to the activated modified matrix obtained in step (2), and after reaction, obtain a zirconium dioxide-based chromatography medium; The chromatography medium has a zirconium dioxide matrix, and the surface of the matrix is covered with polyacrylate and cross-linked amino cellulose; the cross-linked amino cellulose is activated and connected to the ligand; the ligand is ProA.

2. The method for producing a zirconium dioxide matrix-based chromatography medium according to claim 1, characterized in that, Step (2) specifically comprises: take the modified matrix obtained in step (1), add propylene oxide, acetone, and NaOH, stir and react at 30-40°C for 1-3h, filter, and obtain the activated modified matrix.

3. The method for producing a zirconium dioxide matrix-based chromatography medium according to claim 1, characterized in that, In step (3), the mass ratio of the ligand to the activated modified matrix is 1: (1.5-2.0).

4. The method for producing a zirconium dioxide matrix-based chromatography medium according to claim 1, characterized in that, Step (3) specifically comprises: take the activated modified matrix obtained in step (2), add a ligand, ethanol, and NaOH, stir and react at 40-45°C for 20-30h, filter, and obtain the zirconium dioxide-based chromatography medium.

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