Nickel ion metal chelate affinity chromatography medium and preparation method thereof

Abstract

The present invention provides a nickel ion metal chelate affinity chromatography medium and a preparation method thereof. The nickel ion metal chelate affinity chromatography medium uses porous dextran gel microspheres as a matrix, and allyl glycidyl ether is bonded to the surface of the dextran gel microspheres. The allyl glycidyl ether is connected to ethylenediaminetetraacetic acid, and the ethylenediaminetetraacetic acid is complexed with nickel ions. The nickel ion metal chelate affinity chromatography medium uses the ethylenediaminetetraacetic acid to bond with allyl glycidyl ether, and the ethylenediaminetetraacetic acid can bind nickel ions. 2+ Carry out complexation and effectively improve the stability of chromatography media.

Description

Technical Field

[0001] The present invention relates to a chromatography medium, in particular to a nickel ion metal chelate affinity chromatography medium and a preparation method thereof. Background Art

[0002] As a method for protein separation and purification, immobilized metal chelate affinity chromatography plays an important role in fields such as pharmaceuticals. The chromatographic medium used in immobilized metal chelate affinity chromatography contains transition metal ions, which can coordinate and bind to tryptophan, cysteine, and histidine on the surface of proteins. These amino acids vary in type, quantity, spatial conformation, and location on different proteins, resulting in varying affinity during coordination and binding, leading to separation and purification during chromatography.

[0003] Nickel ion is one of the commonly used transition metal ions. Current nickel ion metal chelate affinity chromatography media mainly use agarose gel as the matrix material, which has low mechanical strength and poor stability as a chromatography medium. The performance of separating target proteins decreases rapidly after repeated use, and the service life is short. Summary of the Invention

[0004] The object of the present invention is to provide a nickel ion metal chelate affinity chromatography medium and a preparation method thereof, so as to solve the problems of low mechanical strength and poor stability of nickel ion metal chelate affinity chromatography medium in the prior art.

[0005] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0006] A nickel ion metal chelate affinity chromatography medium uses porous dextran gel microspheres as a matrix. Allyl glycidyl ether is bonded to the surface of the dextran gel microspheres, the allyl glycidyl ether is connected to ethylenediaminetetraacetic acid, and the ethylenediaminetetraacetic acid is complexed with nickel ions.

[0007] Preferably, the raw materials for preparing the porous dextran gel microspheres include dextran aqueous solution, vinyltriethoxysilane, silicone oil, and chlorobenzene.

[0008] Preferably, the porous dextran gel microspheres are obtained by the following method: taking an aqueous solution of dextran, adding a mixture of vinyltriethoxysilane, silicone oil and chlorobenzene thereto at 50-80°C, stirring evenly, then adding sodium hydroxide solution thereto, reacting at 85-95°C for 1-5h, filtering to obtain porous dextran gel microspheres.

[0009] Preferably, the volume ratio of the aqueous solution of dextran to the mixed solution of vinyltriethoxysilane, silicone oil and chlorobenzene is 1:(4-8).

[0010] Preferably, the concentration of the dextran aqueous solution is 0.3-0.8 g / ml.

[0011] Preferably, the mass ratio of the vinyltriethoxysilane, silicone oil and chlorobenzene is (1-5): (1-3): 50.

[0012] Preferably, the particle size of the dextran gel microspheres is 100-120 μm.

[0013] Preferably, the nickel ions are complexed with the ethylenediaminetetraacetic acid via a nickel acetate buffer solution.

[0014] The present invention provides a method for preparing a nickel ion metal chelate affinity chromatography medium, comprising the following steps:

[0015] (1) taking porous dextran gel microspheres, adding allyl glycidyl ether thereto, and reacting to obtain microspheres bonded with allyl glycidyl ether;

[0016] (2) adding ethylenediaminetetraacetic acid to the microspheres bonded with allyl glycidyl ether obtained in step (1), and reacting to obtain microspheres bonded with metal chelate ligands;

[0017] (3) Immersing the microspheres bonded with the metal chelate ligand obtained in step (2) in a nickel acetate buffer solution, and after reaction, obtaining a nickel ion metal chelate affinity chromatography medium.

