Gel filtering medium as well as preparation method and application thereof

Abstract

The invention relates to the technical field of chromatography medium preparation, in particular to a gel filtering medium and a preparation method and application thereof. The preparation method of the gel filtering medium provided by the invention comprises the following steps: S1, mixing allyl glucan with N, N '-methylene bisacrylamide and a pore-foaming agent to prepare a water phase; s2, mixing a surfactant and an organic solvent to obtain an oil phase, wherein the organic solvent is white oil; and S3, dispersing the water phase obtained in the step S1 into the oil phase obtained in the step S2, and carrying out reverse-phase polymerization to obtain the gel filtering medium. The white oil is adopted as an organic solvent to be compounded with the surfactant to prepare the oil phase, so that the toxicity of the gel filtering medium can be reduced, the dispersity of the water phase in the oil phase can be improved, the prepared glucan microspheres are smooth in appearance, and the prepared gel filtering medium is high in resolution ratio, high in flow speed and good in pressure resistance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chromatography medium preparation, in particular to a gel filtration medium and a preparation method and application thereof. BACKGROUND

[0002] Gel filtration chromatography, also known as molecular exclusion chromatography, is a liquid chromatography method mainly using the molecular sieving effect of porous gel to separate different molecules according to their size differences. There is no mutual influence between different components in terms of chemical properties, and the separation conditions are mild and the sample recovery rate is high. It is one of the most widely used separation methods in the field of life sciences. With the continuous development of disciplines such as genetic engineering protein, protein engineering, and vaccine engineering and their application fields, large-scale and efficient protein separation and purification have put forward higher requirements for high-throughput chromatography technology. The medium needs to have higher loading capacity, i.e., to separate and purify more target proteins with the same or even less amount of medium. At present, agarose is mostly used as the matrix to purify proteins, which can improve the loading capacity of target substances to a certain extent.

[0003] The prior art discloses a high-loading agarose chromatography medium and a preparation method thereof. The medium uses agarose as the matrix and introduces dextran to form a composite structure to improve the loading capacity of target substances. However, the medium has low resolution in the separation and purification of biological macromolecules, resulting in poor separation effect of target substances. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to overcome the defects of low resolution of the gel filtration medium in the prior art, which leads to poor separation effect of target substances, so as to provide a gel filtration medium and a preparation method and application thereof, which can prepare a medium with high resolution and strong pressure resistance, and improve the separation and purification effect of target substances.

[0005] The present application provides a preparation method of a gel filtration medium, comprising the following steps: S1, mixing allyl dextran, a crosslinking agent, and a pore-forming agent to obtain an aqueous phase, wherein the crosslinking agent is N,N'-methylenebisacrylamide (MBA); S2, mixing a surfactant and an organic solvent to obtain an oil phase, wherein the organic solvent is white oil; S3, dispersing the aqueous phase obtained in step S1 in the oil phase obtained in step S2 to obtain a gel filtration medium by inverse polymerization.

[0006] In one embodiment, the viscosity of the white oil in step S2 is 2-27.5 mm 2 / s.

[0007] Optionally, the white oil includes at least one of No. 3 white oil, No. 7 white oil, No. 15 white oil, and No. 26 white oil.

[0008] In one embodiment, the surfactant includes a non-ionic surfactant.

[0009] Optionally, the surface active agent comprises at least one of hydrophilic surface active agent, lipophilic surface active agent.

[0010] Optionally, the hydrophilic surface active agent comprises at least one of Tween, nonylphenol polyoxyethylene ether, fatty alcohol polyoxyethylene ether, alkyl polyethylene glycol hydroxy ethanol, wherein the nonylphenol polyoxyethylene ether is NP10.

[0011] Preferably, the Tween comprises at least one of Tween 20, Tween 40, Tween 60, Tween 80.

[0012] Optionally, the lipophilic surface active agent comprises at least one of Span, nonylphenol polyoxyethylene ether, oleic acid, octadecyl methacrylate, propylene glycol mono fatty acid ester, ethylene glycol monostearate, PEG-25 propylene glycol stearate, glycerol monostearate, sorbitan sesquioleate, propylene glycol monolaurate, polyoxyethylene stearate, preferably the nonylphenol polyoxyethylene ether is NP4.

[0013] Preferably, the Span comprises at least one of Span 85, Span 80, Span 65, Span 60, 40, Span 20.

[0014] In one embodiment, the preparation method of the surface active agent comprises mixing the hydrophilic surface active agent and the lipophilic surface active agent.

[0015] In one embodiment, the mass ratio of the surface active agent to the organic solvent is (1-10):100.

[0016] In one embodiment, the preparation method of the oil phase in the step S2 further comprises a step of adding an initiator to the oil phase.

[0017] Optionally, the initiator in the step S2 comprises at least one of oil-soluble azo initiator, organic peroxide initiator.

[0018] Optionally, the oil-soluble azo initiator comprises at least one of azobisdimethyl isobutyl cyanide, azobisdimethyl isohexyl cyanide.

[0019] Optionally, the organic peroxide initiator comprises at least one of dibenzoyl peroxide, diisocyanate peroxide.

[0020] In one embodiment, the mass ratio of the initiator to the organic solvent in the oil phase is (0.02-10):100.

[0021] In one embodiment, the temperature of mixing the surface active agent and the organic solvent in the step S2 is 50-80℃.

[0022] In one embodiment, during the preparation of the oil phase, the surfactant, organic solvent, and initiator are mixed at 50–80°C and 400–600 rpm for 0.8–1.2 h.

[0023] Optionally, the preparation of the oil phase may also include a step of allowing the mixed oil phase to stand to allow the materials in the oil phase to mix thoroughly.

[0024] In one embodiment, in step S1, the pore-forming agent includes at least one of deionized water, alcohol solvent, and aprotic polar solvent.

[0025] Optionally, in step S1, the porogen includes at least one of deionized water, methanol, ethanol, dimethyl sulfoxide, N,N'-dimethylformamide, N,N'-dimethylacetamide, and N,N'-dimethylethanolamine.

[0026] Preferably, the porogen in step S1 includes any two of deionized water, methanol, ethanol, dimethyl sulfoxide, N,N'-dimethylformamide, N,N'-dimethylacetamide, and N,N'-dimethylethanolamine.

[0027] In one embodiment, the method for preparing the aqueous phase in step S1 further includes the step of adding an initiator to the aqueous phase.

[0028] Optionally, the initiator in step S1 includes at least one of water-soluble azo initiators and inorganic peroxide initiators.

