Macroporous high-rigidity agarose microspheres and preparation method thereof

Abstract

The application provides a macroporous high-rigidity agarose microsphere and a preparation method thereof. The preparation method comprises the following steps: adding a pore-forming agent which is soluble in water and insoluble in general organic solvents into an agarose aqueous solution, then transferring the sugar solution into an oil phase containing an emulsifier and a stabilizer to perform emulsification, transferring the emulsion into purified water after temperature reduction and solidification, dissolving the pore-forming agent in the purified water through stirring, then removing the upper oil phase to obtain the macroporous agarose microsphere; and then draining water from the obtained macroporous agarose microsphere, adding purified water, anhydrous sodium sulfate, a sodium hydroxide solution and a crosslinking agent, and cleaning repeatedly after reaction to obtain the macroporous high-rigidity agarose microsphere. The product obtained by the application guarantees the activity of biological macromolecules and meets the separation and purification requirements in industry. Moreover, the operation process of the application is simple, waste liquid generation is greatly reduced, and the application is suitable for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of agarose microsphere preparation technology, and relates to a large-pore, high-rigidity agarose microsphere and its preparation method. Background Technology

[0002] With the rapid development of the global biopharmaceutical industry over the past decade, the upstream and downstream production processes of monoclonal antibody drugs, vaccines, and recombinant proteins in the biopharmaceutical field have become mature and stable. With the continuous expansion of the frontiers of biological science and technology and the deepening of research in fields such as genomics, proteomics, and cell engineering, new high-value-added biological products that can be developed into drugs are constantly emerging, such as VLP vaccines, viral vectors, cytokines, plasmids, and PEG-modified proteins.

[0003] Meanwhile, separation and purification technologies, essential "downstream technologies" in biopharmaceutical processes, are facing increasing demands in terms of efficiency, cost, and ease of use. Currently, column chromatography and related consumables (chromatographic media) are the mainstream choices for separation and purification. Because the microscopic size of target proteins can reach tens or even hundreds of nanometers, and the pore size of commercially available traditional agarose chromatography media is generally limited to 5nm-40nm due to manufacturing limitations, this significantly increases the resistance to molecular mass transfer, easily causing molecular structure damage and loss of activity, resulting in a significant decrease in separation efficiency and yield, and increased costs hindering industrialization. Although polymer microspheres can achieve uniform particle size and larger pore sizes, they have disadvantages compared to agarose microspheres, such as poor biocompatibility, high non-specific adsorption, and difficulty in modifying functional groups, greatly limiting their application in the purification of biomolecules. If traditional agarose chromatography media continues to be used, the only option is to reduce the particle size to increase the specific surface area, relying on its surface for adsorption, but this undoubtedly increases the back pressure of the chromatography column and reduces separation efficiency. Therefore, it is necessary to develop an agarose chromatography medium with large pore size, high rigidity, and high flow rate to improve the efficiency of unit separation and purification operations, while shortening the entire downstream process, thereby promoting the overall optimization of modern biotechnology.

[0004] Sinofarm Bioprocess Technology Co., Ltd.'s patent CN113195095A proposes increasing the pore size of microspheres by reducing the agarose content. However, this method results in extremely low microsphere yield, complex preparation processes, and insufficient pressure resistance. Furthermore, the reduced agarose content also decreases the number of binding sites, making functional group modification difficult. Ma Guanghui et al. reported a process for preparing macroporous agarose microspheres based on micellar swelling (CN103055773A), which alters the pore size by adding and adjusting the amount of surfactant. However, the surfactant's swelling effect on the internal pores of agarose is not significant and is affected by factors such as emulsification time, oil phase type, and temperature. The difficulty in cleaning the oil phase and emulsifier necessitates the use of large amounts of organic solvents, generating substantial waste liquid, which is detrimental to large-scale production. Sun Yan et al. used calcium carbonate as a pore-forming agent to control the pore size of microspheres by adjusting the particle size distribution and dosage of calcium carbonate (CN1472002A). However, this method involves repeated soaking and washing with hydrochloric acid, which damages the agarose backbone structure, causing the internal pore structure to collapse, affecting the pore size of microspheres and reducing their service life. Furthermore, the high temperature and rotation speed during emulsification are not conducive to industrial-scale application. Xiao Qiong et al. added cyclic anhydrides to the aqueous phase to induce a single acylation reaction with the agarose chains, and controlled the degree of cross-linking between the anhydrides and agarose chains by temperature to regulate the pore size (CN115612185A). However, the microspheres prepared had small pore sizes, insufficient rigidity, and were not pressure resistant, which could not meet the practical application requirements for the separation and purification of biomacromolecules. In addition, a variety of organic solvents were used in the preparation of microspheres, which had certain toxicity, making them unsuitable for industrial scale-up. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a large-pore, high-rigidity agarose microsphere and its preparation method, which can achieve large-scale production and promotion while reducing waste liquid generation.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a method for preparing large-pore, high-rigidity agarose microspheres, comprising:

