High-rigidity high-flow-rate polysaccharide gel microsphere, preparation method and application

Abstract

The application provides a high-rigidity high-flow-rate polysaccharide gel microsphere, a preparation method and an application, and the method comprises the following steps: S1, dispersing a polysaccharide powder in water to perform heterogeneous modification; S2, emulsifying and preparing a ball after the modified polysaccharide is dissolved; and S3, activation and crosslinking of the modified polysaccharide gel microsphere. The application adopts heterogeneous modification, pre-introduces a certain amount of a crosslinking agent, forms high-rigidity microspheres through crosslinking after solidification into balls, the linear flow rate of the microspheres is above 4000 cm / h, the microspheres have high flow rate and good mechanical strength, and the demand of large-scale separation and purification can be met. In addition, the application adopts heterogeneous modification, simplifies the process step of pre-introducing the crosslinking agent, and the heterogeneous modification makes the modified sites uniformly distributed, is beneficial to controlling the modification degree, and is beneficial to forming a rigid skeleton in the crosslinking process. The crosslinking reaction in the method provided by the application can be continuously performed, the process complexity is reduced, the method has the characteristics of simple operation, and is easy to be enlarged for large-scale industrial production.

Description

Technical Field

[0001] This invention belongs to the field of microsphere preparation technology, and relates to chromatography media, particularly to a high-rigidity, high-flow-rate polysaccharide gel microsphere, its preparation method, and its application. Background Technology

[0002] Natural polysaccharide microspheres, such as cellulose, agarose, and dextran microspheres, which are porous, highly hydrophilic, and biocompatible, are widely used in the purification of biomolecules such as proteins. However, their relatively low mechanical strength limits their application and makes them unable to withstand high pressure.

[0003] There are many methods for cross-linking reactions. Cross-linking agents are generally compounds with two active groups (such as epichlorohydrin, 2,3-dibromopropanol, and butanediol diglycidyl ether). The cross-linking process can be carried out in one step or multiple steps. These methods have improved the mechanical strength of polysaccharide microspheres to some extent. However, with the rapid development of biotechnology, especially the exponential increase in expression levels, the chromatographic purification process of biopharmaceuticals requires continuously increasing demands on the flow rate and pressure resistance of chromatographic media. The technological iteration speed of chromatographic media is slow; current mainstream products have been used for decades, and the performance of chromatographic media can no longer meet the production needs of the biopharmaceutical industry.

[0004] To further improve the mechanical strength of polysaccharide microspheres, US Patent 6602990B1 discloses a novel method for preparing highly cross-linked polysaccharide microspheres. This method introduces a special cross-linking agent before emulsification into microspheres. This agent has an active group at one end and an inert group (such as allyl hydrophobic ether) at the other. After microsphere formation, the inert group is activated, completing the internal cross-linking of the microspheres. However, if the amount of this special cross-linking agent is too high, it can damage the skeletal structure of the polysaccharide microspheres, leading to shrinkage and deformation. Chinese Patent CN103769057A effectively avoids microsphere deformation by mixing polysaccharides modified with a special cross-linking agent with unmodified polysaccharides. However, the drying process of the modified polysaccharides in this method increases the complexity of the process.

[0005] Heterogeneous modification technology for polysaccharides refers to the structural modification of polysaccharide powder or fibers before dissolution, thereby pre-imparting the polysaccharide with corresponding properties. Before dissolution, the polysaccharide is in a coiled or coated state, with only some surface hydroxyl groups effectively activated. By adding appropriate reagents, these hydroxyl groups are modified, pre-introducing functional groups into the polysaccharide. The modified polysaccharide prepared in this way possesses both the gel properties of the polysaccharide itself (the unmodified internal portion easily forms a porous network structure linked by hydrogen bonds) and the special functions of the introduced groups (the externally modified portion allows for structural design). Summary of the Invention

[0006] The first objective of this invention is to provide a method for preparing high-rigidity, high-flow-rate polysaccharide gel microspheres, with the aim of achieving high pressure resistance, high flow rate, and low back pressure performance.

