Macroporous agarose DEAE chromatographic gel as well as preparation method and application thereof

Abstract

The invention discloses macroporous agarose DEAE (diethylaminoethanol) chromatographic gel. The macroporous agarose DEAE chromatographic gel is prepared by the following method: (1) preparing composite microspheres; (2) enzymolysis; (3) introducing a cross-linking agent; and (4) modifying the DEAE group. The macroporous agarose DEAE chromatographic gel is applied to separation and purification of biological macromolecules. The macroporous agarose DEAE chromatographic gel disclosed by the invention has a highly developed pore structure and a remarkably increased pore diameter, has high mechanical strength, relatively strong protein binding capacity, high mass transfer efficiency, high-pressure stability and excellent selectivity, can realize rapid mass transfer of protein in microspheres, and greatly improves the separation and purification efficiency. The macroporous agarose DEAE chromatographic gel disclosed by the invention is expected to become an ideal material in the field of chromatographic separation, and plays an important role in more efficient and diversified separation and purification requirements.

Description

Technical Field

[0001] The invention relates to macroporous agarose DEAE chromatographic gel and a preparation method and application thereof, belonging to the technical field of agarose gel microspheres. Background Art

[0002] With the rapid development of biotechnology, the fields of medicine, food and bioengineering have put forward higher requirements for the separation and purification technology of biomacromolecules. As a commonly used separation and purification method, chromatography technology is key to the performance of chromatographic fillers, which can directly affect the separation efficiency and purification effect. Among the many chromatographic fillers, agarose-based materials are widely used in the separation and purification of biomacromolecules such as proteins due to their excellent hydrophilicity, chemical stability and biocompatibility. However, traditional agarose microspheres are difficult to maintain stability under high pressure conditions due to their small pore size, single pore structure and low mechanical strength, and the mass transfer efficiency is low, which seriously affects the separation and purification efficiency. These problems are particularly prominent under the current demand for efficient and large-scale separation.

[0003] In order to meet the higher requirements for speed and efficiency in modern separation and purification processes, solve the limitations of traditional agarose microspheres, and prepare agarose microspheres with interconnected macroporous structures, it has become the focus of current research. In the separation of macromolecules such as proteins, traditional agarose microspheres are prone to molecular retention and increased mass transfer resistance due to pore size limitations, which ultimately reduces the overall separation effect. The macroporous structure and abundant pores can effectively improve the mass transfer efficiency of microspheres. This is because the larger pore size allows macromolecules to enter and diffuse more easily inside the microspheres, and can also reduce the diffusion resistance during the separation process, thereby reducing the time of molecular transfer, making the separation process smoother and faster, meeting the needs of rapid separation, and the abundant pore structure can more fully separate macromolecular mixtures and improve separation and purification efficiency. In addition, the macroporous structure also improves the performance of microspheres in high flow rate operations. Usually in high-efficiency chromatography systems, higher flow rates are used to speed up the separation speed, and traditional agarose microspheres often exhibit unstable flow behavior at high flow rates, and even blockage occurs, which seriously affects the consistency and repeatability of the separation process. Due to their open pore structure, macroporous microspheres can better maintain a stable flow path, reduce the risk of clogging, and adapt to higher flow rate conditions. This not only improves separation efficiency, but also provides a reliable guarantee for agarose microspheres in continuous separation and purification operations on an industrial scale. Moreover, the introduction of a macroporous structure also gives the microspheres a larger surface area, which helps to enhance the contact opportunities with the target molecules, thereby increasing the binding capacity. In biological separation applications, especially for the separation of large molecular proteins, the larger pore size allows these macromolecules to more fully enter the microspheres and interact with the functionalized groups, thereby achieving higher separation and purification effects.

[0004] It can be seen that it is necessary to develop an agarose-based chromatographic filler with an interconnected macroporous structure, rich pores, high mechanical strength and strong protein binding ability, so as to overcome the problems of traditional agarose microspheres due to their small pore size and single pore structure, resulting in low mass transfer efficiency, poor stability at high flow rates, and insufficient binding capacity in the separation and purification of macromolecules, so as to meet the high-efficiency requirements in the separation and purification of biological macromolecules and improve the separation and purification efficiency of biological macromolecules. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides a macroporous agarose DEAE chromatographic gel and its preparation method and application. The present invention uses water-soluble protein or alcohol-soluble protein (preferably gelatin) as a porogen (protein template method) and performs cross-linking modification and functional modification. The prepared macroporous agarose microspheres have a macroporous structure that is interconnected, rich in pores, good mechanical strength, high mass transfer efficiency, high separation efficiency, and strong flow rate adaptability, which can meet the needs of separation and purification of macromolecular proteins. The present invention is achieved through the following technical solutions: A method for preparing macroporous agarose DEAE chromatographic gel comprises the following steps: (1) Preparing composite microspheres: dissolving water-soluble protein or alcohol-soluble protein and agarose or dextran in water as an aqueous phase, pouring the aqueous phase into an oil phase to prepare a suspension, stirring and cooling to room temperature, centrifuging, and washing to obtain agarose-protein composite microspheres or dextran-protein composite microspheres; (2) Enzymatic hydrolysis: The composite microspheres are fully enzymatically hydrolyzed with protease until all the proteins in the composite microspheres are hydrolyzed, and then centrifuged and washed to obtain macroporous agarose microspheres (AGs) or macroporous dextran microspheres; (3) Introducing a cross-linking agent: cross-linking the macroporous agarose microspheres or macroporous dextran microspheres with epichlorohydrin (ECH) to enhance the mechanical strength of the macroporous agarose microspheres or macroporous dextran microspheres and ensure their stability under high pressure conditions, thereby obtaining cross-linked epoxy-group macroporous agarose microspheres or macroporous dextran microspheres; (4) Modification of diethylaminoethyl (DEAE) groups: DEAE groups are introduced into macroporous agarose microspheres or macroporous dextran microspheres with cross-linked epoxy groups to provide macroporous agarose microspheres or macroporous dextran microspheres with anion exchange function, thereby improving their protein binding ability and selectivity, thereby obtaining macroporous agarose DEAE chromatographic gel or macroporous dextran DEAE chromatographic gel.