[0008] A pore-forming agent soluble in water but insoluble in common organic solvents is added to an aqueous agarose solution. The sugar solution is then transferred to an oil phase containing an emulsifier and a stabilizer for emulsification. After cooling and solidification, the emulsion is transferred to purified water. The pore-forming agent is dissolved in the purified water by stirring. The upper oil phase is then removed to obtain large-pore agarose microspheres. The obtained large-pore agarose microspheres are then drained, and purified water, anhydrous sodium sulfate, sodium hydroxide solution, and a crosslinking agent are added. After the reaction, the microspheres are washed multiple times to obtain large-pore, high-rigidity agarose microspheres.

[0009] Preferably, the agarose aqueous solution is prepared by the following method: 2-10% agarose (by mass fraction of purified water) is added to purified water with a conductivity of 0.01-20 μS / cm; the mixture is heated to 60-100°C, stirred to dissolve, and kept in a sol state until the viscosity reaches 10-500 mPa·s; then the temperature is lowered to 40-60°C to obtain the agarose aqueous solution. In this embodiment, the conductivity of the purified water is more preferably 0.5-10 μS / cm; and the viscosity of the agarose solution is more preferably 50-300 mPa·s.

[0010] Preferably, the agarose used is high gel strength agarose with a gel strength of 750 g / cm³. 2 (1% content) or more, more preferably greater than or equal to 1000 g / cm³ 2 It can be modified agarose, or it can be modified by the user. The preferred modifier is one or two of sodium chloroacetate, ethylene oxide, glycidyl ether, and propylene oxide.

[0011] Preferably, the porogen should have good water solubility and be insoluble in common organic solvents to prevent partial dissolution in organic solvents during emulsification, which would affect emulsification and reduce the pore-forming effect. In addition, it should contain a large number of hydroxyl groups to form hydrogen bonds and bind more agarose micelles, forming micelles with the porogen as the core. During washing, the porogen can be washed away by water, forming pores. Furthermore, when the volume of washing water reaches more than twice the volume of the organic solvent, the porogen can change the surface tension and thicken the water, causing the organic solvent to separate from the water, thus removing the organic solvent and eliminating the need for washing with organic solvents, greatly reducing waste liquid generation. The preferred porogen is hydroxyethyl cellulose, carboxymethyl cellulose, polyvinyl alcohol, polyvinylpyrrolidone, or polyethylene glycol. Since polyethylene glycol also has a certain degree of solubility in common solvents, it can affect the formation of micelles during emulsification, thereby affecting the size of the microsphere pores. Hydroxyethyl cellulose, carboxymethyl cellulose, polyvinyl alcohol, or polyvinylpyrrolidone are more preferred.

[0012] Preferably, 5-10% by mass of agarose aqueous solution is added to the agarose aqueous solution as a pore-forming agent, and the solution is stirred and dissolved for 10-40 minutes.

[0013] Preferably, the oil phase containing emulsifiers and stabilizers is prepared by the following method:

[0014] Emulsifier and stabilizer are added to a water-insoluble organic solvent, and the mixture is heated in a water bath to 30-80°C and stirred until homogeneous. The water-insoluble organic solvent is 1-10 times the mass of the agarose aqueous solution, and the emulsifier and stabilizer are 0.2-5% and 0.1-2% of the mass fraction of the water-insoluble organic solvent, respectively. In this method, the mass of the water-insoluble organic solvent is more preferably 2-6 times the mass of the agarose solution; the amount of emulsifier is more preferably 0.5-2% of the mass fraction of the water-insoluble organic solvent; the amount of stabilizer is more preferably 0.2-1% of the mass fraction of the water-insoluble organic solvent; and the water bath temperature is more preferably 50-70°C, and a temperature difference of not less than 10°C between the water-insoluble organic solvent and the agarose solution must be maintained.