[0018] Preferably, step (1) specifically includes: adding porous dextran gel microspheres to water, adding sodium sulfate and sodium hydroxide thereto, reacting at a temperature of 40-50°C and a stirring speed of 110-150 rpm for 1-2 hours, then adding allyl glycidyl ether thereto, reacting at a temperature of 40-50°C and a stirring speed of 110-150 rpm for 16-20 hours to obtain microspheres bonded with allyl glycidyl ether.

[0019] The dosage of the porous dextran gel microspheres and water is as follows: 20-50 g of the porous dextran gel microspheres are added to every 100 ml of water; the weight ratio of the porous dextran gel microspheres, sodium sulfate, sodium hydroxide, and allyl glycidyl ether is 1000:165:50:(100-300).

[0020] Preferably, step (2) specifically comprises: adding ethylenediaminetetraacetic acid and ethanol to the microspheres bonded with allyl glycidyl ether obtained in step (1), stirring and reacting at 50-65° C. for 10-20 hours to obtain microspheres bonded with metal chelate ligands.

[0021] The weight ratio of the microspheres bonded with allyl glycidyl ether, ethylenediaminetetraacetic acid, and ethanol is 100:(6-10):(100-120).

[0022] Preferably, step (3) specifically comprises: immersing the microspheres bonded with the metal chelate ligand obtained in step (2) in a nickel acetate buffer solution with a concentration of 1.0-1.3 mol / L and a pH value of 4-4.5, stirring the reaction at 40-50° C. for 10-20 hours to obtain a nickel ion metal chelate affinity chromatography medium.

[0023] The above solution of the present invention includes at least the following beneficial effects:

[0024] (1) The nickel ion metal chelate affinity chromatography medium of the present invention is based on porous dextran gel microspheres, on the surface of which allyl glycidyl ether is bonded, and the allyl glycidyl ether is connected to ethylenediaminetetraacetic acid, which is complexed with nickel ions. The nickel ion metal chelate affinity chromatography medium adopts the ethylenediaminetetraacetic acid to bond with allyl glycidyl ether, and the ethylenediaminetetraacetic acid can bind nickel ions. 2+ Carry out complexation and effectively improve the stability of chromatography media.

[0025] (2) The nickel ion metal chelate affinity chromatography medium of the present invention, the raw materials for preparing the porous dextran gel microspheres include an aqueous solution of dextran, vinyltriethoxysilane, silicone oil, and chlorobenzene. The aqueous solution of dextran is the water phase, and the mixed solution of vinyltriethoxysilane, silicone oil, and chlorobenzene is the oil phase. After the two are mixed, the dextran is dispersed in the oil phase through the silicone oil, and then cross-linked by the vinyltriethoxysilane to form porous microspheres. The silanol group of the vinyltriethoxysilane will undergo a condensation reaction with the hydroxyl group of the dextran to form a chemical bond, forming a stable Si-O-Si structure, which increases its ability to resist deformation due to external forces, and the chromatographic medium thus obtained has higher mechanical strength. At the same time, the vinyltriethoxysilane has good compatibility with silicone oil, which is conducive to the uniform dispersion of dextran and vinyltriethoxysilane, and then cross-linking to form a spatial network structure with regular morphology and uniform pore size, so that the chromatographic medium can maintain a stable physical structure when subjected to external forces. In addition, the vinyltriethoxysilane can effectively stabilize the ligand when bonding with allyl glycidyl ether and complexing nickel ions, thereby improving the stability of the chromatography medium and extending the service life of the chromatography medium. DETAILED DESCRIPTION

[0026] In the examples of the present invention, if specific conditions are not specified, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. Reagents or instruments used without specifying the manufacturer are all commercially available conventional products. Raw materials of different manufacturers and types do not affect the implementation of the technical solutions of the present invention and the achievement of the technical effects.