[0006] Furthermore, in step (1), the water-soluble protein is selected from gelatin, and the weight ratio of agarose to gelatin is 3:(1-10), preferably 3:4 or 3:7; and the alcohol-soluble protein is selected from zein.

[0007] Furthermore, in the step (1), the oil phase is liquid paraffin.

[0008] Furthermore, the specific method of step (1) is as follows: (a) Disperse 3 g of agarose powder in 50 ml of water and heat to completely dissolve it to obtain a clear and transparent agarose solution; disperse 1-10 g of gelatin particles in 50 ml of water and heat to completely dissolve it to obtain a clear and transparent gelatin solution; (b) mixing the agarose solution and the gelatin solution to obtain an agarose-gelatin solution as the aqueous phase, wherein the mass concentration of agarose is 3% and the mass concentration of gelatin is 1% to 10%; (c) Pour the agarose-gelatin solution into 400 mL of liquid paraffin (pre-heated to 80°C), add 16 g of Span 80 as an emulsifier, and stir until fully mixed to obtain a suspension; (d) The suspension is emulsified, then cooled to room temperature under stirring, centrifuged, and the microspheres are collected. The microspheres are washed with petroleum ether, isopropanol, and ultrapure water in sequence to obtain agarose-gelatin composite microspheres.

[0009] Furthermore, the specific method of step (2) is as follows: adding agarose-gelatin composite microspheres to a protease solution (mass concentration of 15% to 20%), enzymolysis at 50°C for 72 hours to fully remove the gelatin in the composite microspheres; after enzymolysis, centrifugation is performed to collect the microspheres, and ultrapure water is used to fully wash them to obtain macroporous agarose microspheres.

[0010] Furthermore, the specific method of step (3) is as follows: 5 g of AGs, 70 mL of ECH, 75 mL of dimethyl sulfoxide, 110 mg of sodium borohydride and 150 mL of NAOH solution are mixed, and the mixture is stirred at 40° C. for 12 h; the mixture is thoroughly washed with ultrapure water to obtain macroporous agarose microspheres with cross-linked epoxy groups.

[0011] Furthermore, the specific method of step (4) is as follows: 5 g of AGs-ECH is dispersed in 10 mL of DEAE-HCl solution, and the mixture is stirred at room temperature for 30 min; the mixture is fully washed with ultrapure water to obtain a macroporous agarose DEAE chromatographic gel.

[0012] The macroporous agarose DEAE chromatographic gel prepared by the above method has large pore size, high mechanical strength and excellent protein binding ability.

[0013] The macroporous agarose DEAE chromatographic gel is used as a chromatographic filler in the separation and / or purification of biomacromolecules. Further, the biomacromolecule is a protein, preferably a protein with a molecular weight of 11 to 245 KDa.

[0014] The preparation method of the macroporous agarose DEAE chromatographic gel of the present invention comprises the following steps: firstly, water-soluble protein or alcohol-soluble protein (preferably gelatin) is used as a porogen to prepare macroporous agarose microspheres with uniform spherical structure and highly developed pores, then epoxy crosslinking agent is combined with the macroporous agarose microspheres to prepare macroporous agarose microspheres with high mechanical strength, and then DEAE groups are introduced into the crosslinked microspheres to prepare affinity chromatography fillers suitable for efficient separation and purification. The macroporous agarose affinity chromatographic gel has a highly developed pore structure and a significantly increased pore size, high mechanical strength and strong protein binding ability, high mass transfer efficiency, high pressure stability and excellent selectivity, and can realize rapid mass transfer of proteins inside the microspheres, greatly improving the efficiency of separation and purification, effectively overcoming the disadvantages of traditional agarose microspheres that are prone to molecular retention and increased mass transfer resistance due to pore size limitation, and realizing rapid and efficient separation and purification of biomacromolecules. The macroporous agarose DEAE chromatographic gel of the present invention is expected to become an ideal material in the field of chromatographic separation, and play an important role in meeting the needs of more efficient and diversified separation and purification.