[0015] Preferably, the water-insoluble organic solvent is an aliphatic or aromatic hydrocarbon with six or more carbon atoms, possessing a density not close to that of water, a low boiling point, and easy recovery, including but not limited to heptane, benzene, toluene, xylene, carbon tetrachloride, and dichloroethane. The emulsifier should have good water solubility and be easily washed away by water, such as one or more of polyethylene glycol polyoxyethylene ether phosphate, isotretinoin polyoxyethylene ether phosphate, oleyl alcohol polyether phosphate, sodium dodecyl sulfate, and sodium dodecyl sulfonate. Since a pore-forming agent is added to the agarose solution, it adsorbs agarose micelles together through hydrogen bonding, forming larger, relatively unstable micelles. Therefore, a certain amount of stabilizer needs to be added to the oil phase to protect the micelles. The stabilizer can form a robust film on the surface of the droplets, preventing droplet aggregation and also preventing the decomposition of the pore-forming agent and agarose micelles during emulsification, thus affecting pore formation. Preferred stabilizers include cellulose acetate butyrate, cetyl alcohol, and carrageenan.

[0016] As a preferred method, the mixture is stirred under a top-mounted mixer for 10-30 minutes to emulsify. Once the microsphere particle size meets the requirements, the mixture is cooled and cured. The curing process ends when the internal temperature drops below 20°C, resulting in a microsphere emulsion.

[0017] As a preferred method, after cooling and solidification, the emulsion is poured into 2-10 times its weight of purified water for washing. After thorough stirring and mixing, it is allowed to stand for 12-24 hours. After sedimentation, the supernatant is removed. This process is repeated 4-8 times to obtain large-pore agarose microspheres.

[0018] Preferably, 25-75% purified water (by mass of the microspheres) is added to raise the internal temperature to 30-70°C, and the mixture is stirred until homogeneous. Then, 20-60% anhydrous sodium sulfate (by mass of the microspheres) is added and stirred to dissolve. Next, 15-50% of a 50% sodium hydroxide solution (by mass of the microspheres) is added, followed by dropwise addition of a crosslinking agent (by mass of the microspheres) over 6-8 hours. The reaction is allowed to proceed for 16-20 hours, followed by washing with deionized water 6-8 times. Preferably, the entire process is repeated twice to obtain large-pore, high-rigidity agarose microspheres. In this scheme, it is preferable to raise the internal temperature to 40-60°C; the mass fraction of anhydrous sodium sulfate is more preferably 25-40%; the mass fraction of 50% sodium hydroxide solution is more preferably 20-35%; and the mass fraction of crosslinking agent is more preferably 30-45%. The crosslinking agent can be one or more compounds with bifunctional groups, such as epichlorohydrin, 2,3-dibromopropanol, 1,4-butanediol glycidyl ether, and allyl glycidyl ether. Since the above crosslinking method is known in the art, those skilled in the art can select specific crosslinking conditions as needed.

[0019] The present invention also provides a large-pore, high-rigidity agarose microspheres, which are prepared by the above-described preparation method.

[0020] The large-pore, high-rigidity agarose microspheres prepared by this invention have a maximum pore size of 100-1000 nm and a pressure flow rate of over 500 cm / h.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention involves adding a porogen that is soluble in water but insoluble in common organic solvents to an aqueous agarose solution. After the agarose is completely dissolved into linear molecules, the porogen adsorbs the agarose micelles together through hydrogen bonding, forming a core-shell structure with the porogen as the core and the agarose micelles as the shell. The sugar solution is then transferred to an oil phase containing emulsifiers and stabilizers for emulsification. After cooling and solidification, the emulsion is transferred to a certain amount of pure water, and the porogen is dissolved in the water by stirring. The porogen then separates the oil and water phases by altering the surface tension and thickening the water. The upper oil phase is then removed, yielding large-pore agarose microspheres. These microspheres possess numerous large-sized through-pores that allow the mobile phase to flow in a convective manner, improving mass transfer rates and enabling the substances to be separated to quickly bind with functional groups on the medium, thus increasing separation efficiency. Furthermore, the pore size is more than three times the diameter of biological macromolecules, significantly reducing shear forces during mass transfer, ensuring the normal biological activity of the target analyte, and increasing yield.

