High-rigidity high-flow-velocity polysaccharide gel microspheres, preparation method and application

Abstract

The invention provides a high-rigidity high-flow-velocity polysaccharide gel microsphere, a preparation method and application. The method comprises the following steps: S1, dispersing polysaccharide powder in water for heterogeneous modification; s2, emulsifying and pelletizing after dissolving the modified polysaccharide; and S3, activation and crosslinking of the modified polysaccharide gel microspheres. According to the method, heterogeneous modification is adopted, a certain amount of cross-linking agent is introduced in advance, after curing and balling, high-rigidity microspheres are formed through cross-linking, the linear flow velocity of the microspheres is 4000 cm / h or above, and the microspheres have high flow velocity and good mechanical strength and can meet the requirements of large-scale separation and purification; besides, heterogeneous modification is adopted, so that the process step of introducing a cross-linking agent in advance is simplified, modification sites are uniformly distributed due to heterogeneous modification, the modification degree is favorably controlled, and a rigid framework is favorably formed in the cross-linking process; according to the method provided by the invention, the cross-linking reaction can be continuously carried out, the process complexity is reduced, and the method has the characteristics of simplicity and easiness in operation and is easy to amplify for large-scale industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of microsphere preparation, relates to chromatography media, and particularly to a high-rigidity and high-flow-rate polysaccharide gel microsphere, a preparation method and application thereof. Background Art

[0002] Natural polysaccharide microspheres with loose porosity, strong hydrophilicity and good biocompatibility, such as cellulose, agarose, and dextran microspheres, are widely used in the purification of biological molecules such as proteins. However, what limits their expansion of application is that they have low mechanical strength and are difficult to withstand high pressure.

[0003] There are many methods for cross-linking reactions. The cross-linking agent generally selects compounds with dual active groups (such as epichlorohydrin, 2,3-dibromopropanol, butanediol diglycidyl ether, etc.). The cross-linking process can be carried out in one step or in multiple steps. These methods have improved the mechanical strength of polysaccharide microspheres to a certain extent, but with the rapid development of biotechnology, especially the exponential increase in expression, the chromatographic purification process of biological drugs has continuously improved the flow rate and pressure resistance of the chromatographic medium. The technical iteration speed of the chromatographic medium is slow. The current mainstream products have been used for decades, and the performance of the chromatographic medium can no longer meet the production needs of the biopharmaceutical industry.

[0004] In order to further improve the mechanical strength of polysaccharide microspheres, U.S. Patent No. 6602990B1 discloses a method for preparing a novel highly cross-linked polysaccharide microspheres, wherein a special cross-linking agent is introduced before emulsification into balls, wherein one end of the cross-linking agent has an active group and the other end has an inert group (such as allyl hydrophobic glyceryl ether), and the inert group is activated after balling to complete the internal cross-linking of the microspheres. However, the special cross-linking agent introduced in the method is too high, which will damage the skeleton structure of the polysaccharide microspheres, causing the microspheres to shrink and deform. Chinese Patent CN103769057A effectively avoids the deformation of the microspheres by mixing the polysaccharide modified by the special cross-linking agent with the unmodified polysaccharide. However, the drying and other processes of the modified polysaccharide in the method increase the complexity of the process.

[0005] The heterogeneous modification technology of polysaccharides refers to the structural modification of polysaccharide powder or fiber before the polysaccharide is dissolved, so as to pre-endow the polysaccharide with corresponding properties. Before the polysaccharide is dissolved, it is in a curled or coated state, and only part of the hydroxyl groups on the surface are effectively activated. By adding corresponding reagents, this part of the hydroxyl groups is modified, and functional groups are pre-introduced into the polysaccharide. The modified polysaccharide prepared in this way has both the gel properties of the polysaccharide itself (the unmodified part inside is easy to form a porous network structure connected by hydrogen bonds), and the special functions of the corresponding introduced groups (the modified part outside can be structurally designed). Summary of the invention

[0006] The first object of the present invention is to provide a method for preparing high-rigidity and high-flow-rate polysaccharide gel microspheres, in order to achieve the performance of high pressure resistance, high flow rate and low back pressure.