[0007] Therefore, the above-mentioned objective of the present invention is achieved through the following technical solution:

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

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

[0010] Disperse 2-12 parts of polysaccharide powder in 100 parts of water (to form a polysaccharide suspension, not a solution), heat to 30-60°C to keep the polysaccharide in suspension, so that it does not dissolve or aggregate, add 1-10 parts of 33wt% alkaline solution, slowly add 1-5 parts of bifunctional crosslinking agent, react for 1-24 hours, adjust the pH to neutral, and terminate the reaction.

[0011] The preferred reaction time is 2–6 hours;

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

[0013] The bifunctional crosslinking agent comprises at least one active functional group and at least one inert functional group. The active functional group has the activity of reacting with hydroxyl groups on polysaccharides, and the inert functional group can form a self-crosslinking reaction with hydroxyl groups on polysaccharides after activation.

[0014] S2. Modified polysaccharide dissolved and then emulsified to form pellets:

[0015] The neutralized modified polysaccharide dispersion was heated to dissolve into a clear solution, cooled to no less than 50°C, and kept at this temperature as the aqueous phase for later use. The aqueous phase was poured into the oil phase heated to 50-70°C and stirred to emulsify, so that the aqueous phase was dispersed into droplets of the desired particle size. The emulsion was cooled to allow the aqueous phase droplets to form gel-like microspheres, thus obtaining the modified polysaccharide gel microspheres.

[0016] The oil phase comprises an inert solvent and an emulsifier, wherein 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: The modified polysaccharide gel microspheres are dispersed in pure water and an activation reagent is added to activate the inert functional groups (inert functional groups on the bifunctional crosslinking agent) in the microspheres to obtain 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 a self-crosslinking reaction with hydroxyl groups to obtain self-crosslinked microspheres.

[0021] S33. Using epichlorohydrin to crosslink polysaccharide gel microspheres: Under alkaline conditions, a crosslinking agent is used to crosslink polysaccharide gel microspheres. The volume of the crosslinking agent is 1-20% of the volume of the microspheres to obtain polysaccharide gel microspheres with high rigidity and high flow rate.

[0022] The crosslinking agent contains at least two active functional groups, which have the activity of reacting with hydroxyl groups on the polysaccharide;

[0023] Alternatively, complete step S33 first, then complete steps S31 and S32.

[0024] Alternatively, after step S31 is completed, steps S32 and S33 can be performed consecutively. Continuous performance means that after completion, there is no need to clean the microspheres or change the reaction solution; the reagents or conditions required for the next step can be added directly to the original reactor.

[0025] Alternatively, steps S31 and S32 can be performed consecutively before step S33 is completed.

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

[0027] Alternatively, steps S31, S32, and S33 can be performed consecutively.

[0028] High rigidity means that the microspheres are not easily deformed to affect their performance, and can be used for separation and purification operations at flow rates greater than 2000 cm / h, with a maximum flow rate of over 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 embodiment of the present invention: in step S1, the weight percentage of the alkaline solution is preferably 1 to 4 parts. The alkali is preferably sodium hydroxide or potassium hydroxide. - The concentration is 0.5–5 M. To prevent polysaccharides from decomposing under alkaline conditions, NaBH4 can be added.

[0031] As a preferred embodiment of the present invention: In step S1, the polysaccharide is preferably at least one selected from agar, agarose, cellulose, and dextran, more preferably agarose. It can swell in an alkaline dispersion at a lower temperature, thus facilitating heterogeneous modification. Upon further heating, it can dissolve, and upon cooling, it can form a porous gel-like substance.

[0032] As a preferred technical solution of the present invention: in step S1, the bifunctional crosslinking agent is bromopropylene 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 crosslinking 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, such as Span or Tween, preferably Span 80.

[0036] The concentration of the emulsifier is 0.5–10% (w / v), preferably 1–6% (w / v). The emulsifier concentration 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 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, the cooling curing method is selected, and when the polysaccharide is dextran, the cross-linking curing method is selected.