[0023] The product obtained by this invention has a large pore size and low back pressure. After cross-linking, its rigidity and flow rate are greatly improved, making the separation and purification operation faster and more efficient. It can be used for the separation and purification of biological macromolecules, such as antibodies, viruses, mRNA and recombinant proteins, ensuring the activity of biological macromolecules while meeting the needs of industrial separation and purification.

[0024] The method described in this invention is simple to operate, does not involve high temperature and high pressure reactions, has stable and easily controllable pore size, and only requires pure water rinsing after discharge without the need for organic solvents. The rinsing process is simple, greatly reduces waste liquid generation, and is suitable for large-scale production. Attached Figure Description

[0025] Figure 1 This is an electron microscope image (5 μm) of large-pore, high-rigidity agarose microspheres from Embodiment 1 of the present invention.

[0026] Figure 2 This is an electron microscope image (10 μm) of large-pore, high-rigidity agarose microspheres from Embodiment 1 of the present invention.

[0027] Figure 3 This is an electron microscope image (5 μm) of large-pore, high-rigidity agarose microspheres from Embodiment 2 of the present invention.

[0028] Figure 4 This is an electron microscope image (10 μm) of large-pore, high-rigidity agarose microspheres from Embodiment 2 of the present invention.

[0029] Figure 5 This is an electron microscope image (5 μm) of the large-pore, high-rigidity agarose microspheres in Embodiment 3 of the present invention.

[0030] Figure 6 This is an electron microscope image (10 μm) of the large-pore, high-rigidity agarose microspheres in Example 3 of the present invention.

[0031] Figure 7 This is an electron microscope image (5 μm) of the large-pore, high-rigidity agarose microspheres in Example 4 of this invention.

[0032] Figure 8 This is an electron microscope image (10 μm) of the large-pore, high-rigidity agarose microspheres in Example 4 of this invention.

[0033] Figure 9 The internal pore structure (1 μm) of the large-pore, high-rigidity agarose microspheres in Embodiment 1 of the present invention is shown.

[0034] Figure 10 The internal pore structure (1 μm) of the large-pore, high-rigidity agarose microspheres in Embodiment 2 of the present invention is shown.

[0035] Figure 11 The internal pore structure (1 μm) of the large-pore, high-rigidity agarose microspheres in Embodiment 3 of the present invention is shown.

[0036] Figure 12 The internal pore structure (1 μm) of the large-pore, high-rigidity agarose microspheres in Embodiment 4 of this invention is shown.

[0037] Figure 13 This is the pressure-flow rate curve of the large-pore, high-rigidity agarose microspheres of the present invention. Detailed Implementation

[0038] To more clearly illustrate the present invention, the invention will be further described in detail below with reference to embodiments and accompanying drawings. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0039] The aperture size in the technical solution provided by this invention is obtained by measuring with an electron scanning microscope (Germany ZEISG GeminiSEM 300). The aperture size needs to be compared with a scale bar to obtain the result.

[0040] Example 1

[0041] (1) Add 100 mL of purified water with a conductivity of 10 μS / cm to a three-necked flask, and add 2 g of agarose (gel strength 1000 g / cm). 2 The solid powder was placed in a water bath and heated to 90°C. It was stirred at 200 rpm to dissolve the powder and kept in a sol state until its viscosity was about 50 mPa·S. Then the temperature was lowered to 50°C.

[0042] (2) Add 5g of hydroxyethyl cellulose and dissolve for 10 minutes.

[0043] (3) During the dissolution of agarose, add 0.5g of polyethylene glycol polyoxyethylene ether phosphate to 150g of heptane, then add 0.3g of cellulose acetate butyrate, heat the mixture in a water bath to 50℃, and stir until evenly mixed.

[0044] (4) Quickly transfer the agarose aqueous solution obtained in step (2) to the oil phase in step (3) and mix thoroughly by stirring for 10 min.

