A novel composite chromatography medium and a method for preparing the same

Abstract

The present application belongs to the technical field of chromatography medium, and particularly relates to a novel composite chromatography medium and a preparation method thereof. The medium is formed by blending polysaccharide and polyether polyol polymer to form an interpenetrating network structure, and microspheres are prepared by a suspension polymerization method. The structure significantly improves the mechanical strength of the medium, so that the pressure resistance of the medium can reach 0.5 MPa. Meanwhile, the rich hydroxyl functional groups enable the load capacity of the medium to reach 1-2 times of that of a commercially available similar product (such as sepharose fast flow) after the medium is modified into an ion exchange medium or an affinity chromatography medium or a hydrophobic chromatography medium. The preparation method is simple, low in cost and small in organic solvent usage, and the obtained product is good in mechanical strength and high in load capacity, and is suitable for laboratory and industrial purification of biological macromolecules.

Description

Technical Field

[0001] This invention belongs to the field of chromatography media technology, specifically relating to a novel composite chromatography media and its preparation method. Background Technology

[0002] Chromatographic media are the core materials of chromatographic separation technology, and their performance directly affects separation efficiency, resolution, and cost.

[0003] With the rapid development of biotechnology, the separation and purification of biological products has become crucial. Biomolecules (proteins, peptides), pharmaceuticals, cosmetics, and food all require separation and purification technologies, and chromatography media are a core technology in chromatography. These media need to have higher mechanical strength and higher loading capacity. However, the commonly available Sepharose™ series has low mechanical strength, and even modified ion exchange media, such as Q Sepharose™ FF and CM Sepharose™ FF, have relatively low loading capacities. Patent applications CN201210413412.9 and CN201610806733.3 disclose a pre-crosslinking method to obtain high-flow-rate polysaccharide microspheres. While this improves the mechanical strength of the microspheres, the operation steps are cumbersome and difficult to control. Inventors Wang Shaoyun et al., in their earlier patent application CN201910197946.4, significantly increased the loading capacity of FF series microspheres by grafting dextran onto them, but this method involves many steps, increasing uncontrollable factors in the reaction and resulting in higher costs. Therefore, developing a novel composite chromatography medium that is simple to operate, controllable, environmentally friendly, low-cost, and has good performance is beneficial for industrial production and is a technical problem that urgently needs to be solved in separation and purification. Summary of the Invention

[0004] In order to solve the above-mentioned problems of the prior art, one of the objectives of the present invention is to provide a novel composite chromatography medium, which has the advantages of good mechanical strength and high loading capacity.

[0005] Another object of the present invention is to provide a method for preparing the novel composite chromatography medium.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A novel composite chromatography medium comprises microspheres with a particle size of 20-250 μm, wherein the microspheres are formed by blending polysaccharides and polyether polyol polymers to form an interpenetrating network structure. These microspheres can withstand pressures of at least 0.5 MPa, and when modified into an ion-exchange chromatography medium, their dynamic protein loading capacity is 1-2 times that of commercially available FF series agarose-based ion-exchange chromatography media.

[0007] Preferably, the polysaccharide is selected from one or more of agarose, dextran, and regenerated cellulose; More preferably, the polysaccharide is agarose.

[0008] Preferably, the polyvinyl alcohol derivative polymer is selected from one or more of polyvinyl alcohol, hydroxyethyl polyvinyl alcohol, and hydroxypropyl polyvinyl alcohol.

[0009] More preferably, the polyvinyl alcohol derivative polymer is polyvinyl alcohol with a molecular weight of less than 100,000; Alternatively, hydroxyethyl polyvinyl alcohol or hydroxypropyl polyvinyl alcohol with a molecular weight of less than 200,000.

[0010] The hydroxyl groups on the main chain and / or side chain of the polyether in this application exhibit high chemical reactivity in the strong alkaline environment of this invention, and crosslink with the polysaccharide to form an interpenetrating network.