[0027] Example 1

[0028] The nickel ion metal chelate affinity chromatography medium in this embodiment is based on porous dextran gel microspheres, on the surface of which allyl glycidyl ether is bonded, and the allyl glycidyl ether is connected to ethylenediaminetetraacetic acid, which is complexed with nickel ions.

[0029] The porous dextran gel microspheres are prepared from raw materials including a dextran aqueous solution, vinyltriethoxysilane, silicone oil, and chlorobenzene. The porous dextran gel microspheres are obtained by the following method: adding a mixture of vinyltriethoxysilane, silicone oil, and chlorobenzene to the dextran aqueous solution at 50°C, stirring uniformly, then adding a sodium hydroxide solution, reacting at 85°C for 3 hours, and filtering to obtain the porous dextran gel microspheres.

[0030] The volume ratio of the dextran aqueous solution to the mixture of vinyltriethoxysilane, silicone oil, and chlorobenzene was 1:6. The concentration of the dextran aqueous solution was 0.5 g / ml. The mass ratio of vinyltriethoxysilane, silicone oil, and chlorobenzene was 1:3:50. The particle size of the dextran gel microspheres was approximately 110 μm.

[0031] The preparation method of the nickel ion metal chelate affinity chromatography medium of this embodiment comprises the following steps:

[0032] (1) Porous dextran gel microspheres were added to water, and sodium sulfate and sodium hydroxide were added thereto. The reaction was carried out at a temperature of 50°C and a stirring speed of 110 rpm for 2 h. Allyl glycidyl ether was then added thereto. The reaction was carried out at a temperature of 40°C and a stirring speed of 130 rpm for 16 h to obtain microspheres bonded with allyl glycidyl ether.

[0033] The dosage of the porous dextran gel microspheres and water is as follows: 35 g of the porous dextran gel microspheres are added to every 100 ml of water; the weight ratio of the porous dextran gel microspheres, sodium sulfate, sodium hydroxide, and allyl glycidyl ether is 1000:165:50:100.

[0034] (2) Adding ethylenediaminetetraacetic acid and ethanol to the microspheres bonded with allyl glycidyl ether obtained in step (1), stirring and reacting at 65° C. for 10 hours to obtain microspheres bonded with metal chelate ligands.

[0035] The weight ratio of the microspheres bonded with allyl glycidyl ether, ethylenediaminetetraacetic acid, and ethanol is 100:10:100.

[0036] (3) Immerse the microspheres bonded with the metal chelate ligand obtained in step (2) in a nickel acetate buffer solution with a concentration of 1.3 mol / L and a pH value of 4, and stir the reaction at 50° C. for 10 hours to obtain a nickel ion metal chelate affinity chromatography medium.

[0037] It should be noted that the microspheres bonded with allyl glycidyl ether obtained in step (1), the microspheres bonded with metal chelate ligands obtained in step (2), and the nickel ion metal chelate affinity chromatography medium obtained in step (3) are all obtained by filtering and washing the mixed solution after the reaction.

[0038] Example 2

[0039] The nickel ion metal chelate affinity chromatography medium in this embodiment is based on porous dextran gel microspheres, on the surface of which allyl glycidyl ether is bonded, and the allyl glycidyl ether is connected to ethylenediaminetetraacetic acid, which is complexed with nickel ions.

[0040] The porous dextran gel microspheres are prepared from raw materials including a dextran aqueous solution, vinyltriethoxysilane, silicone oil, and chlorobenzene. The porous dextran gel microspheres are obtained by the following method: adding a mixture of vinyltriethoxysilane, silicone oil, and chlorobenzene to the dextran aqueous solution at 80°C, stirring uniformly, then adding a sodium hydroxide solution, reacting at 95°C for 5 hours, and filtering to obtain the porous dextran gel microspheres.