[0045] (5) After the microspheres meet the size requirements, they are cooled and cured. The curing ends when the internal temperature drops below 20℃.

[0046] (6) Pour the solidified microsphere emulsion into 500g of purified water for washing, stir thoroughly and let stand for 16h. After the sedimentation and stratification are complete, remove the upper liquid. Repeat this process 4 times to obtain large-pore agarose microspheres.

[0047] (7) Drain the water from the large-pore agarose microspheres cleaned in step (6) (approximately 100g), add 25g of purified water, raise the internal temperature to 30℃, and stir at 150rpm. Add 20g of anhydrous sodium sulfate and stir for 30min until completely dissolved. Then add 15g of 50% sodium hydroxide solution and dropwise add 30g of epichlorohydrin over 6h. After the addition is complete, react for 16h and wash 6 times with deionized water.

[0048] (8) Repeat step (7) twice more to obtain large-pore, high-rigidity agarose microspheres. Electron micrographs are shown below. Figure 1 , Figure 2 and Figure 9 As shown, the surface of the microspheres is uniformly covered with large pores with a diameter of 300-500 nm.

[0049] Example 2

[0050] (1) Add 100 mL of purified water with a conductivity of 5 μS / cm to a three-necked flask, and add 4 g of agarose (gel strength 1000 g / cm). 2 The solid powder was placed in a water bath and heated to 88°C. It was stirred at 200 rpm to dissolve the powder and kept in a sol state until its viscosity was about 100 mPa·S. Then the temperature was lowered to 50°C.

[0051] (2) Add 6g of carboxymethyl cellulose and dissolve for 15 minutes.

[0052] (3) During the dissolution of agarose, add 2g of isotridecyl alcohol polyoxyethylene ether phosphate to 200g benzene, then add 0.8g of cellulose acetate butyrate, heat the mixture in a water bath to 40℃, and stir until evenly mixed.

[0053] (4) Quickly transfer the agarose aqueous solution obtained in step (2) to the oil phase in step (3) and mix thoroughly by stirring for 15 min.

[0054] (5) After the microspheres meet the size requirements, they are cooled and cured. The curing ends when the internal temperature drops below 20℃.

[0055] (6) Pour the solidified microsphere emulsion into 900g of purified water for washing, stir thoroughly and let stand for 18h. After the sedimentation and stratification are complete, remove the upper liquid. Repeat this process 5 times to obtain large-pore agarose microspheres.

[0056] (7) Drain approximately 100g of the large-pore agarose microspheres cleaned in step (6), add 50g of purified water, raise the internal temperature to 40℃, and stir at 150rpm. Add 25g of anhydrous sodium sulfate and stir for 40min until completely dissolved. Then add 20g of 50% sodium hydroxide solution and dropwise add 35g of 2,3-dibromopropanol over 6h. After reacting for 17h, wash 6 times with deionized water.

[0057] (8) Repeat step (7) twice more to obtain large-pore, high-rigidity agarose microspheres. Electron micrographs are shown below. Figure 3 , Figure 4 and Figure 10 As shown, the surface of the microspheres is uniformly covered with ultra-large pores with a diameter of 500-1000 nm.

[0058] Example 3

[0059] (1) Add 100 mL of purified water with a conductivity of 1 μS / cm to a three-necked flask, and add 6 g of agarose (gel strength 1200 g / cm). 2 The solid powder was placed in a water bath and heated to 92°C. It was stirred at 200 rpm to dissolve the powder and kept in a sol state until its viscosity was about 200 mPa·S. Then the temperature was lowered to 60°C.

[0060] (2) Add 8g of polyvinyl alcohol and dissolve for 20 minutes.

[0061] (3) During the dissolution of agarose, add 3g of oleyl alcohol polyether phosphate to 300g of toluene, then add 2.5g of cetyl alcohol, heat the water bath to 50℃, and stir to mix evenly.

[0062] (4) Quickly transfer the agarose aqueous solution obtained in step (2) to the oil phase in step (3) and mix thoroughly by stirring for 20 minutes.

[0063] (5) After the microspheres meet the size requirements, they are cooled and cured. The curing ends when the internal temperature drops below 20℃.