[0011] Preferably, the mass ratio of the polysaccharide to the polyether polyol polymer is 20:1 to 1:1.

[0012] More preferably, the mass ratio of the polysaccharide to the polyether polyol polymer is 10:1-2:1.

[0013] Another technical solution of the present invention provides a method for preparing the composite chromatography medium, which includes the following steps: S1: Mix polysaccharides, polyether polyol polymers with water, heat to dissolve, and form a homogeneous aqueous phase; S2: The organic phase and surfactant are mixed to obtain the oil phase; S3: Under stirring, the aqueous phase is added to the oil phase to form a W / O type emulsion, which is then emulsified and cured at low temperature to obtain composite microspheres. S4: The composite microsphere matrix is ​​mixed with alkaline solution, crosslinking agent and salt to carry out crosslinking reaction to obtain the novel composite chromatography medium.

[0014] Preferably, in step S1, the mass ratio of the polysaccharide to the polyether polyol polymer is 1:1 to 10:1.

[0015] Preferably, in step S2, the organic phase is selected from one or more of cyclohexane, liquid paraffin, toluene, and xylene.

[0016] Preferably, in step S3, the emulsification temperature is 50-90℃ and the time is 0.5-2h; the curing temperature is 0-25℃ and the time is 0.5-2h.

[0017] More preferably, the emulsification temperature is 60-80°C.

[0018] Preferably, the crosslinking agent is epichlorohydrin, 1,4-butanediol diglycidyl ether, or allyl glycidyl ether.

[0019] Preferably, the curing temperature is 0-25℃ and the curing time is 0.5-2h.

[0020] Preferably, the mass ratio of the surfactant to the oil phase is 1:50-1:10; More preferably, the mass ratio of the surfactant to the oil phase is 1:30-1:15.

[0021] Preferably, the surfactant is one or more of Span85, Span80, Tween 80, and Span60.

[0022] Preferably, the mass concentration of polysaccharides and polyether polyols in the aqueous phase is 3%-10% relative to the aqueous phase.

[0023] Preferably, the oil phase is liquid paraffin.

[0024] Preferably, the volume ratio of the aqueous phase to the oil phase is 1:5 to 1:2.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: The novel composite chromatography medium provided by this invention has the following performance advantages: Significantly improved mechanical strength By introducing polyether polyol polymers into a traditional polysaccharide matrix and forming an interpenetrating network structure, the physical stability and mechanical strength of the composite microspheres are significantly improved. These microspheres can withstand pressures up to 0.5 MPa, higher than traditional polysaccharide microspheres, making them suitable for high-flow-rate, high-pressure chromatography processes. Significantly enhanced load capacity performance Polyether polyol polymers contain abundant hydroxyl functional groups, which further enhance the functional group density of microspheres when blended with polysaccharides. After modification into ion exchange or affinity media, their dynamic protein loading capacity can reach 1-2 times that of commercially available FF series agarose-based media, significantly improving separation and purification efficiency. Stable structure and strong ligands Interpenetrating network structures enhance the structural stability of the medium, making it less likely for modified functional ligands (such as ion exchange groups and affinity ligands) to detach, which is beneficial for repeated use and long-term stability. The preparation process is simple. The composite chromatography medium provided by this invention has fewer preparation steps, uses less organic solvent, and has low production cost, which is conducive to market promotion. Attached Figure Description

[0026] Figure 1Light micrograph of the novel composite chromatography medium provided in Example 1; Figure 2 Pressure-flow rate curves for the novel composite microspheres in Example 1 and the conventional polysaccharide microspheres in Comparative Example 1 are shown. Figure 3 This example compares the loading capacity of the novel composite CM ion exchange medium in Example 2 with that of the conventional CM in Comparative Example 2. Figure 4 Example 7 compares the novel composite ProA affinity medium with the conventional ProA affinity microspheres in Comparative Example 4. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments are not intended to limit the present invention. In the following embodiments of the present invention, the protein loading was measured using a protein purification instrument (AKTA Explorer).