[0007] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] A method for preparing high-rigidity and high-flow-rate polysaccharide gel microspheres, characterized in that the method comprises the following steps:

[0009] S1. Dispersing polysaccharide powder in water for heterogeneous modification:

[0010] Disperse 2 to 12 parts of polysaccharide powder in 100 parts of water (to form a polysaccharide suspension, not a solution), heat to 30 to 60° C. to keep the polysaccharide suspended, without dissolving or aggregating, add 1 to 10 parts of a 33 wt% alkaline solution, slowly drop 1 to 5 parts of a bifunctional crosslinking agent, react for 1 to 24 hours, adjust the pH to neutral, and terminate the reaction;

[0011] The reaction time is preferably 2 to 6 hours;

[0012] Polysaccharides are natural polysaccharides, typical examples of which are cellulose, agarose, dextran, etc.

[0013] The bifunctional cross-linking agent comprises at least one active functional group and at least one inert functional group, wherein the active functional group has the activity of reacting with the hydroxyl group on the polysaccharide, and the inert functional group can form a self-cross-linking reaction with the hydroxyl group on the polysaccharide after activation;

[0014] S2. Emulsification and balling after dissolving modified polysaccharide:

[0015] The neutralized modified polysaccharide dispersion is heated and dissolved to form a clear solution, cooled to not less than 50°C, and kept warm as a water phase for later use; the water phase is poured into the oil phase heated to 50-70°C, stirred and emulsified, so that the water phase is dispersed into droplets of a desired particle size, and the emulsion is cooled so that the water phase droplets form gel microspheres, thereby obtaining modified polysaccharide gel microspheres;

[0016] The oil phase comprises an inert solvent and an emulsifier, and the inert solvent is immiscible with water;

[0017] The emulsifier is a nonionic surfactant; the emulsifier is a single emulsifier or a compound emulsifier with an HLB value of 3 to 8;

[0018] S3. Activation and cross-linking of modified polysaccharide gel microspheres:

[0019] S31, activation of modified polysaccharide gel microspheres: dispersing the modified polysaccharide gel microspheres in pure water, adding an activation reagent, activating the inert functional groups in the microspheres (the inert functional groups on the bifunctional cross-linking agent), and obtaining activated polysaccharide gel microspheres;

[0020] S32, self-crosslinking of activated polysaccharide gel microspheres: under alkaline conditions, the activated functional groups on the polysaccharide gel microspheres undergo self-crosslinking reaction with hydroxyl groups to obtain self-crosslinked microspheres;

[0021] S33, using epichlorohydrin to cross-link polysaccharide gel microspheres: under alkaline conditions, using a cross-linking agent to cross-link the polysaccharide gel microspheres, the volume amount of the cross-linking agent is 1-20% of the volume of the microspheres, to obtain polysaccharide gel microspheres with high rigidity and high flow rate;

[0022] The cross-linking agent comprises at least two active functional groups, and the active functional groups have the activity of reacting with the hydroxyl groups on the polysaccharide;

[0023] Alternatively, step S33 is completed first, and then steps S31 and S32 are completed.

[0024] Alternatively, after step S31 is completed, step S32 and step S33 can be performed continuously. Continuous performance means that after completion, there is no need to wash the microspheres or replace the reaction solution, and the drugs or conditions required for the next step can be directly added or implemented in the original reactor.

[0025] Alternatively, step S31 and step S32 may be performed continuously, and then step S33 is completed.

[0026] Alternatively, step S33 is completed first, and steps S31 and S32 can be performed continuously.

[0027] Alternatively, step S31, step S32, and step S33 may be performed continuously.

[0028] High rigidity means that the microspheres are not easily deformed in a way that would affect their performance, and can be used for separation and purification at flow rates greater than 2000 cm / h, with a maximum flow rate of more than 4000 cm / h.

[0029] While adopting the above technical solutions, the present invention may also adopt or combine the following technical solutions:

[0030] As a preferred technical solution of the present invention: in step S1, the weight of the alkaline solution is preferably 1 to 4 parts. The alkali is preferably sodium hydroxide or potassium hydroxide. - The concentration is 0.5~5M. NaBH4 can be added to prevent polysaccharides from cleaving under alkaline conditions.

[0031] As a preferred technical solution of the present invention: in step S1, the polysaccharide is preferably at least one of agar, agarose, cellulose and dextran, more preferably agarose. It can swell in an alkaline dispersion at a relatively low temperature, thus facilitating heterogeneous modification. It can dissolve after further heating and form a porous gel-like substance after cooling.

[0032] As a preferred technical solution of the present invention: in step S1, the bifunctional cross-linking agent is allyl bromide or allyl glycidyl ether, preferably allyl glycidyl ether.