[0041] The volume ratio of the dextran aqueous solution to the mixture of vinyltriethoxysilane, silicone oil, and chlorobenzene was 1:4. The concentration of the dextran aqueous solution was 0.8 g / ml. The mass ratio of vinyltriethoxysilane, silicone oil, and chlorobenzene was 5:1:50. The particle size of the dextran gel microspheres was approximately 120 μm.

[0042] The preparation method of the nickel ion metal chelate affinity chromatography medium of this embodiment comprises the following steps:

[0043] (1) Porous dextran gel microspheres were added to water, and sodium sulfate and sodium hydroxide were added thereto. The mixture was reacted at a temperature of 45°C and a stirring speed of 150 rpm for 2 h. Allyl glycidyl ether was then added thereto. The mixture was reacted at a temperature of 50°C and a stirring speed of 150 rpm for 18 h to obtain microspheres bonded with allyl glycidyl ether.

[0044] The dosage of the porous dextran gel microspheres and water is as follows: 50 g of the porous dextran gel microspheres are added to every 100 ml of water; the weight ratio of the porous dextran gel microspheres, sodium sulfate, sodium hydroxide, and allyl glycidyl ether is 1000:165:50:200.

[0045] (2) Adding ethylenediaminetetraacetic acid and ethanol to the microspheres bonded with allyl glycidyl ether obtained in step (1), stirring and reacting at 65° C. for 20 hours to obtain microspheres bonded with metal chelate ligands.

[0046] The weight ratio of the microspheres bonded with allyl glycidyl ether, ethylenediaminetetraacetic acid, and ethanol is 100:8:120.

[0047] (3) Immersing the microspheres bonded with the metal chelate ligand obtained in step (2) in a nickel acetate buffer solution with a concentration of 1.2 mol / L and a pH value of 4.5, stirring and reacting at 45° C. for 15 h, thereby obtaining a nickel ion metal chelate affinity chromatography medium.

[0048] Example 3

[0049] The nickel ion metal chelate affinity chromatography medium in this embodiment is based on porous dextran gel microspheres, on the surface of which allyl glycidyl ether is bonded, and the allyl glycidyl ether is connected to ethylenediaminetetraacetic acid, which is complexed with nickel ions.

[0050] The porous dextran gel microspheres are prepared from raw materials including a dextran aqueous solution, vinyltriethoxysilane, silicone oil, and chlorobenzene. The porous dextran gel microspheres are obtained by the following method: adding a mixture of vinyltriethoxysilane, silicone oil, and chlorobenzene to the dextran aqueous solution at 65°C, stirring uniformly, then adding a sodium hydroxide solution, reacting at 90°C for 1 hour, and filtering to obtain the porous dextran gel microspheres.

[0051] The volume ratio of the dextran aqueous solution to the mixture of vinyltriethoxysilane, silicone oil, and chlorobenzene was 1:8. The concentration of the dextran aqueous solution was 0.3 g / ml. The mass ratio of vinyltriethoxysilane, silicone oil, and chlorobenzene was 3:2:50. The particle size of the dextran gel microspheres was approximately 100 μm.

[0052] The preparation method of the nickel ion metal chelate affinity chromatography medium of this embodiment comprises the following steps:

[0053] (1) Porous dextran gel microspheres were added to water, and sodium sulfate and sodium hydroxide were added thereto. The reaction was carried out at a temperature of 40°C and a stirring speed of 130 rpm for 1 hour. Allyl glycidyl ether was then added thereto. The reaction was carried out at a temperature of 45°C and a stirring speed of 110 rpm for 20 hours to obtain microspheres bonded with allyl glycidyl ether.

[0054] The dosage of the porous dextran gel microspheres and water is as follows: 20 g of the porous dextran gel microspheres are added to every 100 ml of water; the weight ratio of the porous dextran gel microspheres, sodium sulfate, sodium hydroxide, and allyl glycidyl ether is 1000:165:50:300.