[0028] Example 1: Preparation of packing material for large-scale plasmid purification

[0029] (1) Preparation of agarose microspheres In a 500 mL three-necked flask, 200 g of liquid paraffin, 10 g of Span 85, and 2 g of Tween 80 were added to prepare the oil phase, which was then heated to 80 °C and stirred to dissolve. In a 200 mL three-necked flask, 100 mL of deionized water, 5 g of agarose, and 1 g of polyvinyl alcohol were added to prepare the aqueous phase, which was stirred at 95 °C for 2 h to dissolve. Under mechanical stirring, the aqueous phase was added to the oil phase to prepare a W / O emulsion. After emulsification for 1 h, the mixture was cooled to below 15 °C to solidify and prepare microspheres. The microspheres were then washed with 1% SDS to obtain the novel composite microsphere matrix.

[0030] (2) Novel composite chromatography media Add 50g to a 250ml reaction flask, then add 15g sodium sulfate, 25mL 50% NaOH, 0.1g sodium borohydride and 5mL epichlorohydrin to the reaction flask. Stir at 37℃ for 16h. After the reaction is complete, wash with ethanol and pure water to obtain one-crosslinked microspheres. Repeat the crosslinking step once to obtain a novel composite chromatography medium in the form of microspheres.

[0031] To better understand the structure of microspheres, their mechanical strength and particle size were tested, such as... Figure 1 The image shown is a light micrograph of the novel composite chromatography medium provided in Example 1.

[0032] Comparative Example 1: 6% Preparation of Traditional Chromatography Media (1) Preparation of agarose microspheres In a 500 mL three-necked flask, 200 g of liquid paraffin, 10 g of Span 85, and 2 g of Tween 80 were added to prepare the oil phase, which was then heated to 80 °C and stirred to dissolve. In a 200 mL three-necked flask, 100 mL of deionized water and 6 g of agarose were added to prepare the aqueous phase, which was then stirred to dissolve at 95 °C for 2 h. Under mechanical stirring, the aqueous phase was added to the oil phase to prepare a W / O emulsion. After emulsification for 1 h, the mixture was cooled to below 15 °C to solidify and prepare microspheres. The microspheres were then washed with 1% SDS to obtain the novel composite microsphere matrix.

[0033] (2) Traditional microsphere crosslinking Crosslinking was performed according to the method in Example 1 to obtain a conventional chromatography medium. Mechanical strength testing of composite chromatography media

[0034] The mechanical strength of the composite chromatography media was evaluated using a pressure-flow rate test. The chromatography column was ID16mm*30cm, and the protein purification system was AKTA Explorer. The specific methods are as follows: (1) Preparation of the test instrument: Set the flow rate of pump A to 1.0 ml / min, rinse the system with ultrapure water for about 5-10 minutes, connect the inlet line of the chromatographic column to the system, and purge the air bubbles in the inlet line.

[0035] (2) Add an appropriate amount of water to the chromatographic column for detection, remove air bubbles from the chromatographic column and outlet pipeline, weigh 21.3g of the sample to be tested, dissolve it in an appropriate amount of ultrapure water, and pour it into the test chromatographic column at once. After the composite chromatographic medium has settled completely, fill the chromatographic column with ultrapure water and connect it to the protein purification system. (3) At a flow rate of 1.0 ml / min, after running for 20 min, record the height of the medium column bed. Test the flow rate in constant pressure mode, and run at constant pressure under pressure conditions of 0.1 MPa, 0.2 MPa, 0.3 MPa, 0.4 MPa, 0.5 MPa and 0.6 MPa respectively. When the flow rate is stable, run for 3 min and record the corresponding flow rate and column bed height.