[0033] As a preferred technical solution of the present invention: in step S1, the amount of the bifunctional cross-linking agent added is 0.1% to 40% of the polysaccharide suspension, and more preferably 2 to 15%.

[0034] As a preferred technical solution of the present invention: in step S2, the heating temperature of the modified polysaccharide dispersion is preferably 95°C.

[0035] As a preferred technical solution of the present invention: in step S2, the emulsifier is an oil-soluble emulsifier, a Span type or a Tween type, preferably Span 80.

[0036] The concentration of the emulsifier is 0.5 to 10% (w / v), preferably 1 to 6% (w / v). The concentration of the emulsifier refers to the ratio of the mass of the emulsifier to the volume of the oil phase.

[0037] As a preferred technical solution of the present invention: in step S2, the oil phase is a mixture of one or more of liquid paraffin, cyclohexane, petroleum ether, and cottonseed oil, preferably cyclohexane.

[0038] As a preferred technical solution of the present invention: in step S2, when the polysaccharide is agarose, a cooling solidification method is selected, and when the polysaccharide is dextran, a cross-linking solidification method is selected.

[0039] As a preferred technical solution of the present invention: in step S31, the activation reagent is a bromination reagent, preferably bromine water or N-bromosuccinimide.

[0040] The amount of bromine water added is: add bromine water dropwise until the solution turns yellow, and keep it for 1 minute without changing color. After the reaction is completed, add an appropriate amount of sodium formate to remove excess bromine water.

[0041] As a preferred technical solution of the present invention: in step S31, 100 mL of water contains 10 to 100 g of microspheres, preferably 50 g of microspheres.

[0042] As a preferred technical solution of the present invention: in step S32, the cross-linking agent is epichlorohydrin or 1,4-butanediol diglycidyl ether or divinyl sulfone or 2,3-dibromopropanol.

[0043] As a preferred technical solution of the present invention: in step S32, the alkaline condition is OH - The concentration is 0.05 to 5 M, preferably 0.25 to 2.5 M. NaBH4 may be added to prevent polysaccharide from decomposing under alkaline conditions.

[0044] As a preferred technical solution of the present invention: in step S32, the reaction temperature is 25-60°C and the reaction time is 3-20 hours.

[0045] The second object of the present invention is to provide a polysaccharide gel microsphere with high rigidity and high flow rate.

[0046] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0047] A high-rigidity and high-flow-rate polysaccharide gel microsphere, characterized in that the high-rigidity and high-flow-rate polysaccharide gel microsphere is prepared by the preparation method of the high-rigidity and high-flow-rate polysaccharide gel microsphere described above.

[0048] The third object of the present invention is to provide a chromatography medium.

[0049] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0050] A chromatography medium, characterized in that: the chromatography medium is obtained by modifying the high-rigidity and high-flow rate polysaccharide gel microspheres mentioned above with ligands, for example: connecting different functional groups to prepare hydrophobic media, ion exchange media, metal chelate media, and affinity media as chromatographic separation fillers.

[0051] Another object of the present invention is to provide a use of the chromatography medium described above.

[0052] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0053] Based on the application of chromatographic media in the separation and purification of biomacromolecules as described above.

[0054] The invention provides a high-rigidity and high-flow-rate polysaccharide gel microsphere, a preparation method and an application thereof. The invention adopts heterogeneous modification, introduces a certain amount of cross-linking agent in advance, solidifies into a ball, and then forms a high-rigidity microsphere through cross-linking. The linear flow rate of the microsphere is above 4000 cm / h. The microsphere has a high flow rate and good mechanical strength, and can meet the needs of large-scale separation and purification. In addition, the heterogeneous modification is adopted to simplify the process steps of pre-introducing the cross-linking agent, and the heterogeneous modification makes the modified sites evenly distributed, which is conducive to controlling the degree of modification and forming a rigid skeleton during the cross-linking process. In the method provided by the invention, the cross-linking reaction can be carried out continuously, which reduces the process complexity, has the characteristics of being simple and easy to operate, and is easy to be scaled up for large-scale industrial production. The heterogeneous modification will not completely destroy the hydroxyl structure of the polysaccharide, is conducive to the formation of a hydrogen bond skeleton during the gelation process, and effectively avoids the deformation and shrinkage of the microsphere. After cross-linking, the obtained microsphere not only retains the performance advantages of natural polysaccharides, but also can withstand a high operating flow rate, and is an ideal industrial chromatography medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is a microscope photo of the high-rigidity agarose microspheres prepared in Example 1.