[0055] (2) Adding ethylenediaminetetraacetic acid and ethanol to the microspheres bonded with allyl glycidyl ether obtained in step (1), stirring and reacting at 50° C. for 15 hours to obtain microspheres bonded with metal chelate ligands.

[0056] The weight ratio of the microspheres bonded with allyl glycidyl ether, ethylenediaminetetraacetic acid, and ethanol is 100:6:110.

[0057] (3) Immersing the metal chelate-bonded microspheres obtained in step (2) in a nickel acetate buffer solution having a concentration of 1.0 mol / L and a pH value of 4.5, stirring and reacting at 40° C. for 20 h, thereby obtaining a nickel ion metal chelate affinity chromatography medium.

[0058] Example 4

[0059] The nickel ion metal chelate affinity chromatography medium in this embodiment is based on porous dextran gel microspheres, on the surface of which allyl glycidyl ether is bonded, and the allyl glycidyl ether is connected to ethylenediaminetetraacetic acid, which is complexed with nickel ions.

[0060] The porous dextran gel microspheres are prepared from raw materials including a dextran aqueous solution, vinyltriethoxysilane, silicone oil, and chlorobenzene. The porous dextran gel microspheres are obtained by the following method: adding a mixture of vinyltriethoxysilane, silicone oil, and chlorobenzene to the dextran aqueous solution at 70°C, stirring uniformly, then adding a sodium hydroxide solution, reacting at 90°C for 5 hours, and filtering to obtain the porous dextran gel microspheres.

[0061] The volume ratio of the dextran aqueous solution to the mixture of vinyltriethoxysilane, silicone oil, and chlorobenzene was 1:6. The concentration of the dextran aqueous solution was 0.6 g / ml. The mass ratio of vinyltriethoxysilane, silicone oil, and chlorobenzene was 4:2:50. The particle size of the dextran gel microspheres was 100 μm.

[0062] The preparation method of the nickel ion metal chelate affinity chromatography medium of this embodiment comprises the following steps:

[0063] (1) Porous dextran gel microspheres were added to water, and sodium sulfate and sodium hydroxide were added thereto. The reaction was carried out at a temperature of 45°C and a stirring speed of 110 rpm for 2 h. Allyl glycidyl ether was then added thereto. The reaction was carried out at a temperature of 45°C and a stirring speed of 130 rpm for 20 h to obtain microspheres bonded with allyl glycidyl ether.

[0064] The dosage of the porous dextran gel microspheres and water is as follows: 40 g of the porous dextran gel microspheres are added to every 100 ml of water; the weight ratio of the porous dextran gel microspheres, sodium sulfate, sodium hydroxide, and allyl glycidyl ether is 1000:165:50:280.

[0065] (2) Adding ethylenediaminetetraacetic acid and ethanol to the microspheres bonded with allyl glycidyl ether obtained in step (1), stirring and reacting at 65° C. for 20 hours to obtain microspheres bonded with metal chelate ligands.

[0066] The weight ratio of the microspheres bonded with allyl glycidyl ether, ethylenediaminetetraacetic acid, and ethanol is 100:8:120.

[0067] (3) Immersing the microspheres bonded with the metal chelate ligand obtained in step (2) in a nickel acetate buffer solution with a concentration of 1.0 mol / L and a pH value of 4, stirring and reacting at 45° C. for 20 h to obtain a nickel ion metal chelate affinity chromatography medium.

[0068] Comparative Example 1

[0069] In this comparative example, a nickel ion metal chelate affinity chromatography medium was prepared by the same method as in Example 4, except that the ethylenediaminetetraacetic acid was replaced by trihydroxymethylethylenediamine.

[0070] Comparative Example 2

[0071] In this comparative example, the nickel ion metal chelate affinity chromatography medium was prepared by the same method as in Example 4, except that the ethylenediaminetetraacetic acid was omitted.

