Magnetic polysaccharide separation and purification microspheres, protein in-situ purification microspheres and preparation method and application of magnetic polysaccharide separation and purification microspheres and protein in-situ purification microspheres

Abstract

The invention belongs to the technical field of separation and purification, and particularly relates to a magnetic polysaccharide separation and purification microsphere, a protein in-situ purification microsphere, a preparation method and application. Protein in-situ purified microspheres can be obtained by coupling the magnetic polysaccharide separation and purification microspheres provided by the invention with recombinant protein A; in the preparation process of the magnetic polysaccharide separation and purification microspheres, the steps of swelling, incubating, cross-linking and the like are carried out, so that the performance of the magnetic polysaccharide separation and purification microspheres is greatly improved, the protein separation and purification effect of the protein in-situ purification microspheres is improved, the requirements of pharmaceutical enterprises on separation and purification of various biomacromolecules can be met, and the application prospect is wide. The technical problem that a high-performance separation and purification medium is lacked in the prior art is solved.

Description

Technical Field

[0001] The present application belongs to the field of separation and purification technology, and in particular relates to a magnetic polysaccharide separation and purification microsphere, a protein in situ purification microsphere, and a preparation method and application thereof. Background Art

[0002] Column chromatography is a commonly used method for separation and purification of biomacromolecules. The separation and purification process is to fill cross-linked agarose microspheres and other polysaccharide microspheres as liquid chromatography media in the column. When the biomacromolecules flow through the liquid chromatography medium driven by the aqueous phase, different types of molecules will flow out of the column in order from large to small and thus be separated and purified. For column chromatography, cross-linked agarose microspheres and other polysaccharide microspheres are filled as liquid chromatography media in longer and thinner columns, which can adsorb more target proteins and achieve better separation and purification effects. However, longer and thinner columns have greater column pressure, and when used for separation and purification of biomacromolecules with more impurities such as fermentation broth, biomacromolecules with more impurities such as fermentation broth will be blocked in the column, resulting in crushing of cross-linked agarose microspheres and other polysaccharide microspheres filled in the column. Therefore, the method of using column chromatography to separate and purify biomacromolecules is limited, and it is necessary to develop new methods for separation and purification of biomacromolecules.

[0003] Biomagnetic beads are a kind of paramagnetic particles, which are usually prepared by loading nanomagnetic particles in polysaccharide particles. The polysaccharide particles loaded with nanomagnetic particles can undergo rapid aggregation, dispersion and other movements under the control of a magnetic field. After binding to specific ligands on the surface of the biomagnetic beads, they can be used for immunoassays. However, the current biomagnetic beads have a small particle size and are suitable for immunoassays in laboratories. However, biomagnetic beads with small particle sizes are easy to aggregate, which is not conducive to the contact and adsorption of biomacromolecules to be separated by the biomagnetic beads. In addition, the magnetic particle loading effect of the currently prepared biomagnetic beads is not good, resulting in weak magnetism of the biomagnetic beads, increasing the difficulty of controlling movement in a magnetic field, and being not conducive to controlling the contact and adsorption of biomacromolecules by the biomagnetic beads in a magnetic field, which makes the amount of biomacromolecules adsorbed by the current biomagnetic beads not high. In addition, the magnetic retention effect of some current biomagnetic beads is not good, the magnetic particles are easy to fall off, and the biomagnetic beads are difficult to reuse. Therefore, the performance of the current biomagnetic beads is not high, and they are often used for immunoassays, but are not suitable for separation and purification in the industrial field.

[0004] However, in recent years, with the rapid development of biopharmaceuticals such as monoclonal antibodies, fusion proteins, vaccines, insulin, and peptides, the separation and purification of antibody drugs has become the core production link of pharmaceutical companies, which directly determines the purity and quality of drugs. Therefore, it is necessary to develop new separation and purification media to meet the needs of separation and purification in the industrial field. Summary of the invention

[0005] In view of this, the present application provides a magnetic polysaccharide separation and purification microsphere, a protein in situ purification microsphere, and a preparation method and application thereof, which are used to solve the technical problem of the lack of high-performance separation and purification media in the prior art.