[0015] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Scanning electron microscope images, optical microscope images, and particle size distribution diagrams of the macroporous agarose microspheres prepared in Examples 1 to 4, wherein the small images from top to bottom and from left to right are numbered A to T, i.e., the numbers from top to bottom in the leftmost column are A, B, C, D, E, and so on, the numbers from top to bottom in the second column are F, G, H, I, J, the numbers from top to bottom in the third column are K, L, M, N, O, and the numbers from top to bottom in the fourth column (the rightmost column) are P, Q, R, S, T; A, B, C are scanning electron microscope images of 3:1 AGs, F, G, H are scanning electron microscope images of 3:4 AGs, K, L, M are scanning electron microscope images of 3:7 AGs, and P, Q, R are scanning electron microscope images of 3:10 AGs. Scanning electron microscope images of AGs; the scale bars in A, F, K, and P are 50 μm, the scale bars in B, G, L, and Q are 10 μm, and the scale bars in C, H, M, and R are 2 μm. D, I, N, and S are optical microscope images of 3:1 AGs, 3:4 AGs, 3:7 AGs, and 3:10 AGs, respectively. The scale bars in D, I, N, and S are 50 μm. E, J, O, and T are particle size distribution diagrams of 3:1 AGs, 3:4 AGs, 3:7 AGs, and 3:10 AGs, respectively.

[0017] Figure 2: Schematic diagram of the porosity, total pore area, swelling capacity and water holding capacity of the macroporous agarose microspheres prepared in Examples 1 to 4, wherein A is the porosity (A) and the total pore area, B is the swelling capacity, and C is the water holding capacity.

[0018] Figure 3 : Scanning electron microscope images of the cross-linked macroporous agarose microspheres prepared in Examples 1 to 4, wherein A1 to A3 are 3:1 AGs-ECH, A4 to A6 are 3:1 AGs-GTE, and A7 to A9 are 3:1 AGs-PGE; B1 to B3 are 3:4 AGs-ECH, B4 to B6 are 3:4 AGs-GTE, and B7 to B9 are 3:4 AGs-PGE; C1 to C3 are 3:7 AGs-ECH, C4 to C6 are 3:7 AGs-GTE, and C7 to C9 are 3:7 AGs-PGE; D1 to D3 are 3:10 AGs-ECH, D4 to D6 are 3:10 AGs-GTE, and D7 to D9 are 3:10 AGs-PGE. The scale bar in A1, A4, A7, B1, B4, B7, C1, C4, C7, D1, D4, D7 is 50 μm; the scale bar in A2, A5, A8, B2, B5, B8, C2, C5, C8, D2, D5, D8 is 10 μm; the scale bar in A3, A6, A9, B3, B6, B9, C3, C6, C9, D3, D6, D9 is 2 μm.

[0019] Figure 4 : Schematic diagram of the pressure-flow measurement results of the macroporous agarose microspheres and cross-linked macroporous agarose microspheres prepared in Examples 1 to 4, wherein A: macroporous agarose microspheres and cross-linked macroporous agarose microspheres when the ratio of agarose to gelatin is 3:1; B: macroporous agarose microspheres and cross-linked macroporous agarose microspheres when the ratio of agarose to gelatin is 3:4; C: macroporous agarose microspheres and cross-linked macroporous agarose microspheres when the ratio of agarose to gelatin is 3:7; D: macroporous agarose microspheres and cross-linked macroporous agarose microspheres when the ratio of agarose to gelatin is 3:10.

[0020] Figure 5: Gel filtration images and SDS-PAGE images of a standard protein mixture solution passing through a chromatographic column containing a chromatographic gel, wherein A: Gel filtration images of a standard protein mixture solution passing through a chromatographic column containing a commercial agarose DEAE chromatographic gel (SFF-DEAE), 3:4AG-ECH-DEAE, and 3:7AG-ECH-DEAE; B: SDS-PAGE image of a standard protein mixture solution; C: SDS-PAGE image of a single protein peak separated after the standard protein mixture was separated using an SFF-DEAE chromatographic column; D: SDS-PAGE image of a single protein peak separated after the standard protein mixture was separated using a 3:4AG-ECH-DEAE chromatographic column; E: SDS-PAGE image of a single protein peak separated after the standard protein mixture was separated using a 3:7AG-ECH-DEAE chromatographic column. M: protein molecular weight marker; S: standard protein mixture solution; lanes 1 to 4 in C, lanes 1 to 5 in D, and lanes 1 to 5 in E: eluted fractions. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0022] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods available in the prior art.

[0023] The agarose used in the present invention was purchased from Biowest Technologies (Spain) with the product number BY-R0100 (1% gel strength: >750 gr / cm 2 ; Gel temperature: 37±1.5℃; Electro-endosmotic pressure: 0.15).