[0072] Specifically, the preparation method of the chromatography medium of this comparative example comprises the following steps:

[0073] (1) Porous dextran gel microspheres were added to water, and sodium sulfate and sodium hydroxide were added thereto. The reaction was carried out at a temperature of 45°C and a stirring speed of 110 rpm for 2 h. Allyl glycidyl ether was then added thereto. The reaction was carried out at a temperature of 45°C and a stirring speed of 130 rpm for 20 h to obtain microspheres bonded with allyl glycidyl ether.

[0074] The dosage of the porous dextran gel microspheres and water is as follows: 40 g of the porous dextran gel microspheres are added to every 100 ml of water; the weight ratio of the porous dextran gel microspheres, sodium sulfate, sodium hydroxide, and allyl glycidyl ether is 1000:165:50:280.

[0075] (2) The microspheres bonded with allyl glycidyl ether obtained in step (1) were immersed in a nickel acetate buffer solution with a concentration of 1.0 mol / L and a pH value of 4, and stirred at 45° C. for 20 h to obtain a chromatography medium.

[0076] Comparative Example 3

[0077] In this comparative example, the nickel ion metal chelate affinity chromatography medium was prepared by the same method as in Example 4, except that the porous dextran gel microspheres were replaced with agarose microspheres of the same particle size.

[0078] Comparative Example 4

[0079] In this comparative example, the nickel ion metal chelate affinity chromatography medium was prepared by the same method as in Example 4, except that silicone oil was not included in the raw materials for preparing the porous dextran gel microspheres.

[0080] Specifically, in this comparative example, the porous dextran gel microspheres were obtained by the following method: taking an aqueous solution of dextran, adding a mixture of vinyltriethoxysilane and chlorobenzene thereto at 70°C, stirring evenly, then adding sodium hydroxide solution thereto, reacting at 90°C for 5h, filtering, and obtaining porous dextran gel microspheres.

[0081] The volume ratio of the dextran aqueous solution to the mixed solution of vinyltriethoxysilane and chlorobenzene is 1:6. The concentration of the dextran aqueous solution is 0.6 g / ml. The mass ratio of the vinyltriethoxysilane and chlorobenzene is 4:50.

[0082] Comparative Example 5

[0083] In this comparative example, the nickel ion metal chelate affinity chromatography medium was prepared by the same method as in Example 4, with the only difference being that the amount of vinyltriethoxysilane used in the preparation of the porous dextran gel microspheres was different.

[0084] Specifically, in this comparative example, the porous dextran gel microspheres were obtained by the following method: taking an aqueous solution of dextran, adding a mixture of vinyltriethoxysilane, silicone oil, and chlorobenzene thereto at 70°C, stirring evenly, then adding sodium hydroxide solution thereto, reacting at 90°C for 5h, filtering, and obtaining porous dextran gel microspheres.

[0085] The volume ratio of the dextran aqueous solution to the mixture of vinyltriethoxysilane, silicone oil, and chlorobenzene was 1:6. The concentration of the dextran aqueous solution was 0.6 g / ml. The mass ratio of vinyltriethoxysilane, silicone oil, and chlorobenzene was 0.1:2:50. The particle size of the dextran gel microspheres was 100 μm.

[0086] Comparative Example 6

[0087] In this comparative example, the nickel ion metal chelate affinity chromatography medium was prepared by the same method as in Example 4, with the only difference being that the amount of vinyltriethoxysilane used in the preparation of the porous dextran gel microspheres was different.

[0088] Specifically, in this comparative example, the porous dextran gel microspheres were obtained by the following method: taking an aqueous solution of dextran, adding a mixture of vinyltriethoxysilane, silicone oil, and chlorobenzene thereto at 70°C, stirring evenly, then adding sodium hydroxide solution thereto, reacting at 90°C for 5h, filtering, and obtaining porous dextran gel microspheres.