[0064] (6) Pour the solidified microsphere emulsion into 1500g of purified water for washing, stir thoroughly and let stand for 20h. After the sedimentation and stratification are complete, remove the upper liquid. Repeat this process 6 times to obtain large-pore agarose microspheres.

[0065] (7) Drain approximately 100g of the large-pore agarose microspheres cleaned in step (6), add 75g of purified water, raise the internal temperature to 50℃, and stir at 200rpm. Add 30g of anhydrous sodium sulfate and stir for 50min until completely dissolved. Then add 25g of 50% sodium hydroxide solution and dropwise add 40g of 1,4-butanediol glycidyl ether over 7h. Allow the reaction to proceed for 18h, and then wash 6 times with deionized water.

[0066] (8) Repeat step (7) twice more to obtain large-pore, high-rigidity agarose microspheres. Electron micrographs are shown below. Figure 5 , Figure 6 and Figure 11 As shown, the surface of the microspheres is uniformly distributed with large pores with a diameter of 200-300 nm.

[0067] Example 4

[0068] (1) Add 100 mL of purified water with a conductivity of 0.5 μS / cm to a three-necked flask, and add 8 g of agarose (gel strength 1200 g / cm). 2 The solid powder was placed in a water bath and heated to 92°C. It was stirred at 200 rpm to dissolve the powder and kept in a sol state until its viscosity was about 300 mPa·S. Then the temperature was lowered to 60°C.

[0069] (2) Add 10g of polyvinylpyrrolidone and dissolve for 30 minutes.

[0070] (3) During the dissolution of agarose, add 5g of sodium dodecyl sulfate to 400g xylene, then add 4g of carrageenan, heat the water bath to 60℃, and stir to mix evenly.

[0071] (4) Quickly transfer the agarose aqueous solution obtained in step (2) to the oil phase in step (3) and mix thoroughly by stirring for 30 minutes.

[0072] (5) After the microspheres meet the size requirements, they are cooled and cured. The curing ends when the internal temperature drops below 20℃.

[0073] (6) Pour the solidified microsphere emulsion into 2500g of purified water for washing, stir thoroughly and let stand for 20h. After the sedimentation and stratification are complete, remove the upper liquid. Repeat this process 6 times to obtain large-pore agarose microspheres.

[0074] (7) Drain the water from the large-pore agarose microspheres cleaned in step (6) (approximately 100g), add 100g of purified water, raise the internal temperature to 60℃, and stir at 200rpm. Add 35g of anhydrous sodium sulfate and stir for 50min until completely dissolved. Then add 30g of 50% sodium hydroxide solution and dropwise add 45g of allyl glycidyl ether. The addition is completed in 8h, and the reaction is allowed to proceed for 18h. Wash 6 times with deionized water.

[0075] (8) Repeat step (7) twice more to obtain large-pore, high-rigidity agarose microspheres. Electron micrographs are shown below. Figure 7 , Figure 8 and Figure 12 As shown, the surface of the microspheres is uniformly distributed with relatively large pores with a diameter of 100-200 nm.

[0076] Detection example

[0077] 1. Weigh 100g of the microspheres prepared in Example 2 of this invention and add them to 100mL of pure water. Mix thoroughly and pour into an XK26 / 30 chromatography column. Allow to settle naturally for 15min.

[0078] 2. Press the column at 0.1 MPa for 6 minutes and record the column height h.

[0079] 3. Flow rate was measured under different pressures. The time required to collect 500 mL of outflow liquid at different pressures was measured in seconds.

[0080] Record the temperature T of the effluent (corresponding to different viscosity coefficients i under different temperature conditions).

[0081] 4. Calculation formula:

[0082] 5. Summarize the recorded data and create a pressure-flow rate curve, such as... Figure 13 As shown in the graph, the linear flow rate of the large-pore, high-rigidity agarose microspheres can reach 700 cm / h at 1 bar (0.1 MPa), which is more than 50% higher than the pressure flow rate of currently commercially available large-pore agarose microspheres. Furthermore, the back pressure does not change significantly with the increase of flow rate.