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

[0040] The amount of bromine water added is as follows: add bromine water dropwise until the solution turns yellow and remains unchanged for 1 minute. After the reaction is complete, 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, 100mL of water contains 10-100g of microspheres, preferably 50g of microspheres.

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

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

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

[0045] The second objective of this invention is to provide a high-rigidity, high-flow-rate polysaccharide gel microsphere.

[0046] Therefore, the above-mentioned objective of the present invention is achieved through the following technical solution:

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

[0048] The third objective of this invention is to provide a chromatography medium.

[0049] Therefore, the above-mentioned objective of the present invention is achieved through the following technical solution:

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

[0051] Another object of the present invention is to provide the application of the chromatography media described above.

[0052] Therefore, the above-mentioned objective of the present invention is achieved through the following technical solution:

[0053] Based on the application of chromatography media in the separation and purification of biological macromolecules as described above.

[0054] This invention provides a high-rigidity, high-flow-rate polysaccharide gel microsphere, its preparation method, and its application. The method employs heterogeneous modification, pre-introducing a certain amount of cross-linking agent. After curing into spheres, high-rigidity microspheres are formed through cross-linking. The linear flow rate of the microspheres exceeds 4000 cm / h. The microspheres possess high flow rate and good mechanical strength, meeting the requirements for large-scale separation and purification. Furthermore, the heterogeneous modification simplifies the pre-introduction of the cross-linking agent process, and the heterogeneous modification ensures a uniform distribution of modification sites, facilitating control of the modification degree and promoting the formation of a rigid framework during cross-linking. The cross-linking reaction in the method provided by this invention can be carried out continuously, reducing process complexity and offering simplicity and ease of operation, making it easy to scale up for large-scale industrial production. The heterogeneous modification does not completely destroy the hydroxyl structure of the polysaccharide, promoting the formation of hydrogen-bonded frameworks during gelation and effectively preventing microsphere deformation and shrinkage. After cross-linking, the resulting microspheres retain the performance advantages of natural polysaccharides while also withstanding high operating flow rates, making them ideal industrial chromatography media. Attached Figure Description

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

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

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

[0058] Figure 4 The pressure / flow rate characteristic curves of intermediate products during the microsphere crosslinking process are shown. Among them, intermediate product 1 is uncrosslinked microspheres, intermediate product 2 is microspheres that have only undergone self-crosslinking, and intermediate product 3 is microspheres that have only undergone epichlorohydrin crosslinking. Detailed Implementation

[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, with stepwise crosslinking.

[0061] (a) Heterogeneous modification of agarose

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

[0063] (b) Emulsification and pelletizing after dissolution

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

[0065] (c) Activation of allyl agarose microspheres

[0066] Disperse 100g of allyl agarose microspheres (after draining excess water) in 100mL of pure water, and add 5g of sodium acetate. After stirring for 10min, add bromine water dropwise until the solution turns yellow and the color remains unchanged for 1min. Continue the reaction for 20min, then add an appropriate amount of sodium formate to remove excess bromine water. After the reaction is complete, wash the microspheres with plenty of pure water until neutral.

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

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

[0069] (e) Epichlorohydrin crosslinking

[0070] 100g of self-crosslinked allyl agarose microspheres, after draining excess water, were dispersed in 33mL of pure water. The mixture was heated to 40℃, and 45g of anhydrous sodium sulfate was added. After stirring and dissolving for 1 hour, 2mL of 33% (w / w) NaOH and 0.1g of sodium borohydride were added. Stirring was continued for 30 minutes, and the temperature was raised to 50℃. Over 3 hours, 10mL of epichlorohydrin and 12mL of 33% (w / w) NaOH were added dropwise simultaneously. After the addition was complete, the reaction was continued for 16 hours. After the reaction was complete, the microspheres were washed with plenty of pure water until neutral.

[0071] Microspheres were sieved to an average particle size of approximately 90 μm using 100-mesh and 250-mesh sieves, and their pressure / flow rate characteristics were measured using the following method.