[0056] Figure 2 This is a microscope photo of the high-rigidity agarose microspheres prepared in Example 2.

[0057] Figure 3 The pressure / flow rate characteristic curves of the microspheres prepared in Examples 1-5 and the microspheres in Comparative Examples 1 and 2.

[0058] Figure 4 The pressure / flow rate characteristic curve of the intermediate product in the microsphere cross-linking process, wherein: intermediate product 1 is an uncross-linked microsphere, intermediate product 2 is a microsphere that has only undergone self-cross-linking, and intermediate product 3 is a microsphere that has only undergone epichlorohydrin cross-linking. DETAILED DESCRIPTION

[0059] The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

[0060] Example 1 Preparation of high-rigidity agarose microspheres by heterogeneous modification method, step-by-step cross-linking

[0061] (a) Heterogeneous modification of agarose

[0062] Weigh 7g of agarose powder, disperse it in 96mL of water, and make agarose suspension by mechanical stirring. Heat to 45℃, add 1.5mL of 33% (mass fraction) NaOH, 0.1g of sodium borohydride, stir for 30min, slowly add 3mL of allyl glycidyl ether (0.5h to complete), and continue the reaction for 4h after the addition is complete. After the reaction time is reached, use HCl (6M) to adjust the pH to 7-8 to terminate the reaction.

[0063] (b) Dissolve and emulsify to make balls

[0064] In the original reactor, the modified agarose suspension obtained in step (a) is directly heated to 95°C to dissolve it. After complete dissolution, the temperature is lowered to 85°C and kept warm, and poured into 200mL cyclohexane containing 2g of Span 80 for emulsification for 10min, and the emulsification speed is 800rpm. After the emulsification is completed, the temperature is lowered to below 20°C at a rate of 2°C / min and maintained for 30min to solidify the agarose into balls. After filtering out the oil phase, it is washed with 3 volumes of ethanol and 10 volumes of pure water in turn (the cleaning of the microspheres can be observed under a microscope during the process, and ethanol and pure water can be appropriately increased or decreased according to the actual situation) to obtain agarose microspheres modified with allyl groups, and obtain intermediate product 1.

[0065] (c) Activation of allyl agarose microspheres

[0066] Disperse 100g of allyl agarose microspheres in 100mL of pure water and add 5g of sodium acetate. After stirring for 10min, add bromine water until the solution turns yellow and does not fade for 1min. After continuing the reaction for 20min, add an appropriate amount of sodium formate to remove excess bromine water. After the reaction is completed, wash the microspheres with a large amount of pure water until they are neutral.

[0067] (d) Self-crosslinking of activated microspheres

[0068] 100 g of activated allyl agarose microspheres were drained and dispersed in 100 mL of pure water. 10 g of anhydrous sodium sulfate was added and stirred for 20 min to dissolve. Then, 12 mL of 33% (mass fraction) NaOH and 0.1 g of sodium borohydride were added. The mixture was heated to 40° C. and stirred for 16 h. After the reaction was completed, the microspheres were washed with a large amount of pure water until neutral to obtain intermediate product 2.

[0069] (e) Epichlorohydrin crosslinking

[0070] Disperse 100g of self-crosslinked allyl agarose microspheres in 33mL pure water, heat to 40°C, add 45g of anhydrous sodium sulfate, stir and dissolve for 1h, add 2mL of 33% (mass fraction) NaOH, 0.1g of sodium borohydride, continue stirring for 30min, heat to 50°C, and simultaneously add 10mL of epichlorohydrin and 12mL of 33% (mass fraction) NaOH over 3h. After the addition is complete, continue the reaction for 16h. After the reaction is completed, wash the microspheres with a large amount of pure water until they are neutral.

[0071] The microspheres were sieved with 100-mesh and 250-mesh sieves to an average particle size of about 90 μm, and their pressure / flow rate characteristics were measured by the following method.