[0006] The first aspect of the present application provides a method for preparing magnetic polysaccharide separation and purification microspheres, the preparation method comprising the steps of:

[0007] Step S1, adding polysaccharide into water and heating to dissolve, to obtain a polysaccharide aqueous solution;

[0008] Step S2, adding the magnetic particle suspension to the polysaccharide aqueous solution and stirring evenly to obtain a magnetic polysaccharide aqueous solution;

[0009] Step S3, emulsifying the magnetic polysaccharide aqueous solution into the oil phase component containing the emulsifier to obtain a water-in-oil emulsion in which the magnetic polysaccharide aqueous droplets are dispersed;

[0010] Step S4, solidifying the dispersed phase magnetic polysaccharide water droplets in the water-in-oil emulsion into gel to obtain a suspension of dispersed magnetic polysaccharide gel microspheres;

[0011] Step S5, sedimenting and stratifying the suspension containing dispersed magnetic polysaccharide gel microspheres to obtain magnetic polysaccharide gel microspheres;

[0012] Step S6, swelling the magnetic polysaccharide gel microspheres in an aqueous solution to obtain magnetic polysaccharide gel microspheres with modified surface area;

[0013] Step S7, mixing and incubating the surface area modified magnetic polysaccharide gel microspheres and the magnetic nanoparticle suspension to obtain magnetically modified magnetic polysaccharide gel microspheres;

[0014] Step S8, mixing the magnetically modified magnetic polysaccharide gel microspheres and the cross-linking agent and performing a cross-linking reaction to obtain magnetic polysaccharide separation and purification microspheres.

[0015] Preferably, in step S4, the curing to form gel is performed by lowering the temperature to form gel or adding a cross-linking agent to form gel.

[0016] Preferably, in step S4, lowering the temperature comprises lowering the temperature to 20-30° C.; adding a cross-linking agent comprises adding at least one cross-linking agent selected from epichlorohydrin, 1-chloro-2,3-epoxypropane, butanediol glycidyl ether or divinyl sulfone.

[0017] Preferably, in step S1 to step S3, the temperature of the polysaccharide aqueous solution, magnetic polysaccharide aqueous solution and water-in-oil emulsion is 60-100° C.;

[0018] Preferably, in step S2 and step S7, the particle size of the magnetic nanoparticles in the magnetic nanoparticle suspension is 30-100 nm;

[0019] The particle size of the surface area modified magnetic polysaccharide gel microspheres is 30-200 μm.

[0020] Preferably, in step S1, the polysaccharide is selected from at least one of agarose, dextran and cellulose.

[0021] Preferably, the polysaccharide is selected from agarose.

[0022] Preferably, in step S3, the magnetic nanoparticles are selected from at least one of ferroferric oxide nanoparticles, cobalt oxide nanoparticles, and nickel oxide nanoparticles.

[0023] Preferably, in step S3, the emulsifier is selected from at least one of Tween 80, Tween 60, Tween 40, Tween 20, Span 65, Span 85 and Span 80;

[0024] The oil phase component is selected from at least one of liquid paraffin, toluene, petroleum ether, n-hexane, n-heptane or n-octane.

[0025] Preferably, in step S8, the cross-linking agent is selected from at least one of epichlorohydrin, epibromohydrin, 2,3-dibromopropanol, 1,4-butanediol diglycidyl ether, and glutaraldehyde.

[0026] Preferably, in step S1, the mass volume concentration of the polysaccharide aqueous solution is 3-10% (w / v).

[0027] Preferably, in step S2, the mass volume concentration of the magnetic particle suspension is 1-4% (w / v);

[0028] The volume ratio of the magnetic particle suspension to the polysaccharide aqueous solution is 1:2-1:5.

[0029] Preferably, in step S3, the mass volume concentration of the oil phase component containing the emulsifier is 0.1-5% (w / v).

[0030] Preferably, in step S6, the swelling time is 8 to 24 hours.

[0031] Preferably, in step S7, the incubation time is 8 to 24 hours.

[0032] The second aspect of the present application provides a protein in situ purification microsphere, comprising the magnetic polysaccharide separation and purification microsphere and recombinant protein A described in the first aspect;

[0033] The recombinant protein A is coupled to the surface of the magnetic polysaccharide separation and purification microspheres.