[0029] Optionally, the water-soluble azo initiator includes azobisisobutyramidine hydrochloride.

[0030] Optionally, the inorganic peroxide initiator includes at least one of ammonium persulfate and potassium persulfate.

[0031] In one embodiment, the mass ratio of crosslinking agent, pore-forming agent, initiator and allyl dextran in the aqueous phase is (0.2-5):(0.5-10):(0.01-0.1):1.

[0032] In one embodiment, the temperature during mixing of allyl dextran, MBA, and the porogen is 40–60°C, preferably 50°C, and the dissolution time is 4–8 h, preferably 4 h.

[0033] In one embodiment, the mass concentration of allyl dextran in the aqueous phase is 5-60%.

[0034] In one embodiment, in step S3, the reaction temperature for reverse polymerization is 50–80°C, and the reaction time is 1–20 h.

[0035] In one embodiment, the volume ratio of the oil phase to the water phase is 1:(2-10).

[0036] In one embodiment, the oil phase and the water phase are mixed under stirring, wherein the stirring speed is 100-800 rpm and the stirring time is 10-15 min, wherein the stirring speed when mixing the water phase and the oil phase affects the particle size of the microspheres.

[0037] Optionally, the aqueous phase is added to the oil phase at a rate of 8–12 mL / s to form a mixture of the oil and aqueous phases.

[0038] In one embodiment, in step S1, the method for preparing allyl dextran includes coupling a compound containing double bonds with a dextran solution, followed by drying to obtain allyl dextran.

[0039] In one embodiment, the allyl dextran was prepared by adding glacial acetic acid to adjust the pH of the reaction solution to neutral to terminate the reaction.

[0040] Optionally, the mass ratio of glacial acetic acid to dextran is (0.8-1.2):2, preferably 1:2.

[0041] In one embodiment, the reaction temperature for coupling the compound containing the double bond with the dextran solution is 30–80°C, and the reaction time is 1–10 h.

[0042] In one embodiment, the compound containing a double bond includes an organic compound containing an allyl group.

[0043] Optionally, the allyl-containing organic compound includes at least one of allyl glycidyl ether, allyl bromide, allyl chloride, allylamine, and diallylamine.

[0044] In one embodiment, the method for preparing the dextran solution includes the step of mixing dextran and a reducing agent under alkaline conditions.

[0045] In one embodiment, the reducing agent comprises a metal hydride, optionally including at least one of potassium borohydride, sodium borohydride, and lithium borohydride.

[0046] In one embodiment, the molecular weight of the dextran is 1000 to 2 × 10⁻⁶. 7 .

[0047] In one embodiment, the mass ratio of the dextran to the reducing agent is 100:(1-20).

[0048] In one embodiment, the mass ratio of the compound containing the double bond to the dextran is (0.1–5):1.

[0049] In one embodiment, the drying step in the preparation method of allyl dextran includes freeze drying. The freeze drying conditions are: pretreatment at -55 to -60°C for 10 to 14 hours, followed by vacuum freeze drying for 48 to 72 hours to obtain allyl dextran dry powder. The moisture content of the allyl dextran dry powder is below 2%.

[0050] In one embodiment, before the drying step, there is a step of removing impurities from the allyl dextran. The step of removing impurities includes adding glacial acetic acid to the allyl dextran, washing it with anhydrous ethanol, removing the supernatant, dissolving it with ultrapure water, and repeating the washing step 3 to 10 times to obtain allyl dextran with high purity.

[0051] In one embodiment, the method for preparing the gel filter medium further includes the steps of washing and sieving the obtained gel filter medium.

[0052] In one embodiment, the washing step includes sequentially washing the gel filter media with toluene, acetone, and deionized water.

[0053] Optionally, the washing process is performed at least once. Preferably, the washing step includes washing the gel filter medium three times in sequence with toluene, acetone, and deionized water.

[0054] On the other hand, the present invention provides a gel filter medium prepared by the above preparation method, which is capable of separating and purifying proteins.

[0055] The technical solution of this invention has the following advantages:

[0056] 1. The preparation method of the gel filter medium provided by the present invention includes the following steps: S1, mixing allyl dextran with N,N'-methylenebisacrylamide and a pore-forming agent to obtain an aqueous phase; S2, mixing a surfactant and an organic solvent to obtain an oil phase, wherein the organic solvent is white oil; S3, dispersing the aqueous phase obtained in step S1 in the oil phase obtained in step S2, and performing reverse polymerization to obtain the gel filter medium. The present invention uses white oil as an organic solvent and a surfactant to prepare the oil phase, which not only reduces the toxicity of the gel filter medium but also improves the dispersibility of the aqueous phase in the oil phase, resulting in smooth-looking dextran microspheres. The prepared gel filter medium has high resolution, fast flow rate, good pressure resistance, and good separation and purification effect on the target analyte.

[0057] Meanwhile, this invention uses N,N'-methylenebisacrylamide as a crosslinking agent. On the one hand, the "#"-shaped structural unit formed by the crosslinking of MBA and allyl dextran can effectively improve the stability of the dextran microsphere structure compared to the "H"-shaped structural unit formed by the crosslinking of hydroxyl dextran. On the other hand, the use of N,N'-methylenebisacrylamide, which has higher rigidity, can improve the pressure resistance of the dextran microspheres.

[0058] 2. The method for preparing gel filter media provided by the present invention includes a nonionic surfactant. The present invention uses a nonionic surfactant combined with the organic solvent white oil, which can improve the structural stability of the prepared gel filter media, thereby improving the separation efficiency and sensitivity of the gel filter media for target analytes.

[0059] 3. The preparation method of the gel filter medium provided by the present invention includes a surfactant formulation method comprising a mixture of hydrophilic and lipophilic surfactants. The surfactant added in the present invention is a combination of hydrophilic and lipophilic surfactants, which enables uniform dispersion of the aqueous phase containing allyl dextran in the oil phase, and contributes to the stability of the dextran microsphere structure during the polymerization reaction in both the aqueous and oil phases.

[0060] 4. In the preparation method of the gel filter medium provided by the present invention, in step S1, the porogen is at least two of deionized water, methanol, ethanol, dimethyl sulfoxide, N,N'-dimethylformamide, N,N'-dimethylacetamide, and N,N'-dimethylethanolamine. The use of at least two materials as porogens in this invention can effectively make the pore structure of dextran microspheres more uniformly distributed, which helps to improve the stability of the dextran microparticle structure.