[0036] Using the above method, the maximum pressure resistance of the novel composite chromatography medium provided in Example 1 was measured to be 0.5 MPa, and its flow rate reached 1500 cm / h. The maximum pressure resistance of the novel composite chromatography medium provided in Comparative Example 1 was measured to be 0.3 MPa, and its flow rate reached 600 cm / h.

[0037] Example 2: Preparation of a novel composite CM ion exchange chromatography medium Weigh 50g of the composite chromatography medium prepared in step (2) of Example 1 and place it in a 250mL reactor. Add 50mL of DMSO and 10mL of 45% NaOH (add 0.15g of sodium borohydride dissolved in NaOH solution, freshly prepared). After pre-activation at 60℃ and 120rpm for 30min, add 1.5g of chloroacetic acid solid particles and react at 120rpm for 16h. After the reaction is complete, wash with 0.2M acetic acid solution until neutral (pH=7); then soak with twice the volume of deionized water each time (soaking for 15min each time), and then dry. Repeat 3 times to obtain the novel composite CM ion exchange chromatography medium. Performance testing

[0038] I. Ion exchange capacity test, the method is as follows: (1) Weigh 6.0g of the sample to be tested, place it in a disposable cup, weigh two portions, and record W1 and W2; (2) Wet the sample to be tested with an appropriate amount of ultrapure water, fix the 100mL glass exchange chromatography column on the iron stand, place the waste liquid bottle at the filtrate outlet of the chromatography column, open the bottom knob of the chromatography column, add a small amount of water, wait until the water level is 0.5-1.0cm, shake the sample well, use a dropper to transfer the sample to be tested into the chromatography column, rinse the disposable cup and dropper in small amounts several times, transfer the remaining sample into the chromatography column, rinse each glass component of the chromatography column with ultrapure water to wash off the medium adhering to the wall; (3) Measure 30 mL of 0.5 M HCl solution to clean the medium, and control the flow rate at 0.5-0.75 mL / min (drops / 8-12 seconds); (4) Wash with ultrapure water until the filtrate is neutral (about 200 ml of ultrapure water is required): Collect 5-10 drops of filtrate in a 1.5 ml centrifuge tube, add 2-3 drops of methyl orange indicator, and it should not change color (blank control: transfer an appropriate amount of ultrapure water for washing and add 2-3 drops of methyl orange indicator). (5) Take 25 ml of 0.1M NaOH standard solution to rinse the medium and collect the rinsing solution; (6) Take 50 mL of 1M NaCl rinsing medium and collect the rinsing solution, and combine it with the rinsing solution from operation (5); (7) Add 2-3 drops of methyl orange indicator to the collected solution, shake well, and then titrate with 0.1M HCl standard solution. The titration endpoint is when the solution changes from yellow to orange. Record the volume of HCl standard solution consumed.

[0039] C = (C NaOH标液 V NaOH标液 - C HCl标液 V HCl标液 ) / (1.42*W) mmol / mL In the formula: C — Total exchange capacity of the medium, unit: mmol / ml; C represents the concentration of HCl standard solution, in mol / L. C. NaOH standard solution — Concentration of NaOH standard solution, unit: mol / L; V HCl standard solution — the volume of HCl standard solution transferred from the elution medium, in mL; VNaOH standard solution — the volume of NaOH standard solution consumed during titration of the filtrate, in mL; W— The accurate mass of the measured medium, in grams; 1.42 — Mass-to-volume conversion factor of the measured medium, unit: ml / g.