[0024] The gelatin used in the present invention was purchased from Sinopharm Chemical Reagent Co., Ltd. (China) with the item number 10010328 (colorless or light yellow to yellow solid, almost odorless, tasteless, a mixture of high molecular weight water-soluble proteins, which can swell to 5 to 10 times its original weight in cold water, pH 3.8 to 7.6).

[0025] The protease used in the present invention was purchased from Ningbo Yinuo Biotechnology Co., Ltd. (China).

[0026] Example 1 Preparation of macroporous agarose DEAE chromatographic gel Here are the steps: (1) Preparation of agarose-gelatin composite microspheres (a) Disperse 3 g of agarose powder in 50 ml of water, heat to completely dissolve it, heat to 80°C and keep warm for 30 min until the solid is completely dissolved, and obtain a clear and transparent agarose solution; disperse 1 g of gelatin particles in 50 ml of water, heat to completely dissolve it, heat to 80°C and keep warm for 30 min until the solid particles are completely dissolved, and obtain a clear and transparent gelatin solution; (b) mixing the agarose solution and the gelatin solution, stirring at 80°C and 400 r / min for 30 min until the mixture is uniformly mixed, to obtain an agarose-gelatin solution as an aqueous phase, wherein the mass concentration of agarose is 3% and the mass concentration of gelatin is 1%; (c) Pour the agarose-gelatin solution into 400 mL of liquid paraffin (preheated to 80°C), add 16 g of Span 80 as an emulsifier, place in a constant temperature reactor, and stir at 80°C and 400 r / min for 30 min until fully mixed to obtain a suspension; (d) The suspension was emulsified at 80°C and 600 r / min for 30 min; then cooled to room temperature under stirring at 200 r / min, separated by centrifugation (2000 r / min, 10 min), and the microspheres were collected and washed with petroleum ether, isopropanol, and ultrapure water in sequence to obtain agarose-gelatin composite microspheres.

[0027] (2) Enzymatic hydrolysis of gelatin to obtain macroporous agarose microspheres: agarose-gelatin composite microspheres were added to an excess of protease solution (mass concentration of 15%) and enzymatic hydrolyzed at 50°C for 72 h to fully remove the gelatin in the composite microspheres; after enzymatic hydrolysis, the microspheres were centrifuged (2000 r / min, 10 min) to separate and collect the microspheres, washed thoroughly with ultrapure water, and sieved with 50-mesh and 300-mesh standard sieves to obtain AGs, in which the initial ratio of agarose to gelatin was 3:1, referred to as 3:1 AGs.

[0028] (3) Introduction of cross-linking agent: 5 g of freeze-dried AGs, 70 mL of ECH, 75 mL of dimethyl sulfoxide, 110 mg of sodium borohydride and 150 mL of NAOH solution (1 mol / L) were added to a 250 mL flask and stirred at 40 °C for 12 h; the mixture was washed thoroughly with ultrapure water to obtain 3:1 AGs-ECH. The same method was used to cross-link glycerol trianhydroglyceryl ether (GTE) and pentaerythritol glycidyl ether (PGE) to obtain 3:1 AGs-GTE and 3:1 AGs-PGE.

[0029] (4) Modification of DEAE group: Disperse 5 g of freeze-dried AGs-ECH in 10 mL of DEAE-HCl solution (1 mol / L) and stir at room temperature for 30 min. After the reaction, wash thoroughly with ultrapure water to obtain the macroporous agarose DEAE chromatographic gel AGs-ECH-DEAE.

[0030] Example 2 Preparation of macroporous agarose DEAE chromatographic gel Here are the steps: (1) Preparation of agarose-gelatin composite microspheres (a) Disperse 3 g of agarose powder in 50 ml of water, heat to completely dissolve it, heat to 80°C and keep warm for 30 min until the solid is completely dissolved, and obtain a clear and transparent agarose solution; disperse 4 g of gelatin particles in 50 ml of water, heat to completely dissolve it, heat to 80°C and keep warm for 30 min until the solid particles are completely dissolved, and obtain a clear and transparent gelatin solution; (b) mixing the agarose solution and the gelatin solution, stirring at 80°C and 400 r / min for 30 min until the mixture is uniformly mixed, to obtain an agarose-gelatin solution as an aqueous phase, wherein the mass concentration of agarose is 3% and the mass concentration of gelatin is 4%; (c) Pour the agarose-gelatin solution into 400 mL of liquid paraffin (preheated to 80°C), add 16 g of Span 80 as an emulsifier, place in a constant temperature reactor, and stir at 80°C and 400 r / min for 30 min until fully mixed to obtain a suspension; (d) The suspension was emulsified at 80°C and 600 r / min for 30 min; then cooled to room temperature under stirring at 200 r / min, separated by centrifugation (2000 r / min, 10 min), and the microspheres were collected and washed with petroleum ether, isopropanol, and ultrapure water in sequence to obtain agarose-gelatin composite microspheres.