[0072] Pressure / Flow Rate Characteristic Test of Microspheres

[0073] Instrument: SCG-100 protein chromatography system protein purification instrument

[0074] Chromatography column: cytivaTricorn 10 / 100 Column

[0075] Mobile phase: pure water

[0076] Test: Load 8mL of microspheres into the chromatography column, start the flow rate from 0.5mL / min, and detect the pressure; gradually increase the flow rate every 5 minutes until the system pressure rises sharply to 3MPa, indicating that the sample collapses and the flow rate cannot continue to increase, and the test is ended. The order of increasing flow rate is 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 12.0, 14.0, 16.0, 18.0, ..., 66.0mL / min. The volume flow rate is converted into linear velocity: V = (60×V_v) / S, V is the linear velocity (cm / h), V_v is the volume flow rate (mL / min), and S is the cross-sectional area of ​​the chromatography column 0.785cm 2 The flow rate and column pressure in the last section of stable pressure before the pressure rises sharply are defined as the maximum flow rate and pressure resistance of the microspheres.

[0077] The average particle size of the microspheres in Example 1 is 87 μm, the maximum flow rate is above 5000 cm / h, and the pressure resistance is above 0.69 MPa.

[0078] Example 2 Preparation of high-rigidity agarose microspheres by heterogeneous modification method, continuous cross-linking (steps c, d, e are continuous)

[0079] (a) Heterogeneous modification of agarose

[0080] The method is the same as that of Example 1 (a).

[0081] (b) Emulsification and balling after dissolution

[0082] The method is the same as that of Example 1 (b).

[0083] (c) Activation of allyl agarose microspheres

[0084] Disperse 100g of allyl agarose microspheres after draining water in 15mL of pure water, add 2g of sodium acetate, stir for 10min, add bromine water dropwise until the solution turns yellow and does not fade for 1min, continue to react for 20min, and then add appropriate amount of sodium formate to remove excess bromine water.

[0085] (d) Self-crosslinking of activated microspheres

[0086] To the reaction system obtained in step (c), add 5 g of anhydrous sodium sulfate, stir and dissolve for 20 min, then add 4 mL of 33% (mass fraction) NaOH and 0.1 g of sodium borohydride, heat to 40° C., and stir to react for 16 h.

[0087] (e) Epichlorohydrin crosslinking

[0088] Keeping at 40°C, add 40g of anhydrous sodium sulfate to the reaction system obtained in step (d), stir and dissolve for 1h, then heat to 50°C, and simultaneously drop 10mL of epichlorohydrin and 12mL of 33% (mass fraction) NaOH over 3h. After the dropwise addition is completed, continue the reaction for 16h. After the reaction is completed, wash the microspheres with a large amount of pure water until they are neutral.

[0089] The average particle size of the microspheres in Example 2 is 86 μm, the maximum flow rate is 4300 cm / h, and the pressure resistance is 0.84 MPa.

[0090] Example 3 Preparation of high-rigidity agarose microspheres by heterogeneous modification, partial continuous crosslinking 1 (steps d and e are continuous)

[0091] (a) Heterogeneous modification of agarose

[0092] The method is the same as that of Example 1 (a).

[0093] (b) Dissolve and emulsify to make balls

[0094] The method is the same as that of Example 1 (b).

[0095] (c) Activation of allyl agarose microspheres

[0096] Disperse 100g of allyl agarose microspheres in 100mL of pure water and add 5g of sodium acetate. After stirring for 10min, add bromine water until the solution turns yellow and does not fade for 1min. After continuing the reaction for 20min, add an appropriate amount of sodium formate to remove excess bromine water. After the reaction is completed, wash the microspheres with a large amount of pure water until they are neutral.

[0097] (d) Self-crosslinking of activated microspheres

[0098] 100 g of activated allyl agarose microspheres were drained and dispersed in 33 mL of pure water. 5 g of anhydrous sodium sulfate was added and stirred to dissolve for 20 min. Then, 4 mL of 33% (mass fraction) NaOH and 0.1 g of sodium borohydride were added. The mixture was heated to 40° C. and stirred to react for 16 h.

[0099] (e) Epichlorohydrin crosslinking

[0100] Keeping at 40°C, add 40g of anhydrous sodium sulfate to the reaction system obtained in step (d), stir and dissolve for 1h, then heat to 50°C, and simultaneously drop 10mL of epichlorohydrin and 12mL of 33% (mass fraction) NaOH over 3h. After the dropwise addition is completed, continue the reaction for 16h. After the reaction is completed, wash the microspheres with a large amount of pure water until they are neutral.

[0101] The average particle size of the microspheres in Example 3 is 88 μm, the maximum flow rate is 4700 cm / h, and the pressure resistance is 0.89 MPa.