[0083] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing a large-pore high-rigidity agarose microsphere, comprising: adding a pore-forming agent selected from hydroxyethyl cellulose, carboxymethyl cellulose, polyvinyl alcohol or polyvinylpyrrolidone to an agarose aqueous solution, then transferring the sugar solution to an oil phase containing an emulsifier and a stabilizer for emulsification, transferring the emulsion to purified water after temperature reduction and solidification, dissolving the pore-forming agent in the purified water by stirring, then removing the upper oil phase to obtain a large-pore agarose microsphere; and then draining the water from the obtained large-pore agarose microsphere, adding purified water, anhydrous sodium sulfate, a sodium hydroxide solution and a crosslinking agent, and repeatedly washing the obtained large-pore high-rigidity agarose microsphere after reaction.

2. The method of claim 1, wherein the agarose microspheres have a diameter of 100- 500 μm and a rigidity of 1.0-2.

0. The agarose aqueous solution is prepared by adding agarose with a mass fraction of 2-10% to purified water with a conductivity of 0.01-20 μS / cm, heating to 60-100 ℃, stirring and dissolving, maintaining the sol state until the viscosity is 10-500 mPa·S, and then reducing the temperature to 40-60 ℃ to obtain the agarose aqueous solution.

3. The method of claim 1, wherein the agarose microspheres have a diameter of 100- 500 μm and a rigidity of 1.0-2.

0. The pore-forming agent with a mass fraction of 5-10% of the agarose aqueous solution is added to the agarose aqueous solution, and stirred and dissolved for 10-40 minutes.

4. The method of claim 1, wherein the agarose microspheres have a diameter of 100- 500 μm and a rigidity of 1.0-2.

0. The oil phase containing an emulsifier and a stabilizer is prepared by: adding an emulsifier and a stabilizer to a water-insoluble organic solvent, heating to 30-80 ℃ in a water bath, and stirring and uniformly mixing; the water-insoluble organic solvent is 1-10 times the mass of the agarose aqueous solution, and the emulsifier and the stabilizer are 0.2-5% and 0.1-2%, respectively, of the mass fraction of the water-insoluble organic solvent.

5. The method of claim 4, wherein the agarose microspheres have a diameter of 100- 500 μm and a rigidity of 1.0-2.

0. The water-insoluble organic solvent is selected from one or more of heptane, benzene, toluene, xylene, carbon tetrachloride and dichloroethane; and / or the emulsifier is selected from one or more of polyethylene glycol polyoxyethylene ether phosphate, isomeric tridecanol polyoxyethylene ether phosphate, oleyl alcohol polyether phosphate, sodium dodecyl sulfate and sodium dodecyl sulfonate; and / or the stabilizer is selected from one or more of cellulose acetate butyrate, cetyl alcohol and carrageenan.

6. The method for preparing large-pore, high-rigidity agarose microspheres according to claim 1, characterized in that, The emulsion is obtained by stirring and uniformly mixing for 10-30 minutes, and the microspheres are cooled and solidified after the particle size meets the requirements; the solidification is completed when the internal temperature is reduced to below 20 ℃, and the microsphere emulsion is obtained.

7. The method of claim 1, wherein the agarose microspheres have a diameter of 100- 500 μm and a rigidity of 1.0-2.

0. After the emulsion is cooled and solidified, the emulsion is poured into 2-10 times the mass of purified water, stirred and uniformly mixed, and then left to stand for 12-24 hours; the upper liquid is removed after sedimentation, and the process is repeated 4-8 times to obtain the large-pore agarose microsphere.

8. The method for preparing large-pore, high-rigidity agarose microspheres according to claim 1, characterized in that, The purified water with a mass fraction of 25-75% of the microspheres is added to increase the internal temperature to 30-70 ℃, and then stirred and uniformly mixed; the anhydrous sodium sulfate with a mass fraction of 20-60% of the microspheres is added and stirred and dissolved; the 50% sodium hydroxide solution with a mass fraction of 15-50% of the microspheres is added, and the crosslinking agent with a mass fraction of 30-60% of the microspheres is added dropwise; the dropwise addition is completed within 6-8 hours, and the reaction is performed for 16-20 hours; the obtained large-pore high-rigidity agarose microsphere is washed with deionized water for 6-8 times; the whole process is repeated twice. 9.A large-pore high-rigidity agarose microsphere prepared by the method for preparing a large-pore high-rigidity agarose microsphere according to any one of claims 1-8.

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