[0061] 5. The preparation method of the gel filter medium provided by the present invention, wherein in step S1, the preparation method of the allyl dextran includes coupling a compound containing double bonds with a dextran solution, followed by drying, to obtain allyl dextran. The present invention modifies dextran by coupling double-bonded functional groups, resulting in a uniform and densely loaded double-bonded structure, improving the stability of the backbone structure formed by the polymerization of the obtained allyl dextran and the crosslinking agent N,N'-methylenebisacrylamide, and resulting in a more uniform porosity distribution, thereby improving the pressure resistance and resolution of the gel filter medium.

[0062] 6. The gel filter medium prepared by the method of the present invention has good pressure resistance, high resolution, and can quickly separate and purify proteins with similar molecular weights with high sensitivity. Attached Figure Description

[0063] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0064] Figure 1 This is a process flow diagram of the preparation method of gel filter medium in Embodiment 1 of the present invention. Detailed Implementation

[0065] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0066] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0067] In this embodiment of the invention, the white oil was purchased from Ruifu Lubricating Oil Co., Ltd. and Hesen Special Oils Co., Ltd.

[0068] Dextran was purchased from Hanbang Huanyu and Shanghai Huamao, with a molecular weight of 1000–2×10⁻⁶. 7 .

[0069] Example 1

[0070] See Figure 1 As shown in the figure, this embodiment provides a method for preparing a gel filter medium, and the specific steps and parameters are as follows:

[0071] (1) Synthesis of allyl dextran: Weigh 100g dextran T100, 500g water, 36g sodium hydroxide and 3.6g potassium borohydride and mix them. Stir at 60℃ for 2h to completely dissolve the above materials to obtain a dextran solution.

[0072] Add 100g of allyl glycidyl ether to the above dextran solution and react at 60°C for 4 hours. After the reaction is complete, immediately add 50g of glacial acetic acid.

[0073] Then wash with anhydrous ethanol, remove the supernatant, and dissolve in ultrapure water. Repeat the washing steps 8-10 times to obtain the sugar solution.

[0074] The sugar solution was freeze-dried to obtain allyl dextran powder. The freeze-drying conditions were: pretreatment at -60℃ for 12 hours, followed by treatment in a vacuum freeze dryer for 72 hours, so that the moisture content of allyl dextran was below 2%.

[0075] (2) Preparation of aqueous phase: Mix 10g allyl dextran dry powder, 8g N,N'-methylenebisacrylamide (MBA), 40g deionized water and 32g methanol, and dissolve at 50℃ for 4h. After the above raw materials are completely dissolved, add 0.5g ammonium persulfate to obtain the aqueous phase.

[0076] (3) Preparation of oil phase: Mix 500g of No. 3 white oil with 15g of Span 85 and 7.5g of Tween 60, heat to 60℃, stir at 500rpm for 1h, stop stirring and let stand for 30min to obtain oil phase.

[0077] (4) According to the volume ratio of water phase to oil phase 1:5, add the water phase obtained in step (2) to the oil phase obtained in step (3) at a speed of 10 mL / s, adjust the stirring speed to 300 rpm, so that the water phase is dispersed in the oil phase, and water phase droplets with a particle size of about 50 μm can be formed. Heat to 70℃ and then stir at a constant temperature for 2 hours to obtain dextran microspheres.

[0078] (5) Transfer the dextran microspheres to a sand core funnel and wash them with 1L of toluene each time for 8-10 times. Then wash them with 1L of acetone for 8-10 times to remove the white oil and surfactant on the dextran microspheres. Finally, wash them with 20L of deionized water to remove the residual acetone.

[0079] (6) Use sieves with pore sizes of 25 μm and 75 μm to sieve the dextran microspheres to obtain dextran microspheres with a particle size range of 25-75 μm, and store them in a 20% ethanol solution.

[0080] Example 2

[0081] This embodiment provides a method for preparing a gel filter medium, the specific steps and parameters of which are as follows:

[0082] (1) Synthesis of allyl dextran: Weigh 100g dextran T100, 500g water, 36g sodium hydroxide and 3.6g potassium borohydride and mix them. Stir at 60℃ for 2h to completely dissolve the above materials to obtain a dextran solution.

[0083] Add 100g of allyl bromide to the above dextran solution and react at 60℃ for 4h. After the reaction is complete, immediately add 50g of glacial acetic acid.

[0084] Wash with anhydrous ethanol, remove the supernatant, then dissolve in ultrapure water. Repeat the washing steps 8-10 times to obtain the sugar solution.

[0085] The sugar solution was freeze-dried to obtain allyl dextran powder. The freeze-drying conditions were: pretreatment at -60℃ for 12 hours, followed by treatment in a vacuum freeze dryer for 72 hours, so that the moisture content of allyl dextran was below 2%.

[0086] (2) Preparation of aqueous phase: Mix 10g allyl dextran dry powder, 8g MBA, 40g deionized water and 32g DMF, and dissolve at 50℃ for 4h. After the above raw materials are completely dissolved, add 0.5g potassium persulfate to obtain the aqueous phase.

[0087] (3) Preparation of oil phase: Mix 500g of No. 3 white oil, 15g of Span 60 and 15g of octadecyl methacrylate, heat to 60℃, stir at 500rpm for 1h, stop stirring and let stand for 30min to obtain oil phase.

[0088] (4) According to the volume ratio of water phase to oil phase 1:5, the water phase obtained in step (2) is added to the oil phase obtained in step (3) at a speed of 10 mL / s. Stir at 300 rpm to disperse the water phase in the oil phase and form water phase droplets with a particle size of about 50 μm. Heat to 70 °C and stir at a constant temperature for 2 h to obtain dextran microspheres.

[0089] (5) Transfer the dextran microspheres to a sand core funnel and wash them with 1L of toluene each time for 8-10 times. Then wash them with 1L of acetone for 8-10 times to remove the white oil and surfactant on the dextran microspheres. Finally, wash them with 20L of deionized water to remove the residual acetone.

[0090] (6) Agarose microspheres were sieved using sieves with pore sizes of 25 μm and 75 μm to obtain dextran microspheres with a particle size range of 25-75 μm, and stored in a 20% ethanol solution.

[0091] Example 3

[0092] This embodiment provides a method for preparing a gel filter medium, the specific steps and parameters of which are as follows:

[0093] (1) Synthesis of allyl dextran: Weigh 100g dextran T200, 500g water, 36g sodium hydroxide and 3.6g potassium borohydride and mix them. Stir at 60℃ for 2h to completely dissolve the above materials to obtain a dextran solution.