[0034] The third aspect of the present application provides a method for preparing protein in situ purification microspheres, which can be used to prepare the protein in situ purification microspheres described in the second aspect. The preparation method comprises the steps of:

[0035] Step S9, subjecting the separated and purified magnetic polysaccharide microspheres to an activation reaction to obtain activated polysaccharide magnetic microspheres with epoxy groups grafted on the surface;

[0036] Step S10, coupling reaction is performed on the activated polysaccharide magnetic microspheres with epoxy groups grafted on the surface and the recombinant protein A to obtain protein in situ purified microspheres.

[0037] Preferably, in step S9, the activation reaction process is: subjecting the magnetic polysaccharide separation and purification microspheres, epibromohydrin, 1,4-butanediol diglycidyl ether and sodium borohydride to an activation reaction under alkaline conditions to obtain activated polysaccharide magnetic microspheres with epoxy groups grafted on the surface.

[0038] Preferably, in step S10, the coupling reaction process is: reacting the activated polysaccharide magnetic microspheres with epoxy groups grafted on the surface and the recombinant protein A in a buffer system with a pH value of 7.5-8.5 to obtain protein in situ purified microspheres.

[0039] Preferably, after step S10, the method further comprises the step of using an ethanolamine solution to block unreacted epoxy groups on the surface of the protein in situ purified microspheres.

[0040] The fourth aspect of the present application provides the use of the protein in situ purification microspheres described in the third aspect in the field of protein purification or detection.

[0041] The fifth aspect of the present application provides a method for in situ purification of a protein, the method comprising the steps of:

[0042] Step A, adding a protein in situ purification microsphere and a protein fermentation liquid as described above into a purification container;

[0043] Step B, applying a moving magnetic field around the purification container to control the cyclic movement of the protein in situ purification microspheres in the purification container to obtain the protein in situ purification microspheres that adsorb the target protein;

[0044] Step C: applying a constant magnetic field to separate and collect the protein in situ purification microspheres adsorbing the target protein, and eluting the protein in situ purification microspheres adsorbing the target protein to obtain the purified target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0046] Figure 1 Schematic diagram of the separation and purification process provided in Experimental Example 1 of this application. DETAILED DESCRIPTION

[0047] The present application provides a magnetic polysaccharide separation and purification microsphere, a protein in situ purification microsphere, and a preparation method and application thereof, which are used to solve the technical problem of the lack of high-performance separation and purification media in the prior art.

[0048] The technical solution of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0049] In view of the low performance of currently prepared biological magnetic beads with small particle size and weak magnetism, it is difficult to meet the requirements of pharmaceutical companies for separation and purification of monoclonal antibodies, fusion proteins, vaccines, insulin, peptides and other biological macromolecules; the present application provides a method for preparing magnetic polysaccharide separation and purification microspheres, the preparation method comprising: firstly adding polysaccharide into water and heating to dissolve to obtain polysaccharide aqueous solution; then adding magnetic particle suspension into polysaccharide aqueous solution and stirring evenly to obtain magnetic polysaccharide aqueous solution; then adding magnetic polysaccharide aqueous solution into oil phase component containing emulsifier to emulsify to obtain oil-in-water emulsion with dispersed magnetic polysaccharide water droplets; then The dispersed phase magnetic polysaccharide water droplets in the oil-in-water emulsion are solidified into a gel to obtain a suspension of dispersed magnetic polysaccharide gel microspheres; the suspension of dispersed magnetic polysaccharide gel microspheres is then settled and layered to obtain magnetic polysaccharide gel microspheres; the magnetic polysaccharide gel microspheres are then swelled in an aqueous solution to obtain surface area modified magnetic polysaccharide gel microspheres; the surface area modified magnetic polysaccharide gel microspheres and a magnetic nanoparticle suspension are then mixed and incubated to obtain magnetically modified magnetic polysaccharide gel microspheres; and the magnetically modified magnetic polysaccharide gel microspheres and a cross-linking agent are mixed and cross-linked to obtain magnetic polysaccharide separation and purification microspheres.