[0072] Microsphere pressure / flow rate characteristic test

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

[0074] Chromatography column: Cytiva Tricorn 10 / 100 Column

[0075] Mobile phase: pure water

[0076] Test: 8 mL of microspheres were packed into the chromatography column. The flow rate was started at 0.5 mL / min, and the pressure was monitored. The flow rate was gradually increased every 5 min until the system pressure rapidly increased to 3 MPa, indicating sample collapse. The flow rate could not be increased further, and the test was terminated. The order of flow rate increases was 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, ..., 66.0 mL / min. The volumetric flow rate was converted to linear velocity: V = (60 × V_v) / S, where V is the linear velocity (cm / h), V_v is the volumetric flow rate (mL / min), and S is the cross-sectional area of ​​the chromatography column (0.785 cm²). 2 The flow velocity and column pressure during the final stage of pressure stabilization before a sharp increase in pressure are defined as the maximum flow velocity and pressure resistance of the microspheres.

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

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

[0079] (a) Heterogeneous modification of agarose

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

[0081] (b) Emulsification and pelletizing after dissolution

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

[0083] (c) Activation of allyl agarose microspheres

[0084] Disperse 100g of allyl agarose microspheres (after draining excess water) in 15mL of pure water, and add 2g of sodium acetate. After stirring for 10min, add bromine water dropwise until the solution turns yellow and remains so for 1min. Continue the reaction for 20min, then add an appropriate amount of sodium formate to remove excess bromine water.

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

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

[0087] (e) Epichlorohydrin crosslinking

[0088] Maintaining the temperature at 40°C, add 40g of anhydrous sodium sulfate to the reaction system obtained in step (d). After stirring and dissolving for 1 hour, raise the temperature to 50°C and simultaneously add 10mL of epichlorohydrin and 12mL of 33% (mass fraction) NaOH dropwise over 3 hours. After the addition is complete, continue the reaction for 16 hours. After the reaction is complete, wash the microspheres with plenty of pure water until neutral.

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

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

[0091] (a) Heterogeneous modification of agarose

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

[0093] (b) Emulsification and pelletizing after dissolution

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

[0095] (c) Activation of allyl agarose microspheres

[0096] Disperse 100g of allyl agarose microspheres (after draining excess water) in 100mL of pure water, and add 5g of sodium acetate. After stirring for 10min, add bromine water dropwise until the solution turns yellow and the color remains unchanged for 1min. Continue the reaction for 20min, then add an appropriate amount of sodium formate to remove excess bromine water. After the reaction is complete, wash the microspheres with plenty of pure water until neutral.

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

[0098] Disperse 100g of activated allyl agarose microspheres (after draining off excess water) in 33mL of pure water, add 5g of anhydrous sodium sulfate, stir to dissolve for 20min, then add 4mL of 33% (mass fraction) NaOH and 0.1g of sodium borohydride, heat to 40℃, and stir to react for 16h.

[0099] (e) Epichlorohydrin crosslinking

[0100] Maintaining the temperature at 40°C, add 40g of anhydrous sodium sulfate to the reaction system obtained in step (d). After stirring and dissolving for 1 hour, raise the temperature to 50°C and simultaneously add 10mL of epichlorohydrin and 12mL of 33% (mass fraction) NaOH dropwise over 3 hours. After the addition is complete, continue the reaction for 16 hours. After the reaction is complete, wash the microspheres with plenty of pure water until neutral.

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

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

[0103] (a) Heterogeneous modification of agarose

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

[0105] (b) Emulsification and pelletizing after dissolution

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

[0107] (c) Activation of allyl agarose microspheres

[0108] Disperse 100g of allyl agarose microspheres (after draining excess water) in 80mL of pure water, and add 5g of sodium acetate. After stirring for 10min, add bromine water dropwise until the solution turns yellow and remains so for 1min. Continue the reaction for 20min, then add an appropriate amount of sodium formate to remove excess bromine water.