[0031] (2) Enzymatic hydrolysis of gelatin to obtain macroporous agarose microspheres: agarose-gelatin composite microspheres were added to an excess of protease solution (mass concentration of 16%) and enzymatic hydrolyzed at 50°C for 72 h to fully remove the gelatin in the composite microspheres; after enzymatic hydrolysis, the microspheres were centrifuged (2000 r / min, 10 min) to separate and collect the microspheres, washed thoroughly with ultrapure water, and sieved with 50-mesh and 300-mesh standard sieves to obtain AGs, in which the initial ratio of agarose to gelatin was 3:4, referred to as 3:4 AGs.

[0032] (3) Introduction of cross-linking agent: 5 g of freeze-dried AGs, 70 mL of ECH, 75 mL of dimethyl sulfoxide, 110 mg of sodium borohydride and 150 mL of NAOH solution (1 mol / L) were added to a 250 mL flask and stirred at 40 °C for 12 h; washed thoroughly with ultrapure water to obtain 3:4 AGs-ECH. GTE and PGE were cross-linked using the same method to obtain 3:4 AGs-GTE and 3:4 AGs-PGE.

[0033] (4) Modification of DEAE group: Disperse 5 g of freeze-dried AGs-ECH in 10 mL of DEAE-HCl solution (1 mol / L) and stir at room temperature for 30 min. After the reaction, wash thoroughly with ultrapure water to obtain the macroporous agarose DEAE chromatographic gel AGs-ECH-DEAE.

[0034] Example 3 Preparation of macroporous agarose DEAE chromatographic gel Here are the steps: (1) Preparation of agarose-gelatin composite microspheres (a) Disperse 3 g of agarose powder in 50 ml of water, heat to completely dissolve it, heat to 80°C and keep warm for 30 min until the solid is completely dissolved, and obtain a clear and transparent agarose solution; disperse 7 g of gelatin particles in 50 ml of water, heat to completely dissolve it, heat to 80°C and keep warm for 30 min until the solid particles are completely dissolved, and obtain a clear and transparent gelatin solution; (b) mixing the agarose solution and the gelatin solution, stirring at 80°C and 400 r / min for 30 min until the mixture is uniformly mixed, to obtain an agarose-gelatin solution as an aqueous phase, wherein the mass concentration of agarose is 3% and the mass concentration of gelatin is 7%; (c) Pour the agarose-gelatin solution into 400 mL of liquid paraffin (preheated to 80°C), add 16 g of Span 80 as an emulsifier, place in a constant temperature reactor, and stir at 80°C and 400 r / min for 30 min until fully mixed to obtain a suspension; (d) The suspension was emulsified at 80°C and 600 r / min for 30 min; then cooled to room temperature under stirring at 200 r / min, separated by centrifugation (2000 r / min, 10 min), and the microspheres were collected and washed with petroleum ether, isopropanol, and ultrapure water in sequence to obtain agarose-gelatin composite microspheres.

[0035] (2) Enzymatic hydrolysis of gelatin to obtain macroporous agarose microspheres: agarose-gelatin composite microspheres were added to an excess of protease solution (mass concentration of 18%) and enzymatic hydrolyzed at 50°C for 72 h to fully remove the gelatin in the composite microspheres; after enzymatic hydrolysis, the microspheres were centrifuged (2000 r / min, 10 min) to separate and collect the microspheres, washed thoroughly with ultrapure water, and sieved with 50-mesh and 300-mesh standard sieves to obtain AGs, in which the initial ratio of agarose to gelatin was 3:7, referred to as 3:7 AGs.

[0036] (3) Introduction of cross-linking agent: 5 g of freeze-dried AGs, 70 mL of ECH, 75 mL of dimethyl sulfoxide, 110 mg of sodium borohydride and 150 mL of NAOH solution (1 mol / L) were added to a 250 mL flask and stirred at 40 °C for 12 h; washed thoroughly with ultrapure water to obtain 3:7 AGs-ECH. GTE and PGE were cross-linked using the same method to obtain 3:7 AGs-GTE and 3:7 AGs-PGE.

[0037] (4) Modification of DEAE group: Disperse 5 g of freeze-dried AGs-ECH in 10 mL of DEAE-HCl solution (1 mol / L) and stir at room temperature for 30 min. After the reaction, wash thoroughly with ultrapure water to obtain the macroporous agarose DEAE chromatographic gel AGs-ECH-DEAE.