[0050] In the preparation method of magnetic polysaccharide separation and purification microspheres provided in the present application, after obtaining the magnetic polysaccharide aqueous solution, it is necessary to add an oil phase component containing an emulsifier to emulsify it, so that the oil phase component covers the magnetic polysaccharide aqueous solution component, and the magnetic polysaccharide aqueous solution is dispersed in the oil phase continuous phase in the form of droplets to form an oil-in-water emulsion; then the dispersed phase magnetic polysaccharide water droplets in the emulsion are solidified into gel and doped with magnetic particles to obtain solidified magnetic polysaccharide gel microspheres, and then sedimentation and stratification are performed to separate them from the oil phase to obtain magnetic polysaccharide gel microspheres; and the present application also swells the magnetic polysaccharide gel microspheres, and the swelling process will increase the particle size of the magnetic polysaccharide gel microspheres and the external surface area. At the same time, the pores rich in the magnetic polysaccharide gel microspheres themselves will also expand, so that the internal surface area is increased, and surface-area-modified magnetic polysaccharide gel microspheres are obtained by swelling. The surface-area-modified magnetic polysaccharide gel microspheres have an increased external surface area, which increases the surface energy of the magnetic polysaccharide gel microspheres, makes them less likely to aggregate, and are easy to interact with biological macromolecules. The contact and adsorption increase the adsorption amount of biomacromolecules, and the increased internal surface area enables the magnetic polysaccharide gel microspheres to load more magnetic particles during the incubation process, thereby enhancing the magnetic properties of the magnetic polysaccharide gel microspheres, and improving the ability to control the contact and adsorption of biological magnetic beads with biomacromolecules in a magnetic field, thereby increasing the adsorption amount of biomacromolecules; at the same time, after the magnetic polysaccharide gel microspheres are swollen and incubated, the present application also adds a cross-linking agent to carry out a cross-linking reaction step, and after the cross-linking reaction, the magnetic polysaccharide gel microspheres are compressed, and the magnetic nanoparticles can be stably fixed in the pores of the magnetic polysaccharide gel microspheres, which is beneficial to the long-term maintenance of the magnetic properties of the magnetic polysaccharide gel microspheres. The magnetic nanoparticles are not easy to fall off, and the magnetic polysaccharide gel microspheres can be reused many times. Therefore, the preparation method of the magnetic polysaccharide separation and purification microspheres provided in the present application can prepare high-performance magnetic polysaccharide separation and purification microspheres, which can meet the requirements of pharmaceutical companies for separation and purification of monoclonal antibodies, fusion proteins, vaccines, insulin, polypeptides and other biomacromolecules.

[0051] Preferably, in the preparation method of magnetic polysaccharide separation and purification microspheres provided in the present application, it is necessary to control the temperature so that the polysaccharide molecules are first dissolved and then solidified into a gel, and the temperature of the polysaccharide aqueous solution, the magnetic polysaccharide aqueous solution and the oil-in-water emulsion needs to be controlled at 60-100°C; and the subsequent solidification into a gel, when the polysaccharide dispersed in the oil-in-water emulsion is agarose, it is necessary to lower the temperature to 20-30°C to solidify it into a gel, and when the dispersed polysaccharide is dextran, it is necessary to add a cross-linking agent such as epichlorohydrin to solidify it.

[0052] Preferably, in the preparation method of magnetic polysaccharide separation and purification microspheres provided in the present application, the particle size of the magnetic polysaccharide gel microspheres and the magnetic nanoparticles needs to be controlled so that the surface area of ​​the microspheres is as large as possible and loads more magnetic nanoparticles. The particle size of the magnetic polysaccharide gel microspheres after swelling is controlled to be 30~200um, and the particle size of the magnetic nanoparticles is controlled to be 30~100nm.

[0053] Preferably, in the preparation method of magnetic polysaccharide separation and purification microspheres provided in the present application, different polysaccharide molecules have different hydrophilicities. In order to improve the biocompatibility of the microspheres and facilitate their use in protein separation and purification, polysaccharide molecules with good hydrophilicity should be selected as much as possible, such as agarose, dextran or cellulose. Since the hydrophilicity of cellulose is average, agarose is further preferred as the polysaccharide.

[0054] Preferably, in the preparation method of magnetic polysaccharide separation and purification microspheres provided in the present application, in order to ensure that the surface area of ​​the magnetic polysaccharide gel microspheres is fully increased during the swelling process, the swelling time cannot be too short; it needs to be controlled within 8 to 24 hours.