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

[0110] Add 10 g of anhydrous sodium sulfate to the reaction system obtained in step (c), stir to 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 complete, wash the microspheres with plenty of pure water until neutral.

[0111] (e) Epichlorohydrin crosslinking

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

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

[0114] Example 5: Preparation of high-rigidity agarose microspheres by heterogeneous modification, with partial continuous cross-linking 3 (step e advanced).

[0115] (a) Heterogeneous modification of agarose

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

[0117] (b) Emulsification and pelletizing after dissolution

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

[0119] (e) Epichlorohydrin crosslinking

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

[0121] (c) Activation of allyl agarose microspheres

[0122] Disperse 100g of epichlorohydrin-crosslinked allyl agarose microspheres (after draining off excess water) in 80mL of pure water, and add 5g of sodium acetate. After stirring for 10min, add bromine water dropwise until the solution turns yellow and remains so for 1min. Continue the reaction for 20min, then add an appropriate amount of sodium formate to remove excess bromine water.

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

[0124] Add 10 g of anhydrous sodium sulfate to the reaction system obtained in step (c), stir to 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 complete, wash the microspheres with plenty of pure water until neutral.

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

[0126] Example 6: High-rigidity agarose microspheres applied to ion exchange media

[0127] SP packing material for ion exchange

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

[0129] 2. Modification with allyl hydrophobic ether. 15 mL of allyl hydrophobic ether was then added, and the mixture was stirred at 50 °C for 16 h. After the reaction was completed, the mixture was washed with pure water.

[0130] Its 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. React at 40℃ with shaking for 16h. After the reaction is complete, wash with pure water.

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

[0133] Example 7: High-rigidity agarose microspheres applied to ion exchange media

[0134] Q-packing material for ion exchange media

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

[0136] 2. Solution preparation. Take 28.5g of glycidyltrimethylammonium chloride (ETA), add 9mL of isopropanol and 9mL of pure water, and stir to dissolve. Add the prepared ETA solution dropwise to the reaction flask, controlling the addition to be completed in about 4 hours. After the addition is complete, raise the temperature to 50℃ and continue the reaction for 6 hours.

[0137] 3. Cleaning. After the reaction is complete, repeatedly clean the container using a vacuum filtration funnel until the filtrate is neutral and odorless. Then, pack the packing material into a 50 mL gravity column and rinse with five volumes of 1 mol / L sodium chloride solution.

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

[0139] Example 8: High-rigidity agarose microspheres applied to hydrophobic media

[0140] Butyl packing material for hydrophobic media

[0141] 1. Take 20g of the sieved microspheres (average particle size of 75μm) prepared in Example 1 into a reaction flask; 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 h.

[0143] 3. After the reaction is complete, first use a vacuum pump to remove the reaction solution, and then wash with anhydrous ethanol and pure water in sequence.

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

[0145] Example 9: High-rigidity agarose microspheres applied to hydrophobic media

[0146] Phenyl filler for hydrophobic media

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

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

[0149] 3. After the reaction is complete, first use a vacuum pump to remove the reaction solution, and then wash with anhydrous ethanol and pure water in sequence.

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

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

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

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

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

[0155] Discussion of Results:

[0156] Figure 3The pressure / flow rate characteristic curves of Examples 1-5 and Comparative Examples 1 and 2 reveal three characteristics of the embodiments of the present invention: 1) The maximum flow rate of the microspheres in the embodiments of the present invention is higher than 4000 cm / h, even exceeding 5000 cm / h, far higher than the approximately 1500 cm / h of commercially available products, but less than 2000 cm / h; 2) The pressure resistance of the microspheres in the embodiments of the present invention is 0.7-0.9 MPa, higher than the approximately 0.30 MPa of commercially available products (0.31 MPa and 0.29 MPa respectively); 3) The back pressure of the microspheres in the embodiments of the present invention is low, meaning lower pressure at the same flow rate, especially Example 4, which exhibits particularly outstanding performance, with a back pressure of less than 0.1 MPa at a flow rate of 1000 cm / h. Furthermore, as... Figure 4 As shown, the microspheres in Example 4 are far superior to those that are not cross-linked or cross-linked only once, demonstrating the advantages of the microspheres provided by the present invention in terms of pressure / flow rate.