[0089] The volume ratio of the dextran aqueous solution to the mixture of vinyltriethoxysilane, silicone oil, and chlorobenzene was 1:6. The concentration of the dextran aqueous solution was 0.6 g / ml. The mass ratio of vinyltriethoxysilane, silicone oil, and chlorobenzene was 6:2:50. The particle size of the dextran gel microspheres was 100 μm.

[0090] Effect Experiment Example

[0091] To verify the technical effect of the nickel ion metal chelate affinity chromatography medium of the present invention, the following experiments were performed:

[0092] The nickel ion metal chelate affinity chromatography media prepared in Examples 1-4 and Comparative Examples 1-6 were tested for pressure resistance and stability.

[0093] Effect Experiment Example 1: Pressure Resistance Test

[0094] 1. Instrument / Equipment

[0095] Circulating water vacuum pump, G3 sand core funnel extraction and washing system, chromatography equipment, chromatography column (Φ1.60cm×20.00cm)

[0096] Step 2

[0097] 1. Sample processing

[0098] The nickel ion metal chelate affinity chromatography media prepared in Examples 1-4 and Comparative Examples 1-6 were used as the test filler. 20-25 mL of the test filler was placed in a 50 mL G3 fritted funnel extraction and washing system. 20 mL of deionized water was added and the vacuum pump was turned on to perform extraction and washing until no liquid dripped from the fritted funnel within 1 minute. This extraction and washing process was repeated five times.

[0099] 2. Sample loading

[0100] The column was packed according to the standard operating procedures for prepacked columns, and the packing bed height was controlled to be 10 ± 0.2 cm.

[0101] 3. Sample testing

[0102] 1) Connect the packed chromatography column to the chromatography equipment.

[0103] 2) Set a certain flow rate Vx (mL / min) starting from 0 (V1 is 1 mL / min, Vx = x mL / min), maintain this flow rate for 5 minutes, and record the system display pressure Px (MPa) at this time.

[0104] 3) Increase the flow rate continuously in a gradient of 1 mL / min, repeat the process in 2), and record the Px at the corresponding Vx.

[0105] 4) Continue until the system pressure exceeds 1.2 MPa. Record the corresponding flow rate at this point as V (mL / min), which is the maximum flow rate. If the system pressure increases sharply when the flow rate is increased at a certain moment, it means that the packing has reached its pressure tolerance limit. Record the flow rate at the previous moment as the maximum flow rate V, and the corresponding pressure as the maximum pressure P.

[0106] 5) Bypass the chromatography column and record the instrument background pressure Py at the same flow rate Vx (mL / min) using the above method.

[0107] 4. Calculation results

[0108] Calculate Vx and the corresponding column pressure Pc according to the formula. The formula is as follows:

[0109] Calculation formula of Vx corresponding to column pressure Pc

[0110] P c =P x -P y

[0111] Where:

[0112] Pc——the corresponding column pressure at flow rate Vx (MPa);

[0113] Px——corresponding system pressure at flow rate Vx (MPa);

[0114] Py——Background pressure of the instrument corresponding to the flow rate Vx (MPa).

[0115] Effect Experiment Example 2: Chromatographic Medium Stability Test

[0116] The chromatographic medium to be tested was used for chromatography experiments using recombinant protein A with six consecutive histidine tags as the target protein. After each chromatography experiment, the medium was rinsed with two column volumes of a mixture of 0.5 mol / L sodium hydroxide and 0.15 mol / L sodium chloride, followed by equilibration with five column volumes of equilibration solution. The above chromatography experiments were repeated. When the immobilization capacity of the chromatographic medium for the target protein A was less than 70% of the immobilization capacity of the target protein A in the first chromatography experiment, the experiment was stopped. The number of reuses of the chromatographic medium was recorded. A higher number of reuses indicates better stability of the chromatographic medium.