[0102] Example 4 Preparation of high-rigidity agarose microspheres by heterogeneous modification, partial continuous cross-linking 2 (steps c and d are continuous)

[0103] (a) Heterogeneous modification of agarose

[0104] The method is the same as that of Example 1 (a).

[0105] (b) Dissolve and emulsify to make balls

[0106] The method is the same as that of Example 1 (b).

[0107] (c) Activation of allyl agarose microspheres

[0108] Disperse 100g of allyl agarose microspheres after draining water in 80mL of pure water, add 5g of sodium acetate, stir for 10min, add bromine water dropwise until the solution turns yellow and does not fade for 1min, continue to react for 20min, and then add appropriate amount of sodium formate to remove excess bromine water.

[0109] (d) Self-crosslinking of activated microspheres

[0110] To the reaction system obtained in step (c), add 10 g of anhydrous sodium sulfate, stir and dissolve for 20 min, then add 12 mL of 33% (mass fraction) NaOH and 0.1 g of sodium borohydride, heat to 40°C, and stir to react for 16 h. After the reaction is completed, wash the microspheres with a large amount of pure water until they are neutral.

[0111] (e) Epichlorohydrin crosslinking

[0112] The method is the same as that of Example 1 (e).

[0113] The average particle size of the microspheres in Example 4 is 85 μm, the maximum flow rate is above 5000 cm / h, and the pressure resistance is above 0.42 MPa.

[0114] Example 5 Preparation of high-rigidity agarose microspheres by heterogeneous modification, partial continuous crosslinking 3 (step e in advance)

[0115] (a) Heterogeneous modification of agarose

[0116] The method is the same as that of Example 1 (a).

[0117] (b) Dissolve and emulsify to make balls

[0118] The method is the same as that of Example 1 (b).

[0119] (e) Epichlorohydrin crosslinking

[0120] The method is the same as that of Example 1 (e) to obtain intermediate product 3.

[0121] (c) Activation of allyl agarose microspheres

[0122] Disperse 100g of epichlorohydrin cross-linked allyl agarose microspheres in 80mL of pure water, add 5g of sodium acetate, stir for 10min, add bromine water dropwise until the solution turns yellow and does not fade for 1min, continue to react for 20min, and then add appropriate amount of sodium formate to remove excess bromine water.

[0123] (d) Self-crosslinking of activated microspheres

[0124] To the reaction system obtained in step (c), add 10 g of anhydrous sodium sulfate, stir and dissolve for 20 min, then add 12 mL of 33% (mass fraction) NaOH and 0.1 g of sodium borohydride, heat to 40°C, and stir to react for 16 h. After the reaction is completed, wash the microspheres with a large amount of pure water until they are neutral.

[0125] The average particle size of the microspheres in Example 5 is 86 μm, the maximum flow rate is above 5000 cm / h, and the pressure resistance is above 0.84 MPa.

[0126] Example 6 High Rigidity Agarose Microspheres Applied as Ion Exchange Media

[0127] SP filler for ion exchange media

[0128] 1. Alkalization. Take 30g of the screened microspheres prepared in Example 1 (average particle size of 40μm) into a reaction bottle; add 18mL 33% (mass fraction) NaOH, 0.12g NaBH4, 4.5g Na2SO4 in sequence, and shake at 50℃ for 0.5h;

[0129] 2. Allyl hydrophobic glyceryl ether modification: Then add 15 mL of allyl hydrophobic glyceryl ether and stir the reaction at 50°C for 16 hours; after the reaction, wash with pure water.

[0130] The allyl density was measured to be 136 μmol / mL.

[0131] 3. SP reaction: Take 20g of the allyl microspheres described in step 2, add 6mL of 4M sodium acetate-acetic acid buffer solution (pH=5), 4g of Na2S2O5, and then adjust the pH to 5 with 33% (mass fraction) NaOH, and shake the reaction at 40°C for 16h. After the reaction is completed, wash with pure water.

[0132] The ion exchange capacity was measured to be 169 μmol / mL, and the loading capacity for lysozyme was 101 mg / mL.

[0133] Example 7 High Rigidity Agarose Microspheres Applied as Ion Exchange Media

[0134] Ion exchange media Q filler

[0135] 1. Add 30 g of the sieved microspheres (average particle size of 75 μm) prepared in Example 1 into a reaction bottle; add 9 mL of isopropanol, 3.3 mL of 33% (mass fraction) NaOH, 0.06 g of NaBH4, set the temperature to 45°C, and stir for 1 h.