[0094] Add 100g of allyl glycidyl ether to the above dextran solution and react at 60℃ for 5h. After the reaction is completed, immediately add 50g of glacial acetic acid.

[0095] Then wash with anhydrous ethanol, remove the supernatant, and dissolve in ultrapure water. Repeat the washing steps 8-10 times to obtain the sugar solution.

[0096] The sugar solution was freeze-dried to obtain allyl dextran powder. The freeze-drying conditions were: pretreatment at -60℃ for 12 hours, followed by treatment in a vacuum freeze dryer for 72 hours, so that the moisture content of allyl dextran was below 2%.

[0097] (2) Preparation of aqueous phase: Weigh 10g of allyl dextran dry powder, 8g of MBA, 40g of deionized water and 32g of DMF and mix them. Dissolve them at a constant temperature of 50℃ for 4h to completely dissolve the above raw materials and obtain the aqueous phase.

[0098] (3) Preparation of oil phase: Heat 500g white oil, 15g Span 60 and 15g octadecyl methacrylate to 60℃, stir at 500rpm for 1h and then add 1g azobisisobutyronitrile (AIBN), stir evenly to obtain oil phase.

[0099] (4) According to the volume ratio of water phase to oil phase 1:5, the water phase obtained in step (2) is added to the oil phase obtained in step (3) at a speed of 10 mL / s. Stir at 300 rpm for 10 min to disperse the water phase in the oil phase and form water phase droplets with a particle size of about 50 μm. Heat to 70℃ and stir for 2 h to obtain dextran microspheres.

[0100] (5) Transfer the dextran microspheres to a sand core funnel and wash them with 1L of toluene each time for 8-10 times. Then wash them with 1L of acetone for 8-10 times to remove the white oil and surfactant on the dextran microspheres. Finally, wash them with 20L of deionized water to remove the residual acetone.

[0101] (6) Agarose microspheres were sieved using sieves with pore sizes of 25 μm and 75 μm to obtain dextran microspheres with a particle size range of 25-75 μm, and stored in a 20% ethanol solution.

[0102] Example 4

[0103] This embodiment provides a method for preparing a gel filter medium, the specific steps and parameters of which are as follows:

[0104] (1) Synthesis of allyl dextran: Weigh 100g dextran T100, 500g water, 36g sodium hydroxide and 3.6g potassium borohydride and mix them. Stir at 60℃ for 2h to completely dissolve the above materials to obtain a dextran solution.

[0105] Add 80g allyl glycidyl ether and 20g allyl bromide to the above dextran solution and react at 60℃ for 4h. After the reaction is completed, immediately add 50g glacial acetic acid.

[0106] Then wash with anhydrous ethanol, remove the supernatant, and dissolve in ultrapure water. Repeat the washing steps 8-10 times to obtain the sugar solution.

[0107] The sugar solution was freeze-dried to obtain allyl dextran powder. The freeze-drying conditions were: pretreatment at -60℃ for 12 hours, followed by treatment in a vacuum freeze dryer for 72 hours, so that the moisture content of allyl dextran was below 2%.

[0108] (2) Preparation of aqueous phase: Mix 10g allyl dextran, 8g MBA, 40g deionized water and 32g methanol, and dissolve at 50°C for 4 hours. After the above raw materials are completely dissolved, add 0.5g ammonium persulfate to obtain the aqueous phase.

[0109] (3) Preparation of oil phase: Heat 500g white oil, 15g Span 85 and 7.5g Tween 60 to 60℃, stir at 500rpm for 1h, stop stirring and let stand for 30min to obtain oil phase.

[0110] (4) According to the volume ratio of water phase to oil phase 1:5, the water phase obtained in step (2) is added to the oil phase obtained in step (3) at a speed of 10 mL / s. The rotation speed is adjusted to 300 rpm to disperse the water phase in the oil phase, which can form water phase droplets with a particle size of about 50 μm. The temperature is raised to 70℃ and stirred for 2 hours to obtain dextran microspheres.

[0111] (5) Transfer the dextran microspheres to a sand core funnel and wash them with 1L of toluene each time for 8-10 times. Then wash them with 1L of acetone for 8-10 times to remove the white oil and surfactant on the dextran microspheres. Finally, wash them with 20L of deionized water to remove the residual acetone.

[0112] (6) Agarose microspheres were sieved using sieves with pore sizes of 25 μm and 75 μm to obtain dextran microspheres with a particle size range of 25-75 μm, and stored in a 20% ethanol solution.

[0113] Example 5

[0114] This embodiment provides a method for preparing a gel filter medium, the specific steps and parameters of which are as follows:

[0115] (1) Synthesis of allyl dextran: Weigh 100g dextran T500, 500g water, 72g sodium hydroxide and 7.2g potassium borohydride and mix them. Stir at 60℃ for 2h to completely dissolve the above materials to obtain a dextran solution.

[0116] Add 100g of allyl glycidyl ether to the above dextran solution and react at 60°C for 6 hours. After the reaction is complete, immediately add 100g of glacial acetic acid.

[0117] Then wash with anhydrous ethanol, remove the supernatant, and dissolve in ultrapure water. Repeat the washing steps 8-10 times to obtain the sugar solution.

[0118] The sugar solution was freeze-dried to obtain allyl dextran powder. The freeze-drying conditions were: pretreatment at -60℃ for 12 hours, followed by treatment in a vacuum freeze dryer for 72 hours, so that the moisture content of allyl dextran was below 2%.

[0119] (2) Preparation of aqueous phase: Mix 10g allyl dextran, 8g MBA, 40g deionized water and 32g methanol, stir at 50℃ for 4h, and add 0.5g ammonium persulfate to obtain aqueous phase.

[0120] (3) Preparation of oil phase: Mix 500g white oil, 15g Span 85 and 7.5g Tween 60, heat to 60℃, stir at 500rpm for 1h, stop stirring and let stand for 30min to obtain oil phase.

[0121] (4) According to the volume ratio of water phase to oil phase 1:5, add the water phase obtained in step (2) to the oil phase obtained in step (3) at a speed of 8 mL / s, adjust the stirring speed to 300 rpm, so that the water phase is dispersed in the oil phase, and water phase droplets with a particle size of about 50 μm can be formed. Heat to 70℃ and then stir at a constant temperature for 2 hours to obtain dextran microspheres.

[0122] (5) Transfer the dextran microspheres to a sand core funnel and wash them with 1L of toluene each time for 8-10 times. Then wash them with 1L of acetone for 8-10 times to remove the white oil and surfactant on the dextran microspheres. Finally, wash them with 20L of deionized water to remove the residual acetone.