[0055] Preferably, in the preparation method of magnetic polysaccharide separation and purification microspheres provided in the present application, in order to promote the stable fixation of magnetic nanoparticles in the microspheres during the cross-linking reaction, it is necessary to control the type of cross-linking agent. The present application selects cross-linking agents such as epichlorohydrin, epibromohydrin, 2,3-dibromopropanol, 1,4-butanediol diglycidyl ether, and glutaraldehyde.

[0056] At the same time, for the specific application of the magnetic polysaccharide separation and purification microspheres provided in this application, this application selects them for protein purification, and the magnetic polysaccharide separation and purification microspheres are coupled with recombinant protein A to obtain protein in situ purification microspheres.

[0057] Furthermore, the present application also provides the use of protein in situ purification microspheres for protein purification or detection.

[0058] Furthermore, the specific use of protein purification is: adding protein in situ purification microspheres to protein fermentation liquid, then applying a mobile magnetic field to make the protein in situ purification microspheres circulate in the protein fermentation liquid, contact and adsorb the protein, and obtain protein in situ purification microspheres adsorbing the target protein; then applying a constant magnetic field to separate and collect the protein in situ purification microspheres adsorbing the target protein, and then eluting the target protein to obtain the purified target protein. And because the protein in situ purification microspheres provided by the present application undergo a cross-linking reaction so that the magnetic nanoparticles are not easy to fall off, the protein in situ purification microspheres can be reused.

[0059] The magnetic polysaccharide separation and purification microspheres and protein in situ purification microspheres provided in the present application will be specifically described below in conjunction with embodiments and experimental examples.

[0060] Example 1

[0061] Example 1 of the present application provides a method for preparing magnetic polysaccharide separation and purification microspheres, which includes the steps of preparing a polysaccharide aqueous solution, preparing a magnetic polysaccharide aqueous solution, preparing an oil-in-water emulsion, preparing a suspension of dispersed magnetic polysaccharide gel microspheres, sedimentation and stratification, surface area modification, incubation of loaded magnetic nanoparticles, and cross-linking reaction.

[0062] The step of preparing the polysaccharide aqueous solution includes: weighing 50g of agarose solid and dissolving it in 1000mL of water at 90°C with magnetic stirring, and then cooling to 80°C to prepare an agarose aqueous solution with a mass volume concentration of 5% (w / v).

[0063] The steps of preparing the magnetic polysaccharide aqueous solution include: dissolving 10g of ferroferric oxide nanoparticles (50nm) in 500mL of water to obtain a ferroferric oxide magnetic nanoparticle suspension with a mass volume concentration of 2% (w / v), then adding the ferroferric oxide magnetic nanoparticle suspension to the agarose aqueous solution, and stirring at 80°C for 30min to form a magnetic agarose solution.

[0064] The steps of preparing the water-in-oil emulsion include: stirring and dissolving 50g of Span 80 and 50g of Tween 20 in 2000mL of toluene solution to obtain an oil phase component containing an emulsifier, then adding the magnetic agarose solution to the oil phase component containing the emulsifier, mechanically stirring and emulsifying and dispersing at 80°C for 1h to obtain the water-in-oil emulsion, wherein the continuous phase in the water-in-oil emulsion is toluene, and the dispersed phase is magnetic polysaccharide water droplets.

[0065] The steps of preparing a suspension of dispersed magnetic polysaccharide gel microspheres include: lowering the temperature of the water-in-oil emulsion to 30°C, solidifying agarose into gel and encapsulating magnetic nanoparticles, and obtaining a suspension of dispersed magnetic polysaccharide gel microspheres.

[0066] The step of sedimentation and stratification includes: sedimentation and stratification of the suspension containing dispersed magnetic polysaccharide gel microspheres, separation of the magnetic polysaccharide gel microspheres from the suspension, and obtaining the magnetic polysaccharide gel microspheres.

[0067] The step of surface area modification includes: adding magnetic polysaccharide gel microspheres into water and swelling them for 8 hours to obtain magnetic polysaccharide gel microspheres with modified surface area.

[0068] The step of incubating the loaded magnetic nanoparticles includes: dissolving 10 g of ferroferric oxide nanoparticles (50 nm) in 500 mL of water to obtain a ferroferric oxide magnetic nanoparticle suspension with a mass volume concentration of 2% (w / v), and then adding surface area modified magnetic polysaccharide gel microspheres to the ferroferric oxide magnetic nanoparticle suspension and incubating for 8 hours, so that the magnetic nanoparticles enter the microsphere pores by electrostatic adsorption to obtain magnetically modified magnetic polysaccharide gel microspheres.