[0136] 2. Prepare the solution. Take 28.5g of glycidyl trimethylammonium chloride (ETA), add 9mL of isopropanol and 9mL of pure water, and stir to dissolve. Add the prepared ETA solution to the reaction bottle, and control the dripping to be completed in about 4 hours. After the dripping is completed, heat it to 50℃ and continue the reaction for 6 hours.

[0137] 3. Washing. After the reaction is completed, wash repeatedly with a suction filter funnel until the filtrate is neutral and has no odor. Then load the filler into a 50mL gravity column and elute with 5 volumes of 1mol / L sodium chloride solution.

[0138] Its ion exchange capacity was measured to be 120 μmol / mL.

[0139] Example 8 High Rigidity Agarose Microspheres Applied in Hydrophobic Media

[0140] Butyl filler for hydrophobic media

[0141] 1. Take 20g of the screened microspheres prepared in Example 1 (average particle size of 75μm) into a reaction bottle; add 3g of pure water, 10g of 33% (mass fraction) NaOH, 0.01g of NaBH4, and 3.3g of Na2SO4, and stir at 50℃ for 1h.

[0142] 2. Add 2.4 mL of n-butyl glycidyl ether (BGE) and react at 50°C for 20 hours.

[0143] 3. After the reaction is completed, first use a vacuum pump to remove the reaction liquid, and then wash it with anhydrous ethanol and pure water in turn.

[0144] The ligand density was measured to be 55 μmol / mL.

[0145] Example 9 High Rigidity Agarose Microspheres Applied in Hydrophobic Media

[0146] Hydrophobic medium phenyl filler

[0147] 1. Take 20g of the screened microspheres prepared in Example 1 (average particle size of 75μm) into a reaction bottle; add 3.67mL 33% (mass fraction) NaOH, 3.67mL dioxane, 0.067g NaBH4, and stir at 50℃ for 1h.

[0148] 2. Add 2.67 mL of phenyl glycidyl ether dropwise for about 4 hours and react at 50°C for 16 hours.

[0149] 3. After the reaction is completed, first use a vacuum pump to remove the reaction liquid, and then wash it with anhydrous ethanol and pure water in turn.

[0150] The ligand density was measured to be 26 μmol / mL.

[0151] Comparative Example 1: Commercially available Sepharose 6 Fast Flow.

[0152] The average particle size of the microspheres in Comparative Example 1 is 91 μm, the maximum flow rate is 1500 cm / h, and the pressure resistance is 0.31 MPa.

[0153] Comparative Example 2: Commercially available NUPharose 6FF.

[0154] The average particle size of the microspheres in Comparative Example 2 is 91 μm, the maximum flow rate is 1700 cm / h, and the pressure resistance is 0.29 MPa.

[0155] Results and Discussion:

[0156] Figure 3It is the pressure / flow rate characteristic curve of Examples 1 to 5 and Comparative Examples 1 and 2. It can be found that the embodiments of the present invention have three characteristics: 1) The maximum flow rate of the microspheres in the embodiments of the present invention is higher than 4000cm / h, or even above 5000cm / h, which is much higher than the commercially available products of about 1500cm / h and less than 2000cm / h; 2) The pressure resistance of the microspheres in the embodiments of the present invention is 0.7 to 0.9MPa, which is higher than the commercially available products of about 0.30MPa (0.31MPa and 0.29MPa, respectively); 3) The back pressure of the microspheres in the embodiments of the present invention is low, that is, the pressure is lower at the same flow rate, especially the performance of Example 4 is particularly outstanding, at a flow rate of 1000cm / h, the back pressure is less than 0.1MPa. And as Figure 4 As shown, it is shown that the microspheres in Example 4 are far superior to the samples that are not cross-linked or cross-linked only once, showing the advantages of the microspheres provided by the present invention in terms of pressure / flow rate.