[0040] II. Protein dynamic loading test, the test procedure is as follows: (1) Column packing: Pack 1 mL of pre-packed column; (2) Equilibration: Equilibrate the chromatography column with Buffer A (20mM PB, pH=7.0) at a flow rate of 3mL / min until the UV conductivity is level, then change the flow rate to 0.4mL / min and continue until the UV conductivity is level; (3) Sample loading: Use 3 mg / mL lysozyme solution for sample loading at a flow rate of 0.4 mL / min. Stop sample loading when UV reaches 10% UVmax, and then rinse with Buffer A at a flow rate of 2 mL / min to the baseline. (4) Elute with Buffer B (20mM PB + 1M NaCl, pH=7.0) at a flow rate of 1mL / min and collect the elution peak; (5) Instrument dead volume (V0) test: Using the same method as in (3) above, load the protein dissolved in Buffer B at a flow rate of 1 mL / min, collect the eluent until the UV peak appears, and stop immediately. The volume of the eluent at this time is the dead volume.

[0041] 10% flow rate = C0*(V1-V0) / V_glue Where: C0 — protein concentration (mg / mL); V1—The volume of protein loaded when the UV signal reaches a maximum of 10% (mL). V0 — Dead volume of the pipeline (mL); V 胶 — Medium volume (mL).

[0042] Using the above method, the ion exchange capacity of the weak cation medium provided in Example 2 was measured to be 0.22 mmol / mL, and its loading was 120 mg / mL.

[0043] Comparative Example 2: Preparation of Traditional Ion Exchange Chromatography Media The difference between this comparative example and Example 2 is that the conventional chromatography medium obtained in step (2) of Comparative Example 1 is used as the filter medium. The ion exchange capacity of the weak cation medium provided in Comparative Example 2 was measured to be 0.13 mmol / mL using the above method, and its loading was 89 mg / mL.

[0044] Example 3: Preparation of a novel composite CM ion exchange chromatography medium The main difference between this embodiment and Example 2 is that: (1) the preparation steps of agarose microspheres are as follows: In a 500 mL three-necked flask, 200 g of liquid paraffin, 10 g of Span 85, and 2 g of Tween 80 were added to prepare the oil phase, which was then heated to 80 °C and stirred to dissolve. In a 200 mL three-necked flask, 100 mL of deionized water, 4 g of agarose, and 2 g of polyvinyl alcohol were added to prepare the aqueous phase, which was stirred at 95 °C for 2 h to dissolve. Under mechanical stirring, the aqueous phase was added to the oil phase to prepare a W / O emulsion. After emulsification for 1 h, the mixture was cooled to below 15 °C to solidify and prepare microspheres. The microspheres were then cleaned with 1% SDS to obtain the novel composite microsphere matrix.

[0045] Using the ion exchange capacity and protein loading test method described in Example 2 above, the ion exchange capacity of the composite CM ion exchange chromatography medium provided in this example was measured to be 0.25 mmol / mL, and its loading was 148 mg / mL.

[0046] Example 4: Preparation of a novel composite CM ion exchange chromatography medium The main difference between this embodiment and embodiment 3 is that: (1) the preparation steps of agarose microspheres are as follows: In a 500 mL three-necked flask, 200 g of liquid paraffin, 10 g of Span 85, and 2 g of Tween 80 were added to prepare the oil phase, which was then heated to 80 °C and stirred to dissolve. In a 200 mL three-necked flask, 100 mL of deionized water, 4 g of agarose, and 2 g of hydroxyethyl polyvinyl alcohol were added to prepare the aqueous phase, which was stirred at 95 °C for 2 h to dissolve. Under mechanical stirring, the aqueous phase was added to the oil phase to prepare a W / O emulsion. After emulsification for 1 h, the mixture was cooled to below 15 °C to solidify and prepare microspheres. The microspheres were then cleaned with 1% SDS to obtain the novel composite microsphere matrix.

[0047] The composite CM ion exchange chromatography medium provided in this embodiment has an ion exchange capacity of 0.20 mmol / mL and a loading of 130 mg / mL.