[0123] (6) Agarose microspheres were sieved using sieves with pore sizes of 25 μm and 75 μm to obtain dextran microspheres with a particle size range of 25-75 μm, and stored in a 20% ethanol solution.

[0124] Example 6

[0125] This embodiment provides a method for preparing a gel filter medium, the specific steps and parameters of which are as follows:

[0126] (1) Synthesis of allyl dextran: Weigh 100g dextran T500, 500g water, 72g sodium hydroxide and 7.2g potassium borohydride and mix them. Stir at 60℃ for 2h to completely dissolve the above materials to obtain a dextran solution.

[0127] Add 100g of allyl bromide to the above dextran solution and react at 60°C for 6 hours. After the reaction is complete, immediately add 100g of glacial acetic acid.

[0128] Then wash with anhydrous ethanol, remove the supernatant, dissolve in ultrapure water, and repeat washing 8-10 times to obtain sugar solution;

[0129] The sugar solution was freeze-dried to obtain allyl dextran powder. The freeze-drying conditions were: pretreatment at -60℃ for 12 hours, followed by treatment in a vacuum freeze dryer for 72 hours, so that the moisture content of allyl dextran was below 2%.

[0130] (2) Preparation of aqueous phase: Mix 10g allyl dextran dry powder, 8g MBA, 40g deionized water and 32g DMF, and dissolve at a constant temperature of 50℃ for 4h. After the above raw materials are completely dissolved, add 0.5g ammonium persulfate to obtain the aqueous phase.

[0131] (3) Preparation of oil phase: Mix 500g white oil, 15g Span 85 and 7.5g Tween 60, heat to 60℃, stir at 500rpm for 1h, add 0.1g AIBN and stir evenly, let stand for 30min to obtain oil phase.

[0132] (4) According to the volume ratio of water phase to oil phase 1:5, the water phase obtained in step (2) is added to the oil phase obtained in step (3) at a speed of 12 mL / s. The rotation speed is adjusted to 300 rpm so that the water phase is dispersed in the oil phase, which can form water phase droplets with a particle size of about 50 μm. The temperature is raised to 70 °C and then stirred at a constant temperature for 2 h to obtain dextran microspheres.

[0133] (5) Transfer the dextran microspheres to a sand core funnel and wash them with 1L of toluene each time for 8-10 times. Then wash them with 1L of acetone for 8-10 times to remove the white oil and surfactant on the dextran microspheres. Finally, wash them with 20L of deionized water to remove the residual acetone.

[0134] (6) Agarose microspheres were sieved using sieves with pore sizes of 25 μm and 75 μm to obtain dextran microspheres with a particle size range of 25-75 μm, and stored in a 20% ethanol solution.

[0135] Example 7

[0136] This embodiment provides a method for preparing a gel filter medium, the specific steps and parameters of which are as follows:

[0137] (1) Synthesis of allyl dextran: Weigh 100g dextran (molecular weight 1000), 500g water, 36g potassium hydroxide and 1g sodium borohydride and mix them. Stir at 50℃ for 2.5h to completely dissolve the above materials to obtain a dextran solution.

[0138] Add 2g allyl chloride and 8g allyl glycidyl ether to the above dextran solution and react at 30°C for 10h. After the reaction is complete, immediately add 50g glacial acetic acid.

[0139] Wash with anhydrous ethanol, remove the supernatant, then dissolve in ultrapure water. Repeat the washing steps 8-10 times to obtain the sugar solution.

[0140] The sugar solution was freeze-dried to obtain allyl dextran powder. The freeze-drying conditions were: pretreatment at -60℃ for 12 hours, followed by treatment in a vacuum freeze dryer for 72 hours, so that the moisture content of allyl dextran was below 2%.

[0141] (2) Preparation of aqueous phase: Mix 5g allyl dextran dry powder, 2g MBA, 23g deionized water and 10g N,N'-dimethylacetamide, and dissolve at a constant temperature of 50℃ for 4h. After the above raw materials are completely dissolved, add 0.5g potassium persulfate to obtain the aqueous phase.

[0142] (3) Preparation of oil phase: 500g of No. 7 white oil, 1g of NP4, and 4g of propylene glycol monofatty acid ester are heated to 50℃ and stirred at 500rpm for 1h. Then, 0.1g of diisolactone peroxide is added and mixed. The mixture is stirred evenly to obtain the oil phase.

[0143] (4) According to the volume ratio of water phase to oil phase 1:2, add the water phase obtained in step (2) to the oil phase obtained in step (3) at a speed of 10 mL / s, adjust the rotation speed to 100 rpm, so that the water phase is dispersed in the oil phase, and water phase droplets with a particle size of about 50 μm can be formed. Heat to 80 °C and then stir at a constant temperature for 1 h to obtain dextran microspheres.

[0144] (5) Transfer the dextran microspheres to a sand core funnel and wash them with 1L of toluene each time for 8-10 times. Then wash them with 1L of acetone for 8-10 times to remove the white oil and surfactant on the dextran microspheres. Finally, wash them with 20L of deionized water to remove the residual acetone.

[0145] (6) Agarose microspheres were sieved using sieves with pore sizes of 25 μm and 75 μm to obtain dextran microspheres with a particle size range of 25-75 μm, and stored in a 20% ethanol solution.

[0146] Example 8

[0147] This embodiment provides a method for preparing a gel filter medium, the specific steps and parameters of which are as follows:

[0148] (1) Synthesis of allyl dextran: Weigh 100g of dextran (molecular weight 2×10⁻⁶) 5 Mix 500g of water, 36g of sodium hydroxide, and 20g of lithium borohydride, and stir at 60℃ for 2 hours to completely dissolve the above materials to obtain a dextran solution.

[0149] Add 200g allylamine and 300g allyl glycidyl ether to the above dextran solution and react at 80℃ for 1h. After the reaction is completed, immediately add 50g glacial acetic acid.

[0150] Wash with anhydrous ethanol, remove the supernatant, then dissolve in ultrapure water. Repeat the washing steps 8-10 times to obtain the sugar solution.

[0151] The sugar solution was freeze-dried to obtain allyl dextran powder. The freeze-drying conditions were: pretreatment at -60℃ for 12 hours, followed by treatment in a vacuum freeze dryer for 72 hours, so that the moisture content of allyl dextran was below 2%.