[0038] Example 4 Preparation of macroporous agarose DEAE chromatographic gel Here are the steps: (1) Preparation of agarose-gelatin composite microspheres (a) Disperse 3 g of agarose powder in 50 ml of water, heat to completely dissolve it, heat to 80°C and keep warm for 30 min until the solid is completely dissolved, and obtain a clear and transparent agarose solution; disperse 10 g of gelatin particles in 50 ml of water, heat to completely dissolve it, heat to 80°C and keep warm for 30 min until the solid particles are completely dissolved, and obtain a clear and transparent gelatin solution; (b) mixing the agarose solution and the gelatin solution, stirring at 80°C and 400 r / min for 30 min until the mixture is uniformly mixed, to obtain an agarose-gelatin solution as an aqueous phase, wherein the mass concentration of agarose is 3% and the mass concentration of gelatin is 10%; (c) Pour the agarose-gelatin solution into 400 mL of liquid paraffin (preheated to 80°C), add 16 g of Span 80 as an emulsifier, place in a constant temperature reactor, and stir at 80°C and 400 r / min for 30 min until fully mixed to obtain a suspension; (d) The suspension was emulsified at 80°C and 600 r / min for 30 min; then cooled to room temperature under stirring at 200 r / min, separated by centrifugation (2000 r / min, 10 min), and the microspheres were collected and washed with petroleum ether, isopropanol, and ultrapure water in sequence to obtain agarose-gelatin composite microspheres.

[0039] (2) Enzymatic hydrolysis of gelatin to obtain macroporous agarose microspheres: agarose-gelatin composite microspheres were added to an excess of protease solution (mass concentration of 20%) and enzymatic hydrolyzed at 50°C for 72 h to fully remove the gelatin in the composite microspheres; after enzymatic hydrolysis, the microspheres were centrifuged (2000 r / min, 10 min) to separate and collect the microspheres, washed thoroughly with ultrapure water, and sieved with 50-mesh and 300-mesh standard sieves to obtain AGs, in which the initial ratio of agarose to gelatin was 3:10, referred to as 3:10 AGs.

[0040] (3) Introduction of cross-linking agent: 5 g of freeze-dried AGs, 70 mL of ECH, 75 mL of dimethyl sulfoxide, 110 mg of sodium borohydride and 150 mL of NAOH solution (1 mol / L) were added to a 250 mL flask and stirred at 40 °C for 12 h; washed thoroughly with ultrapure water to obtain 3:10 AGs-ECH. GTE and PGE were cross-linked using the same method to obtain 3:10 AGs-GTE and 3:10 AGs-PGE.

[0041] (4) Modification of DEAE group: Disperse 5 g of freeze-dried AGs-ECH in 10 mL of DEAE-HCl solution (1 mol / L) and stir at room temperature for 30 min. After the reaction, wash thoroughly with ultrapure water to obtain the macroporous agarose DEAE chromatographic gel AGs-ECH-DEAE.

[0042] Experimental performance verification (1) The macroporous agarose microspheres prepared in Examples 1 to 4 (i.e., 3:1 AGs, 3:4 AGs, 3:7 AGs, and 3:10 AGs) were characterized by scanning electron microscopy, optical microscopy, and particle size distribution. The results are as follows: Figure 1 shown.

[0043] The results show that the macroporous agarose microspheres prepared in Examples 1 to 4 are all spherical and evenly distributed. With the increase of gelatin concentration, the color of AGs shows a trend of gradually deepening under an optical microscope. It is speculated that this is because the transparency of the microspheres increases at low gelatin concentrations, which reduces the scattering of the water phase in the hydrogel. Under an optical microscope, obvious brown spots can be seen inside the AGs, indicating that they have a macroporous structure. In addition, the particle size of the microspheres is normally distributed, and its range gradually expands and the mean moves to a higher direction ( Figure 1 E, J, O, T).

[0044] Scanning electron microscopy revealed that AGs exhibited a highly developed porous structure with dense pore distribution ( Figure 1 AC, FH, KM, PR). During the preparation process, the surface pore size of AGs increased with the increase of gelatin concentration. This phenomenon can be attributed to the fact that the gelatin ratio in agarose-gelatin composite microspheres is positively correlated with the gelatin concentration. Therefore, after gelatin hydrolysis, a relatively loose network skeleton structure is left in the microspheres. This process gives the microspheres a mass transfer advantage. Compared with traditional agarose microspheres, the large pores in AGs are sufficient to allow target molecules to pass through the gel better, thereby significantly improving the mass transfer efficiency to achieve faster separation and purification of complex samples.

[0045] (2) The physical properties of the macroporous agarose microspheres (i.e., 3:1 AGs, 3:4 AGs, 3:7 AGs, and 3:10 AGs) prepared in Examples 1 to 4 were characterized by porosity, total pore area, swelling capacity, and water holding capacity. The results are as follows: Figure 2 shown.

[0046] Figure 2 The A shows that the use of gelatin as a pore-forming agent can significantly increase the porosity and total pore area of ​​the microspheres. From 3:1AGs to 3:7AGs, the porosity and total pore area of ​​the microspheres increased significantly, and the results of SEM analysis ( Figure 1 This was also confirmed by the AC, FH, KM, and PR of the agarose-gelatin composite microspheres. This increase was mainly attributed to the high gelatin content, which resulted in a large number of microporous channels. The micropores contribute greatly to the surface area, while the macropores and mesopores mainly provide the pore volume. In contrast, from 3:7 AGs to 3:10 AGs, the porosity and total pore area decreased with the continuous increase in the proportion of gelatin. This decrease may be due to the excess gelatin in the agarose-gelatin composite microsphere system. After gelatin was degraded by enzymes, the porosity of the microspheres reached a critical point, which had a negative impact on their mechanical stability and caused the collapse of the pore structure. Therefore, 3:4 AGs and 3:7 AGs are the best agarose to gelatin ratios, which are essential for producing AGs with effective pore structures and larger pore areas. These results indicate that AGs provide broad channels and abundant adsorption sites for protein diffusion, thereby achieving high adsorption efficiency.