[0117] Effect experiment results:

[0118] After experimentation, the results are as follows:

[0119]

[0120] According to the comparison of Examples 1-4 and Comparative Examples 1-2, the ethylenediaminetetraacetic acid used in the nickel ion metal chelate affinity chromatography medium of the present invention has a better complexing effect on nickel ions and shows higher stability. In particular, in the comparison of Example 4 and Comparative Example 1, the pressure resistance of the ethylenediaminetetraacetic acid for the chromatography medium is also slightly improved. According to the comparison of Examples 1-4 and Comparative Example 3, the porous dextran gel microspheres used in the nickel ion metal chelate affinity chromatography medium of the present invention are significantly improved compared to agarose microspheres, and the stability is also significantly improved. According to the comparison of Examples 1-4 and Comparative Example 4, in the absence of silicone oil, the mechanical strength and stability of the chromatography medium are significantly reduced. According to the comparison between Examples 1-4 and Comparative Examples 5-6, it can be seen that when the amount of vinyltriethoxysilane is too high or too low, the optimal mechanical strength and stability cannot be achieved. When the volume ratio of the aqueous solution of dextran to the mixed solution of vinyltriethoxysilane, silicone oil and chlorobenzene is 1:6, and the mass ratio of vinyltriethoxysilane, silicone oil and chlorobenzene is 4:2:50, the maximum pressure of the pressure test of the obtained chromatography medium can reach 1.317 MPa, and the number of reuses can reach 94 times.

[0121] It is understood from common technical knowledge that the present invention may be implemented by other embodiments that do not depart from its spirit or essential features. Therefore, the embodiments disclosed above are, in all respects, merely illustrative and not exclusive. All modifications within the scope of the present invention or equivalent to the scope of the present invention are intended to be encompassed by the present invention.

Claims

1. A nickel ion metal chelate affinity chromatography medium, characterized in that The porous dextran gel microspheres are used as a matrix, allyl glycidyl ether is bonded to the surface of the dextran gel microspheres, the allyl glycidyl ether is connected to ethylenediaminetetraacetic acid, and the ethylenediaminetetraacetic acid is complexed with nickel ions; The raw materials for preparing the porous dextran gel microspheres include dextran aqueous solution, vinyltriethoxysilane, silicone oil and chlorobenzene; The porous dextran gel microspheres are obtained by the following method: taking a dextran aqueous solution, adding a mixture of vinyltriethoxysilane, silicone oil, and chlorobenzene to the solution at 50-80° C., stirring evenly, then adding a sodium hydroxide solution to the solution, reacting at 85-95° C. for 1-5 hours, and filtering to obtain porous dextran gel microspheres; The volume ratio of the dextran aqueous solution to the mixed solution of vinyltriethoxysilane, silicone oil and chlorobenzene is 1:(4-8); the concentration of the dextran aqueous solution is 0.3-0.8 g / ml; and the mass ratio of the vinyltriethoxysilane, silicone oil and chlorobenzene is (1-5):(1-3):

50.

2. The nickel ion metal chelate affinity chromatography medium according to claim 1, characterized in that The particle size of the dextran gel microspheres is 100-120 μm.

3. The nickel ion metal chelate affinity chromatography medium according to claim 1, characterized in that The nickel ions are complexed with the ethylenediaminetetraacetic acid through a nickel acetate buffer solution.

4. A method for preparing the nickel ion metal chelate affinity chromatography medium according to any one of claims 1 to 3, characterized in that: The steps include: (1) taking porous dextran gel microspheres, adding allyl glycidyl ether thereto, and reacting to obtain microspheres bonded with allyl glycidyl ether; (2) adding ethylenediaminetetraacetic acid to the microspheres bonded with allyl glycidyl ether obtained in step (1), and reacting to obtain microspheres bonded with metal chelate ligands; (3) Immersing the microspheres bonded with the metal chelate ligand obtained in step (2) in a nickel acetate buffer solution, and after reaction, obtaining a nickel ion metal chelate affinity chromatography medium.

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