[0152] (2) Preparation of aqueous phase: Mix 30g allyl dextran dry powder, 5g MBA, 7g N,N'-dimethylformamide and 8g dimethyl sulfoxide, and dissolve at a constant temperature of 50℃ for 4h. After the above raw materials are completely dissolved, add 0.5g azobisisobutyramidine hydrochloride to obtain the aqueous phase.

[0153] (3) Preparation of oil phase: Mix 500g of No. 15 white oil, 25g of NP10 and 25g of ethylene glycol monostearate, heat to 80℃, stir at 500rpm for 1h, add 0.1g of azobisisobutyronitrile and mix evenly to obtain oil phase.

[0154] (4) According to the volume ratio of water phase to oil phase 1:10, the water phase obtained in step (2) is added to the oil phase obtained in step (3) at a speed of 10 mL / s. The rotation speed is adjusted to 800 rpm so that the water phase is dispersed in the oil phase, which can form water phase droplets with a particle size of about 50 μm. The temperature is raised to 50 °C and then stirred at a constant temperature for 20 h to obtain dextran microspheres.

[0155] (5) Transfer the dextran microspheres to a sand core funnel and wash them with 1L of toluene each time for 8-10 times. Then wash them with 1L of acetone for 8-10 times to remove the white oil and surfactant on the dextran microspheres. Finally, wash them with 20L of deionized water to remove the residual acetone.

[0156] (6) Agarose microspheres were sieved using sieves with pore sizes of 25 μm and 75 μm to obtain dextran microspheres with a particle size range of 25-75 μm, and stored in a 20% ethanol solution.

[0157] Example 9

[0158] This embodiment provides a method for preparing a gel filter medium, the specific steps and parameters of which are as follows:

[0159] (1) Synthesis of allyl dextran: Weigh 100g of dextran (molecular weight 2×10⁻⁶) 7 Mix 500g water, 36g sodium hydroxide, and 3.6g potassium borohydride, and stir at 60℃ for 2 hours to completely dissolve the above materials to obtain a dextran solution.

[0160] Add 20g diallylamine and 80g allyl glycidyl ether to the above dextran solution and react at 60℃ for 4h. After the reaction is completed, immediately add 50g glacial acetic acid.

[0161] Wash with anhydrous ethanol, remove the supernatant, then dissolve in ultrapure water. Repeat the washing steps 8-10 times to obtain the sugar solution.

[0162] The sugar solution was freeze-dried to obtain allyl dextran powder. The freeze-drying conditions were: pretreatment at -60℃ for 12 hours, followed by treatment in a vacuum freeze dryer for 72 hours, so that the moisture content of allyl dextran was below 2%.

[0163] (2) Preparation of aqueous phase: Mix 10g allyl dextran dry powder, 50g MBA, 58g deionized water and 42g N,N'-dimethylethanolamine, and dissolve at 50℃ for 4h. After the above raw materials are completely dissolved, add 0.5g ammonium persulfate to obtain the aqueous phase.

[0164] (3) Preparation of oil phase: Mix 500g of No. 26 white oil, 15g of oleic acid and 7.5g of PEG-25 propylene glycol stearate, heat to 60℃, stir at 500rpm for 1h, add 50g of benzoyl peroxide and mix, stir evenly to obtain oil phase.

[0165] (4) According to the volume ratio of water phase to oil phase 1:8, the water phase obtained in step (2) is added to the oil phase obtained in step (3) at a speed of 10 mL / s. The rotation speed is adjusted to 500 rpm so that the water phase is dispersed in the oil phase, which can form water phase droplets with a particle size of about 50 μm. The temperature is raised to 70 °C and then stirred at a constant temperature for 8 h to obtain dextran microspheres.

[0166] (5) Transfer the dextran microspheres to a sand core funnel and wash them with 1L of toluene each time for 8-10 times. Then wash them with 1L of acetone for 8-10 times to remove the white oil and surfactant on the dextran microspheres. Finally, wash them with 20L of deionized water to remove the residual acetone.

[0167] (6) Agarose microspheres were sieved using sieves with pore sizes of 25 μm and 75 μm to obtain dextran microspheres with a particle size range of 25-75 μm, and stored in a 20% ethanol solution.

[0168] Example 10

[0169] This embodiment provides a method for preparing a gel filter medium. The specific steps and parameters are the same as in Example 1. The difference is that in step (3), the oil phase is prepared by mixing 500g of No. 3 white oil, 15g of propylene glycol monolaurate and 15g of polyoxyethylene stearate, heating to 60°C, stirring at 500rpm for 1h, and then letting it stand for 30min after stopping the stirring to obtain the oil phase.

[0170] Example 11

[0171] This embodiment provides a method for preparing a gel filter medium. The specific steps and parameters are the same as in Example 1. The difference is that in step (3), the oil phase is prepared by mixing 500g of white oil, 15g of fatty alcohol polyoxyethylene ether and 15g of alkoxy polyethylene hydroxy ethanol, heating to 60°C, stirring at 500 rpm for 1 hour, and then letting it stand for 30 minutes after stopping the stirring to obtain the oil phase.

[0172] Example 12

[0173] This embodiment provides a method for preparing a gel filter medium. The specific steps and parameters are the same as in Example 1. The difference is that in step (3), the oil phase is prepared by mixing 500g of No. 3 white oil and 30g of sorbitan sesquioleate, heating to 60°C, stirring at 500rpm for 1h, and then letting it stand for 30min after stopping the stirring to obtain the oil phase.

[0174] Example 13

[0175] This embodiment provides a method for preparing a gel filter medium. The specific steps and parameters are the same as in Example 1. The difference is that in step (3), the oil phase is prepared by mixing 500g of No. 3 white oil and 30g of glyceryl monostearate, heating to 60°C, stirring at 500rpm for 1h, and then letting it stand for 30min after stopping the stirring to obtain the oil phase.

[0176] Example 14

[0177] This embodiment provides a method for preparing a gel filter medium. The specific steps and parameters are the same as in Example 1. The difference is that in step (2), the aqueous phase is prepared by mixing 10g of allyl dextran powder, 8g of N,N'-methylenebisacrylamide (MBA), and 72g of methanol and dissolving them at a constant temperature of 50°C for 4 hours. After the above raw materials are completely dissolved, 0.1g of ammonium persulfate is added to obtain the aqueous phase.

[0178] Comparative Example 1

[0179] This comparative example provides a method for preparing a gel filter medium. The specific steps and parameters are the same as in Example 1. The difference is that toluene of equal mass is used instead of white oil. That is, in step (3), the oil phase is prepared by mixing 500g toluene, 15g Span 85 and 7.5g Tween 60, heating to 60°C, stirring at 500rpm for 1h, and then letting it stand for 30min after stopping stirring to obtain the oil phase.