[0048] Example 5: Preparation of a novel composite CM ion exchange chromatography medium The main difference between this embodiment and Example 2 is that: (1) the preparation steps of agarose microspheres are as follows: In a 500 mL three-necked flask, 200 g of liquid paraffin, 10 g of Span 85, and 2 g of Tween 80 were added to prepare the oil phase, which was then heated to 80 °C and stirred to dissolve. In a 200 mL three-necked flask, 100 mL of deionized water, 4 g of agarose, 1 g of hydroxyethyl polyvinyl alcohol, and 1 g of polyvinyl alcohol were added to prepare the aqueous phase, which was stirred at 95 °C for 2 h to dissolve. Under mechanical stirring, the aqueous phase was added to the oil phase to prepare a W / O emulsion. After emulsification for 1 h, the mixture was cooled to below 15 °C to solidify and prepare microspheres. The microspheres were then washed with 1% SDS to obtain novel composite microspheres.

[0049] The composite CM ion exchange chromatography medium provided in this embodiment has an ion exchange capacity of 0.21 mmol / mL and a loading of 136 mg / mL.

[0050] Example 6: Preparation of 4% Novel Composite Chromatography Medium The main difference between this embodiment and Example 1 is that: (1) The preparation steps of agarose microspheres are as follows: In a 500 mL three-necked flask, 200 g of liquid paraffin, 10 g of Span 85, and 2 g of Tween 80 were added to prepare the oil phase, which was then heated to 80 °C and stirred to dissolve. In a 200 mL three-necked flask, 100 mL of deionized water, 3 g of agarose, and 1 g of polyvinyl alcohol were added to prepare the aqueous phase, which was stirred at 95 °C for 2 h to dissolve. Under mechanical stirring, the aqueous phase was added to the oil phase to prepare a W / O emulsion. After emulsification for 1 h, the mixture was cooled to below 15 °C to solidify and prepare microspheres. The microspheres were then washed with 1% SDS to obtain the novel composite microsphere matrix.

[0051] Using the method described in Example 1 above, the maximum pressure resistance of the novel composite chromatography medium provided in Example 6 was measured to be 0.5 MPa, and its flow rate reached 1000 cm / h.

[0052] Comparative Example 3: Preparation of conventional 4% chromatography media The main difference between this comparative example and comparative example 1 is that (1) the preparation steps of agarose microspheres are as follows: In a 500 mL three-necked flask, 200 g of liquid paraffin, 10 g of Span 85, and 2 g of Tween 80 were added to prepare the oil phase, which was then heated to 80 °C and stirred to dissolve. In a 100 mL three-necked flask, 100 mL of deionized water and 4 g of agarose were added to prepare the aqueous phase, which was then stirred to dissolve at 95 °C for 2 h. Under mechanical stirring, the aqueous phase was added to the oil phase to prepare a W / O emulsion. After emulsification for 1 h, the mixture was cooled to below 15 °C to solidify and prepare microspheres. The microspheres were then washed with 1% SDS to obtain the novel composite microsphere matrix.

[0053] Using the above method, the maximum pressure resistance of the 4% composite chromatography medium provided in Comparative Example 3 was measured to be 0.3 MPa, and its flow rate reached 400 cm / h. Example 7: Preparation of a novel composite proA affinity chromatography medium

[0054] (1) Epoxy activation 50g of the novel composite chromatography medium prepared in Example 6 was placed in a 250ml reaction vessel, and 50mL of DMSO and 25mL of epichlorohydrin were added sequentially. The mixture was stirred at 120 rpm for 0.5 h, and then 10mL of 2mol / L NaOH was added dropwise to the system at a uniform rate. The reaction was carried out at 40℃ with a stirring speed of 120 rpm for 6 h. After the reaction was completed, the mixture was washed with ethanol and pure water.

[0055] (2) Coupling protein A Place 50g of epoxy-activated microspheres into a 250ml reaction vessel, add 1.5g of protein A and 50mL of buffer (buffer is 0.2M PB + 1mM EDTA + 0.25M Na2SO4, pH 8.5), and react at 40℃ and 120 rpm for 20 h. After the reaction is complete, wash with pure water.