[0047] The swelling capacity of AGs affects both the exchange efficiency of water-soluble substances and their structural stability. It is positively correlated with porosity, because the higher the porosity, the stronger the water absorption capacity. In addition, porosity is not the only parameter that affects the swelling capacity of AGs; the specific surface area also affects the water absorption capacity. A lower specific surface area prevents the hydrogel network from absorbing water and swelling freely. AGs with higher water holding capacity can form stable suspensions after being immersed in water, which is beneficial for the separation of aqueous samples, especially the separation and purification of biomacromolecules. Figure 2 As shown in Figures B and C, the AGs prepared with gelatin as the pore-forming agent have high swelling capacity and water holding capacity. It can be seen that with the increase of agarose content during the preparation of microspheres, the swelling capacity of AGs will also increase, while the water holding capacity has no obvious change trend.

[0048] (3) The macroporous agarose microspheres cross-linked with different cross-linking agents (ECH, GTE and PGE) prepared in Examples 1 to 4 were characterized by scanning electron microscopy and pressure-flow measurement. Figure 3 , Figure 4 shown.

[0049] Figure 3 The results shown in the figure indicate that due to the steps of cross-linking, pretreatment and freeze-drying, AGs underwent some shrinkage and slight fracture. The surface texture of cross-linked AGs was rough and the structure was more compact. At the same time, the surface of cross-linked AGs was rich in pores, and the macropore density was directly related to the content of gelatin during the preparation process. Compared with uncross-linked AGs ( Figure 1 ), the surface pores of the microspheres treated with the crosslinker were more obvious. This phenomenon may be due to the fact that during the freeze-drying process, the unmodified microspheres had a larger pore size and insufficient mechanical strength, resulting in more collapse of the pore structure, and thus the surface morphology of the AGs was more wrinkled. In addition, it can be observed that the pore size of the AGs became smaller after cross-linking. This may be attributed to the cross-linking process making the microsphere structure more compact. In general, the ECH-cross-linked microspheres showed particularly clear macropores, which may be due to the enhanced mechanical strength of ECH, which enabled the gel to better maintain its shape during the freeze-drying process. In contrast, the microspheres treated with GTE and PGE showed different degrees of collapse, which may be due to their lower rigidity, resulting in deformation of the pores during the freeze-drying process.

[0050] The purpose of introducing a cross-linking agent into macroporous agarose microspheres is to allow the microspheres to maintain tolerance to high pressure. The microspheres are loaded into the chromatography column, and as the pressure in the column increases, the flow rate also increases proportionally until the maximum tolerance of the microspheres is reached. At this point, the flow rate either tends to stabilize or decreases slightly, which reflects the upper limit of the flow rate capacity of agarose gel microspheres. Figure 4The results shown indicate that all three cross-linking methods can significantly improve the pressure resistance of the microspheres. Among them, the pressure resistance of ECH cross-linked microspheres was significantly enhanced and stable compared with the unmodified agarose microspheres. In contrast, the microspheres treated with GTE and PGE did not show significant improvement in pressure resistance. At a flow rate of 2.0 mL / min, the pressure of the 3:1 AG-PGE microspheres changed suddenly, which may be due to the compression of the microspheres in the chromatographic column. This phenomenon is due to the deformation of the spherical shape of the microspheres, resulting in a significant increase in the pressure in the column.

[0051] The above results show that ECH has the best cross-linking effect, can significantly improve the mechanical strength of AGs, and has the greatest pressure resistance and stability.

[0052] (4) Verification of the protein separation and purification effect of the macroporous agarose DEAE chromatographic gel prepared in Examples 1 to 4 AGs-ECH microspheres with a ratio of agarose to gelatin of 3:4 and 3:7 were selected for DEAE group functionalization to prepare agarose-based chromatographic fillers with protein separation function. In order to gain a deeper understanding of the chromatographic properties of these two microspheres, AGs-ECH-DEAE and commercially available agarose DEAE chromatographic gel SFF-DEAE were loaded into chromatographic columns, and a mixed solution of six standard proteins was separated and purified. The separated protein solution was analyzed by SDS-PAGE. The results are shown in Figure 2. Figure 5 shown.