[0180] Comparative Example 2

[0181] This comparative example provides a method for preparing a gel filter medium. The specific steps and parameters are the same as in Example 1. The difference is that an equal mass of liquid paraffin is used to replace white oil. That is, in step (3), the oil phase is prepared by mixing 500g of liquid paraffin, 15g of Span 85 and 7.5g of Tween 60, heating to 60°C, stirring at 500rpm for 1h, and then letting it stand for 30min after stopping stirring to obtain the oil phase.

[0182] Comparative Example 3

[0183] This comparative example provides a method for preparing a gel filter medium. The specific steps and parameters are the same as in Example 1. The difference is that carbon tetrachloride of equal mass is used instead of white oil. That is, in step (3), the oil phase is prepared by mixing 500g carbon tetrachloride, 15g Span 85 and 7.5g Tween 60, heating to 60°C, stirring at 500rpm for 1h, and then letting it stand for 30min after stopping stirring to obtain the oil phase.

[0184] Experimental Example

[0185] The pressure resistance flow rate, non-specific adsorption capacity, and resolution of the gel filter media prepared in Examples 1-14 and Comparative Examples 1-3 were tested.

[0186] The non-specific adsorption capacity detection method is as follows:

[0187] Equipment: Peristaltic pump, 10mm×200mm glass column, protein and nucleic acid detector, ultraviolet spectrophotometer

[0188] Concentrations of buffer solutions and protein solutions:

[0189] Solution A: A mixture of 2.5 mol / L (NH4)2SO4 and 20 mmol / L PBS, pH 7.0. Preparation method: Weigh 330.35 g of (NH4)2SO4 (MW 132.14), 1.73 g of anhydrous disodium hydrogen phosphate (MW 141.96), and 1.22 g of sodium dihydrogen phosphate dihydrate (MW 156.01). Dissolve in deionized water, adjust pH to 7.0, and bring to a final volume of 1000 mL. Shake well.

[0190] Solution B: 20 ​​mmol / L PBS, pH 7.0. The preparation method is as follows: weigh 1.73 g of anhydrous disodium hydrogen phosphate (MW 141.96) and 1.22 g of sodium dihydrogen phosphate dihydrate (MW 156.01), dissolve in deionized water, adjust the pH to 7.0, and make up to 1000 mL. Shake well.

[0191] Protein sample: Bovine serum albumin (BSA) sample solution, prepared at a concentration of 5 mg / mL. The preparation method is to weigh 0.5 g of bovine serum albumin and dissolve it in 100 mL of solution A.

[0192] Sample loading and elution: Take 2 mL of the gel filter media prepared in Examples 1-14 and Comparative Examples 1-3 respectively and fully diffuse them in deionized water. Place the gel suspension in a 5 mL graduated cylinder and let it stand overnight. Accurately read the volume.

[0193] Stir the settled sample thoroughly and pour it into a 10 mm diameter column. Wash with 30 mL of deionized water and connect to the instrument.

[0194] Wash with solution A at 1 mL / min, allowing the solution to flow slowly through the column until the baseline levels out.

[0195] Introduce the protein sample solution and flow it through the column at a rate of 0.5 mL / min. The baseline will start flat, then slowly rise, and finally level off at the high absorption point.

[0196] Wash with solution A at 1 mL / min until the baseline drops and then levels off.

[0197] Elute with solution B at 1 mL / min, collect the protein eluent in a 100 mL volumetric flask, and observe the baseline decrease until it levels off.

[0198] The absorbance of the eluent was calculated using a UV spectrophotometer at a wavelength of 280 nm. The protein adsorption capacity per unit volume of the gel was then calculated, and the results are shown in Table 1.

[0199] The pressure-resistant flow rate testing method is as follows:

[0200] The gel filtration medium was packed into a column, and pressure resistance flow rate tests were conducted using an Incyte protein purification system with pure water as the mobile phase. The column volume was 120 mL, the column inlet pressure and column pressure difference were set to 0.55 MPa, and the flow rate was gradually increased (e.g., 5 mL / min, 10 mL / min, 15 mL / min up to 60 mL / min). The total equilibration time was 10 min, and the pressure values ​​corresponding to different flow rates were obtained. 0.2 MPa was selected as the pressure value, and the flow rates of the gel filtration media prepared in each example and comparative example were measured. The results are shown in Table 1.

[0201] Resolution detection method:

[0202] 1. Column installation: Install the column according to the "Standard Operating Procedure for Column Installation of XK Series", with a column height of 30±0.5cm.

[0203] 2. Mobile phase:

[0204] A: 20mM PB+0.15M NaCl, pH 7.0.

[0205] Protein solution samples: Prepare sample solutions with final concentrations of 5 mg / ml BSA, 2 mg / ml β-lactoglobulin, 2 mg / ml IgG, 2 mg / ml cytochrome C, and 2 mg / ml cytidine.

[0206] 3. The experiment was conducted using the AKTA system programming. The detection wavelengths were set to 280 nm and 215 nm, the column inlet pressure was 0.5 MPa, and the Delta P was 0.3 MPa. The chromatography column was connected to the system, the flow rate was set to 0.8 mL / min, the mobile phase was equilibrated with 0.5 CV, 0.5 mL of protein solution was loaded, and elution was performed with 1.5 CV. The separation effect of mixed proteins was tested in the mobile phase A buffer system. The results are shown in Table 2.

[0207] Table 1 Performance of Gel Filtration Media

[0208]

[0209]

[0210] Table 2. Retention volume of separated mixed proteins

[0211]

[0212]

[0213] " / " indicates that the parts were not separated.

[0214] As shown in Table 1, compared with the results of Comparative Examples 1-3, the gel filter medium prepared in the embodiments of the present invention can reduce the amount of non-specific adsorption, proving its good separation and purification effect on the target analyte. The oil phase prepared by the present invention using white oil as the organic solvent in the oil phase and combining it with a surfactant results in a gel filter medium with a smooth surface, good pore uniformity, large protein adsorption capacity, fast flow rate, and high resolution. Among them, comparing the data of Examples 1 and Examples 12-13, the oil phase using two surfactants can effectively improve the uniformity of the aqueous phase dispersion in the oil phase compared with the oil phase using only one surfactant. The resulting gel filter medium has a more stable structure and performs better in terms of protein adsorption capacity and flow rate. Comparing the data of Examples 1 and 14, the aqueous phase using two porogens can improve the pore uniformity of the resulting gel filter medium compared with the aqueous phase using only one porogen. The structure is more stable and performs better in terms of non-specific adsorption capacity, flow rate, and pressure resistance.