[0157] The above-mentioned specific implementation methods are used to explain the present invention and are only preferred embodiments of the present invention, rather than limiting the present invention. Any modifications, equivalent substitutions, improvements, etc. made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A method for preparing high-rigidity and high-flow-rate polysaccharide gel microspheres, characterized in that: The method comprises the following steps: S1. Dispersing polysaccharide powder in water for heterogeneous modification: Disperse 2 to 12 parts of polysaccharide powder in 100 parts of water, heat to 30 to 60°C, add 1 to 10 parts of 33 wt% alkaline solution, slowly drop 1 to 5 parts of a bifunctional crosslinking agent, react for 1 to 24 hours, adjust the pH to neutral, and terminate the reaction; The bifunctional cross-linking agent comprises at least one active functional group and at least one inert functional group, wherein the active functional group has the activity of reacting with the hydroxyl group on the polysaccharide, and the inert functional group can form a self-cross-linking reaction with the hydroxyl group on the polysaccharide after activation; S2. Emulsification and balling after dissolving modified polysaccharide: The neutralized modified polysaccharide dispersion is heated and dissolved to form a clear solution, cooled to not less than 50°C, and kept warm as a water phase for later use; the water phase is poured into the oil phase heated to 50-70°C, mechanically stirred and emulsified, so that the water phase is dispersed into droplets of a desired particle size, and the emulsion is cooled so that the water phase droplets form gel microspheres, thereby obtaining modified polysaccharide gel microspheres; The oil phase comprises an inert solvent and an emulsifier, and the inert solvent is immiscible with water; The emulsifier is a nonionic surfactant; the emulsifier is a single emulsifier or a compound emulsifier with an HLB value of 3 to 8; S3. Activation and cross-linking of modified polysaccharide gel microspheres: S31, activation of modified polysaccharide gel microspheres: dispersing the modified polysaccharide gel microspheres in pure water, adding an activation reagent, activating the inert functional groups in the microspheres, and obtaining activated polysaccharide gel microspheres; S32, self-crosslinking of activated polysaccharide gel microspheres: under alkaline conditions, the activated functional groups on the polysaccharide gel microspheres undergo self-crosslinking reaction with hydroxyl groups to obtain self-crosslinked microspheres; S33, using epichlorohydrin to cross-link polysaccharide gel microspheres: under alkaline conditions, using a cross-linking agent to cross-link the polysaccharide gel microspheres, the volume amount of the cross-linking agent is 1-20% of the volume of the microspheres, to obtain polysaccharide gel microspheres with high rigidity and high flow rate; The cross-linking agent comprises at least two active functional groups, and the active functional groups have the activity of reacting with the hydroxyl groups on the polysaccharide; Alternatively, step S33 is completed first, and then steps S31 and S32 are completed; Alternatively, after step S31 is completed, steps S32 and S33 are performed continuously; Alternatively, step S31 and step S32 are performed continuously, and then step S33 is completed; Alternatively, step S33 is completed first, and steps S31 and S32 are performed continuously; Alternatively, step S31, step S32, and step S33 are performed continuously.

2. The method for preparing high-rigidity and high-flow-rate polysaccharide gel microspheres according to claim 1, characterized in that: In step S1, the weight portion of the alkaline solution is preferably 1 to 4 parts.

3. The method for preparing high-rigidity and high-flow-rate polysaccharide gel microspheres according to claim 1, characterized in that: In step S1, the polysaccharide is preferably at least one of agar, agarose, cellulose and dextran, more preferably agarose.

4. The method for preparing high-rigidity and high-flow-rate polysaccharide gel microspheres according to claim 1, characterized in that: In step S1, the bifunctional cross-linking agent is allyl bromide or allyl glycidyl ether.

5. The method for preparing high-rigidity and high-flow-rate polysaccharide gel microspheres according to claim 1, characterized in that: In step S2, the heating temperature of the modified polysaccharide dispersion is preferably 95°C.

6. The method for preparing high-rigidity and high-flow-rate polysaccharide gel microspheres according to claim 1, characterized in that: In step S31, the activation reagent is a bromination reagent, preferably bromine water or N-bromosuccinimide.

7. The method for preparing high-rigidity and high-flow-rate polysaccharide gel microspheres according to claim 1, characterized in that: In step S32, the cross-linking agent is epichlorohydrin or 1,4-butanediol diglycidyl ether or divinyl sulfone or 2,3-dibromopropanol.

8. A high-rigidity and high-flow-rate polysaccharide gel microsphere, characterized in that: The high-rigidity and high-flow-rate polysaccharide gel microspheres are prepared by the method for preparing high-rigidity and high-flow-rate polysaccharide gel microspheres according to any one of claims 1 to 7.

9. A chromatography medium, characterized in that: The chromatography medium is obtained by modifying the high-rigidity and high-flow-rate polysaccharide gel microspheres described in claim 8 with ligands.

10. Use of the chromatography medium according to claim 9 in separation and purification of biomacromolecules.

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