[0053] like Figure 5 As shown in the figure, AGs-ECH-DEAE showed better protein resolution compared to SFF-DEAE, especially for proteins with larger molecular weight. The improved resolution may be due to the presence of larger macropores and more complex pore networks in these microspheres, which promote the sequential elution of proteins of different sizes, thereby improving the overall separation efficiency. In addition, both 3:4 AG-ECH-DEAE and 3:7 AG-ECH-DEAE have good separation effects on high molecular weight proteins. However, compared with 3:4 AG-ECH-DEAE, 3:7 AG-ECH-DEAE has a weaker separation effect on low molecular weight proteins ( Figure 5 Peak 5 in D, Figure 5 Peak 5 in E). An increase in large pores generally results in a decrease in small pores. Although the pore size of 3:4 AG-ECH-DEAE is slightly smaller than that of 3:7 AG-ECH-DEAE, its total pore area is larger. The larger area creates a more complex pore network, allowing proteins to pass through the pores more efficiently, thus extending the protein path and improving the separation of high molecular weight proteins without negatively affecting the separation of low molecular weight proteins.

[0054] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A method for preparing macroporous agarose DEAE chromatographic gel, characterized in that: The following steps are involved: (1) Preparing composite microspheres: dissolving water-soluble protein or alcohol-soluble protein and agarose or dextran in water as an aqueous phase, pouring the aqueous phase into an oil phase to prepare a suspension, stirring and cooling to room temperature, centrifuging, and washing to obtain agarose-protein composite microspheres or dextran-protein composite microspheres; (2) Enzymatic hydrolysis: using protease to fully enzymatically hydrolyze the composite microspheres until all the proteins in the composite microspheres are hydrolyzed, centrifuging and washing to obtain macroporous agarose microspheres or macroporous dextran microspheres; (3) Introducing a cross-linking agent: cross-linking the macroporous agarose microspheres or macroporous dextran microspheres with epichlorohydrin to obtain macroporous agarose microspheres or macroporous dextran microspheres with cross-linked epoxy groups; (4) Modification of DEAE groups: Introducing DEAE groups into macroporous agarose microspheres or macroporous dextran microspheres having cross-linked epoxy groups to obtain macroporous agarose DEAE chromatography gel or macroporous dextran DEAE chromatography gel.

2. The method for preparing the macroporous agarose DEAE chromatography gel according to claim 1, characterized in that: In the step (1), the water-soluble protein is selected from gelatin, and the weight ratio of agarose to gelatin is 3:(1-10); and the alcohol-soluble protein is selected from zein.

3. The method for preparing the macroporous agarose DEAE chromatography gel according to claim 1, characterized in that: In the step (1), the oil phase is liquid paraffin.

4. The method for preparing the macroporous agarose DEAE chromatography gel according to claim 1, 2 or 3, characterized in that: The specific method of step (1) is as follows: (a) Disperse 3 g of agarose powder in 50 ml of water and heat to completely dissolve it to obtain a clear and transparent agarose solution; disperse 1-10 g of gelatin particles in 50 ml of water and heat to completely dissolve it to obtain a clear and transparent gelatin solution; (b) mixing the agarose solution and the gelatin solution to obtain an agarose-gelatin solution as the aqueous phase, wherein the mass concentration of agarose is 3% and the mass concentration of gelatin is 1% to 10%; (c) pouring the agarose-gelatin solution into 400 mL of liquid paraffin, adding 16 g of Span 80 as an emulsifier, and stirring until fully mixed to obtain a suspension; (d) The suspension is emulsified, then cooled to room temperature under stirring, centrifuged, and the microspheres are collected. The microspheres are washed with petroleum ether, isopropanol, and ultrapure water in sequence to obtain agarose-gelatin composite microspheres.

5. The method for preparing the macroporous agarose DEAE chromatography gel according to claim 1, characterized in that: The specific method of step (2) is as follows: adding agarose-gelatin composite microspheres to a protease solution, performing enzymatic hydrolysis at 50° C. for 72 h, and fully removing gelatin from the composite microspheres; after enzymatic hydrolysis, centrifugation is performed to collect the microspheres, and the microspheres are fully washed with ultrapure water to obtain macroporous agarose microspheres.

6. The method for preparing the macroporous agarose DEAE chromatography gel according to claim 1, characterized in that: The specific method of step (3) is as follows: 5 g of AGs, 70 mL of ECH, 75 mL of dimethyl sulfoxide, 110 mg of sodium borohydride and 150 mL of NAOH solution are mixed, and the mixture is stirred at 40° C. for 12 h; the mixture is thoroughly washed with ultrapure water to obtain macroporous agarose microspheres with cross-linked epoxy groups.

7. The method for preparing the macroporous agarose DEAE chromatography gel according to claim 1, characterized in that: The specific method of step (4) is as follows: 5 g of AGs-ECH is dispersed in 10 mL of DEAE-HCl solution, and the mixture is stirred at room temperature for 30 min; the mixture is fully washed with ultrapure water to obtain a macroporous agarose DEAE chromatographic gel.

8. Macroporous agarose DEAE chromatography gel prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the macroporous agarose DEAE chromatographic gel according to claim 8 in the separation and / or purification of biomacromolecules.

10. The use according to claim 9, characterized in that: The biological macromolecule is a protein.

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