[0215] As can be seen from the data in Table 2, comparative examples 1-3 could only separate three proteins from a mixed sample of five proteins, while the gel filter medium prepared in the embodiments of the present invention could effectively separate four to five proteins from the mixed protein sample. This proves that the gel filter medium prepared in the embodiments of the present invention has good separation effect, high resolution, and high purity of separated proteins.

[0216] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a gel filtration medium, characterized in that, The method comprises the following steps: S1, mixing allyl dextran, a crosslinking agent and a pore-forming agent to obtain an aqueous phase, wherein the crosslinking agent is N,N'-methylene bisacrylamide; S2, mixing a surfactant and an organic solvent to obtain an oil phase, wherein the organic solvent is white oil; S3, dispersing the aqueous phase obtained in step S1 in the oil phase obtained in step S2 to obtain a gel filtration medium by inverse polymerization.

2. The method for producing a gel filtration medium according to claim 1, characterized by, The viscosity of the white oil in step S2 is 2 to 27.5 mm 2 / s; and / or, The mass ratio of the surfactant to the organic solvent is (1-10):100; and / or, The surfactant comprises a non-ionic surfactant.

3. The method for producing a gel filtration medium according to claim 2, characterized by, In step S2, the white oil comprises at least one of No. 3 white oil, No. 7 white oil, No. 15 white oil and No. 26 white oil; and / or, The surfactant comprises at least one of a hydrophilic surfactant and a lipophilic surfactant; and / or, The preparation method of the oil phase in step S2 further comprises the step of adding an initiator to the oil phase; and / or, The preparation method of the aqueous phase in step S1 further comprises the step of adding an initiator to the aqueous phase.

4. The method for producing a gel filtration medium according to claim 3, characterized by, The initiator in step S2 comprises at least one of an oil-soluble azo initiator and an organic peroxide initiator; and / or, The mass ratio of the initiator to the organic solvent in the oil phase is (0.02-10):100; and / or, The preparation method of the surfactant comprises mixing a hydrophilic surfactant and a lipophilic surfactant; and / or, In step S1, the pore-forming agent comprises at least one of deionized water, an alcohol solvent and an aprotic polar solvent; and / or, The mass concentration of the allyl dextran in the aqueous phase is 5-60%; and / or, The initiator in step S1 comprises at least one of a water-soluble azo initiator and an inorganic peroxide initiator.

5. The method for producing a gel filtration medium according to claim 4, characterized in that, The hydrophilic surfactant comprises at least one of Tween, nonylphenol polyoxyethylene ether, fatty alcohol polyoxyethylene ether and alkoxy polyethylene hydroxy ethanol; and / or, The lipophilic surfactant comprises at least one of Span, nonylphenol polyoxyethylene ether, oleic acid, stearyl methacrylate, propylene glycol monofatty acid ester, ethylene glycol monostearate, PEG-25 propylene glycol stearate, glycerol monostearate, sorbitan sesquioleate, propylene glycol monolaurate and polyoxyethylene stearate; and / or, The oil-soluble azo initiator comprises at least one of azobisisobutyronitrile and azobisisoheptyl nitrile; and / or, The organic peroxide initiator comprises at least one of dibenzoyl peroxide and diisocyanate peroxide; and / or, The water-soluble azo initiator comprises azobisdimethylformamide hydrochloride; and / or, The inorganic peroxide initiator comprises at least one of ammonium persulfate and potassium persulfate; and / or, In step S1, the pore-forming agent comprises at least one of deionized water, methanol, ethanol, dimethyl sulfoxide, N,N'-dimethylformamide, N,N'-dimethylacetamide and N,N'-dimethylethanolamine; and / or, The mass ratio of the crosslinking agent, the pore-forming agent, the initiator and the allyl dextran in the aqueous phase is (0.2-5):(0.5-10):(0.01-0.1):

1.

6. The method for producing a gel filtration medium according to claim 5, characterized in that, The porogen in the step S1 includes at least two of deionized water, methanol, ethanol, dimethyl sulfoxide, N,N'-dimethylformamide, N,N'-dimethylacetamide, N,N'-dimethylethanolamine; and / or, The temperature for mixing the surfactant with the organic solvent in the step S2 is 50-80℃; and / or, In the step S3, the reaction temperature for the reverse phase polymerization is 50-80℃, and the reaction time is 1-20h; and / or, The volume ratio of the oil phase to the water phase is 1:(2-10); and / or, The oil phase and the water phase are mixed under stirring, wherein the stirring speed is 100-800rpm, and the stirring time is 10-15min, Optionally, the water phase is added into the oil phase at a rate of 8-12mL / s to form a mixture of the oil phase and the water phase; and / or, In the step S1, the preparation method of the allyl dextran includes coupling reaction of a double-bond-containing compound with a dextran solution, drying to obtain the allyl dextran, Optionally, the reaction temperature for coupling of the double-bond-containing compound with the dextran solution is 30-80℃, and the reaction time is 1-10h, Optionally, the double-bond-containing compound includes an allyl-containing organic compound.

7. The method of producing a gel filtration medium according to claim 6, wherein The preparation method of the dextran solution includes the step of mixing dextran and a reducing agent in an alkaline environment, Optionally, the reducing agent includes a metal hydride, Optionally, the molecular weight of the dextran is 1000 to 2 x 10 7 ; and / or, The allyl-containing organic compound includes at least one of allyl glycidyl ether, allyl bromide, allyl chloride, allyl amine, and diallyl amine.

8. The method for producing a gel filtration medium according to claim 7, characterized by, The metal hydride includes at least one of potassium borohydride, sodium borohydride, and lithium borohydride; and / or, The mass ratio of the dextran to the reducing agent is 100:(1-20); and / or, The mass ratio of the double-bond-containing compound to the dextran is (0.1-5):1; and / or, The preparation method of the gel filtration medium further includes the steps of washing and sieving the obtained gel filtration medium, Optionally, the step of washing includes sequentially washing the gel filtration medium with toluene, acetone, and deionized water.

9. A gel filtration medium, characterized in that, The gel filtration medium is prepared by the preparation method of the gel filtration medium according to any one of claims 1-8.

10. Use of the gel filtration medium according to claim 9 in protein separation and purification.

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