[0157] The above specific embodiments are used to explain and illustrate the present invention, and are only preferred embodiments of the present invention, not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A method for preparing high-rigidity, high-flow-rate polysaccharide gel microspheres, characterized in that: The method includes the following steps: S1. Disperse polysaccharide powder in water for heterogeneous modification: Disperse 2-12 parts of polysaccharide powder in 100 parts of water, heat to 30-60℃ to form a polysaccharide suspension (non-solution), add 1-10 parts of 33 wt% alkaline solution, slowly add 1-5 parts of bifunctional crosslinking agent, react for 1-24 h, adjust the pH to neutral, and terminate the reaction. The bifunctional crosslinking agent comprises at least one active functional group and at least one inert functional group. The active functional group has the activity of reacting with hydroxyl groups on polysaccharides, and the inert functional group can form a self-crosslinking reaction with hydroxyl groups on polysaccharides after activation. S2. Modified polysaccharide dissolved and then emulsified to form pellets: The neutralized modified polysaccharide dispersion was heated to dissolve into a clear solution, cooled to no less than 50°C, and kept at this temperature as the aqueous phase for later use. The aqueous phase was poured into the oil phase heated to 50-70°C and mechanically stirred to emulsify, so that the aqueous phase was dispersed into droplets of the desired particle size. The emulsion was cooled to allow the aqueous phase droplets to form gel-like microspheres, thus obtaining the modified polysaccharide gel microspheres. The oil phase comprises an inert solvent and an emulsifier, wherein 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: The modified polysaccharide gel microspheres were dispersed in pure water and an activation reagent was added to activate the inert functional groups in the microspheres to obtain 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 a self-crosslinking reaction with hydroxyl groups to obtain self-crosslinked microspheres. S33. Using epichlorohydrin to crosslink polysaccharide gel microspheres: Under alkaline conditions, a crosslinking agent is used to crosslink polysaccharide gel microspheres. The volume of the crosslinking agent is 1-20% of the volume of the microspheres to obtain polysaccharide gel microspheres with high rigidity and high flow rate. The crosslinking agent contains at least two active functional groups, which have the activity of reacting with hydroxyl groups on the polysaccharide; Alternatively, complete step S33 first, then complete steps S31 and S32; Alternatively, after step S31 is completed, steps S32 and S33 are performed consecutively; Alternatively, steps S31 and S32 can be performed consecutively, and then step S33 can be completed. Alternatively, complete step S33 first, and then proceed with steps S31 and S32 consecutively. Alternatively, steps S31, S32, and S33 can be performed consecutively.

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

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

4. The method for preparing high-rigidity, high-flow-rate polysaccharide gel microspheres according to claim 1 or 3, characterized in that: In step S1, the polysaccharide is agarose.

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

6. The method for preparing high-rigidity, 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 95℃.

7. The method for preparing high-rigidity, high-flow-rate polysaccharide gel microspheres according to claim 1, characterized in that: In step S31, the activating agent is a brominating agent.

8. The method for preparing high-rigidity, high-flow-rate polysaccharide gel microspheres according to claim 1 or 7, characterized in that: In step S31, the activating reagent is bromine water or... N - Bromosuccinimide.

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

10. A high-rigidity, high-flow-rate polysaccharide gel microsphere, characterized in that: The high-rigidity, high-flow-rate polysaccharide gel microspheres are prepared by the preparation method of high-rigidity, high-flow-rate polysaccharide gel microspheres as described in any one of claims 1 to 9.

11. A chromatography medium, characterized in that: The chromatography medium is obtained by ligand modification of the high-rigidity, high-flow-rate polysaccharide gel microspheres as described in claim 10.

12. The application of the chromatography medium according to claim 11 in the separation and purification of biological macromolecules.

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