[0056] The protein loading test method is described in Example 2, and the loading was measured to be 70 mg / mL.

[0057] Comparative Example 4: Preparation of Traditional proA Affinity Chromatography Media (1) Epoxy activation 50 g of the conventional chromatography medium prepared in Comparative Example 3 was placed in a 250 ml reaction vessel. 50 ml of DMSO and 25 ml of epichlorohydrin were added sequentially, and the mixture was stirred at 120 rpm for 0.5 h. Then, 10 mL of 2 mol / L NaOH was added dropwise to the system at a uniform rate. The reaction was carried out at 40 °C with stirring at 120 rpm for 6 h. After the reaction was completed, the mixture was washed with ethanol and pure water.

[0058] (2) Coupling protein A Place 50g of epoxy-activated microspheres into a 250ml reaction vessel, add 1.5g of protein A and 50mL of buffer (buffer is 0.2M PB + 1mM EDTA + 0.25M Na2SO4, pH 8.5), and react at 40℃ and 120 rpm for 20 h. After the reaction is complete, wash with pure water.

[0059] The loading of the conventional chromatography medium provided in Comparative Example 4 was determined to be 58 mg / mL using the same test method as in Example 2.

[0060] Figure 3 The loading capacity of the CM ion exchange microspheres provided in Example 2 and Comparative Example 2 was measured. The test results showed that the loading capacity of the novel composite CM in Example 2 was significantly higher than that of the conventional one in Comparative Example 2.

[0061] Figure 4 The loading of ProA affinity microspheres provided in Example 7 and Comparative Example 4 was measured. The test results showed that the loading of the novel composite ProA affinity microspheres provided in Example 7 was significantly higher than that of the traditional ones.

[0062] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A novel composite chromatography medium, characterized in that, The microspheres are 20-250 μm in diameter and are formed by blending polysaccharides and polyether polyol polymers to form an interpenetrating network structure.

2. The novel composite chromatography medium according to claim 1, characterized in that, The polysaccharide is selected from one or more of agarose, dextran, and regenerated cellulose.

3. The novel composite chromatography medium according to claim 1 or 2, characterized in that, The polyether polyol polymer is selected from one or more of polyvinyl alcohol, hydroxyethyl polyvinyl alcohol, and polyoxypropylene glycerol ether.

4. The novel composite chromatography medium according to any one of claims 1-3, characterized in that, The mass ratio of the polysaccharide to the polyether polyol polymer is from 1:1 to 10:

1.

5. A method for preparing the novel composite chromatography medium as described in any one of claims 1-4, characterized in that, Includes the following steps: S1: Mix polysaccharides, polyether polyol polymers with water, heat to dissolve, and form a homogeneous aqueous phase; S2: The organic phase and surfactant are mixed to obtain the oil phase; S3: Under stirring, the aqueous phase is added to the oil phase to form a W / O type emulsion, which is then emulsified and cured at a low temperature not exceeding 25°C to obtain composite microspheres. S4: The composite microsphere matrix is ​​mixed with alkaline solution, crosslinking agent and salt to carry out crosslinking reaction to obtain the novel composite chromatography medium.

6. The method according to claim 5, characterized in that, In step S1, the mass ratio of the polysaccharide to the polyether polyol polymer is 1:1 to 10:

1.

7. The method according to claim 5, characterized in that, In step S2, the organic phase is selected from one or more of cyclohexane, liquid paraffin, toluene, and xylene.

8. The method according to claim 5, characterized in that, In step S3, the emulsification temperature is 50-90℃ and the time is 0.5-2h; the curing temperature is 0-25℃ and the time is 0.5-2h.

9. The method according to claim 5, characterized in that, The mass ratio of the surfactant to the oil phase is 1:50 to 1:

10.

10. The method according to claim 5, wherein the mass concentration of polysaccharides and polyether polyols in the aqueous phase is 3%-10% relative to the aqueous phase.

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