[0069] The steps of the cross-linking reaction include: washing the surface of the magnetically modified magnetic polysaccharide gel microspheres, dispersing 10 g in 20 mL of deionized water, gradually heating to 37° C., reacting for 2 h, then slowly dropping 1.0 mL of epichlorohydrin and 1.2 mL of 40% NaOH solution into the system, wherein the NaOH solution contains 2% NaBH4, and continuing the reaction for 12 h to obtain magnetic polysaccharide separation and purification microspheres.

[0070] Example 2

[0071] Example 2 of the present application provides a method for preparing magnetic polysaccharide separation and purification microspheres, the preparation method comprising the steps of preparing a polysaccharide aqueous solution, preparing a magnetic polysaccharide aqueous solution, preparing an oil-in-water emulsion, preparing a suspension of dispersed magnetic polysaccharide gel microspheres, a sedimentation and stratification step, a surface area modification step, an incubation step for loaded magnetic nanoparticles, and a cross-linking reaction step.

[0072] The step of preparing the polysaccharide aqueous solution includes: weighing 80g of dextran solid and dissolving it in 1000mL of water at 90°C with magnetic stirring, and then cooling to 80°C to prepare a dextran aqueous solution with a mass volume concentration of 8% (w / v).

[0073] The steps of preparing the magnetic polysaccharide aqueous solution include: dissolving 10g of ferroferric oxide nanoparticles (50nm) in 250mL of water to obtain a ferroferric oxide magnetic nanoparticle suspension with a mass volume concentration of 4% (w / v), then adding the ferroferric oxide magnetic nanoparticle suspension to the dextran aqueous solution, and stirring at 80°C for 30 minutes to form a magnetic dextran solution.

[0074] The steps of preparing the water-in-oil emulsion include: stirring and dissolving 20g of Span 85 and 20g of Tween 80 in 2000mL of toluene solution to obtain an oil phase component containing an emulsifier, then adding the magnetic dextran solution to the oil phase component containing the emulsifier and mechanically stirring and emulsifying and dispersing at 80°C for 1h to obtain the water-in-oil emulsion, wherein the magnetic dextran solution is dispersed in the emulsion in the form of droplets.

[0075] The steps of preparing a suspension of dispersed magnetic polysaccharide gel microspheres include: adding epichlorohydrin crosslinking agent to an oil-in-water emulsion to 60 degrees to solidify the dextran into a gel and wrap the magnetic nanoparticles to obtain a suspension of dispersed magnetic polysaccharide gel microspheres.

[0076] The step of sedimentation and stratification includes: sedimentation and stratification of the suspension containing dispersed magnetic polysaccharide gel microspheres, separation of the magnetic polysaccharide gel microspheres from the suspension, and obtaining the magnetic polysaccharide gel microspheres.

[0077] The step of surface area modification includes: adding magnetic polysaccharide gel microspheres into water and swelling them for 12 hours to obtain magnetic polysaccharide gel microspheres with modified surface area.

[0078] The step of incubating the loaded magnetic nanoparticles includes: dissolving 10 g of ferroferric oxide nanoparticles (50 nm) in 250 mL of water to obtain a ferroferric oxide magnetic nanoparticle suspension with a mass volume concentration of 4% (w / v), and then adding surface area modified magnetic polysaccharide gel microspheres to the ferroferric oxide magnetic nanoparticle suspension and incubating for 12 hours, so that the magnetic nanoparticles enter the microsphere pores by electrostatic adsorption to obtain magnetically modified magnetic polysaccharide gel microspheres.

[0079] The steps of the cross-linking reaction include: washing the surface of the magnetically modified magnetic polysaccharide gel microspheres, dispersing 8.5 g in 20 mL of deionized water, gradually heating to 37° C., reacting for 2 h, then slowly dropping 1.0 mL of epichlorohydrin and 1.2 mL of 40% NaOH solution into the system, wherein the NaOH solution contains 2% NaBH4, and continuing the reaction for 12 h to obtain magnetic polysaccharide separation and purification microspheres.

[0080] Comparative Example 1

[0081] Comparative Example 1 of the present application provides a method for preparing magnetic agarose microspheres. The difference between the preparation method and Examples 1-2 is that there is no step of surface area modification and no step of incubating loaded magnetic nanoparticles; the preparation method includes the steps of preparing a polysaccharide aqueous solution, preparing a magnetic polysaccharide aqueous solution, preparing an oil-in-water emulsion, preparing a suspension of dispersed magnetic polysaccharide gel microspheres, a sedimentation and stratification step, and a cross-linking reaction step.

[0082] 50 g of agarose solid was weighed and dissolved in 1000 mL of water at 90° C. with magnetic stirring, and then cooled to 80° C. to prepare an agarose aqueous solution with a mass volume concentration of 5% (w / v).

[0083] The steps of preparing the magnetic polysaccharide aqueous solution include: dissolving 10g of ferroferric oxide nanoparticles (50nm) in 500mL of water to obtain a ferroferric oxide magnetic nanoparticle suspension with a mass volume concentration of 2% (w / v), then adding the ferroferric oxide magnetic nanoparticle suspension to the agarose aqueous solution, and stirring at 80°C for 30min to form a magnetic agarose solution.

[0084] The steps of preparing the water-in-oil emulsion include: stirring and dissolving 50g of Span 80 and 50g of Tween 20 in 2000mL of toluene solution to obtain an oil phase component containing an emulsifier, then adding the magnetic agarose solution to the oil phase component of the emulsifier, mechanically stirring and emulsifying and dispersing at 80°C for 1h, to obtain the water-in-oil emulsion.

[0085] The step of preparing a suspension of dispersed magnetic polysaccharide gel microspheres comprises: lowering the temperature of the water-in-oil emulsion to 30°C, so that agarose solidifies into gel and wraps the magnetic nanoparticles, thereby obtaining a suspension of dispersed magnetic polysaccharide gel microspheres.

[0086] The step of sedimentation and stratification includes: sedimentation and stratification of the suspension containing dispersed magnetic polysaccharide gel microspheres, separation of the magnetic polysaccharide gel microspheres from the suspension, and obtaining the magnetic polysaccharide gel microspheres.

[0087] The steps of the cross-linking reaction include: washing the surface of the magnetically modified magnetic polysaccharide gel microspheres, dispersing 8.5 g in 20 mL of deionized water, gradually heating to 37° C., reacting for 2 h, then slowly dropping 1.0 mL of epichlorohydrin and 1.2 mL of 40% NaOH solution into the system, wherein the NaOH solution contains 2% NaBH4, and continuing the reaction for 12 h to obtain magnetic agarose microspheres.

[0088] Example 3

[0089] Example 3 of the present application provides a method for preparing protein in situ purification microspheres, which includes an activation reaction step and a coupling reaction step.

[0090] Among them, the activation reaction step includes: first, 150g of the microspheres provided in Examples 1-2 and Comparative Example 1 are rinsed with about 10 times the volume of injection water, and then 100mL of 1M NaOH solution, 30mL of epibromopropane and 70mL of 1,4-butanediol diglycidyl ether and 0.2g of sodium borohydride are added, stirred, reacted at 30°C for 1.5h, and after the reaction is completed, rinsed with a large amount of injection water to obtain three epoxy-activated microspheres. The steps of the coupling reaction include: adding 100 mL of the above-mentioned epoxy-activated microspheres and 150 mL of 0.1 mol / L borate buffer to a 500 mL reaction container, respectively, controlling the pH value of the system to 7.5 to 8.5, adding 14 g of genetically engineered recombinant protein A, reacting in a 37°C constant temperature system for 20 hours, stopping the reaction, and obtaining protein in situ purified microspheres; after preparation, it is also necessary to rinse the filler with about 10 times the volume of injection water, after rinsing, add 200 mL of 0.2M ethanolamine solution to block the unreacted epoxy groups on the surface of the microspheres, and react at 20°C for 10 hours. After the reaction is completed, rinse with a large amount of injection water; obtain magnetic microspheres for protein in situ purification, wherein the magnetic microspheres prepared by the microspheres provided in Example 1 for protein in situ purification are recorded as separation and purification microsphere sample 1, the magnetic microspheres prepared by the microspheres provided in Example 2 for protein in situ purification are recorded as separation and purification microsphere sample 2, and the magnetic microspheres prepared by the microspheres provided in Comparative Example 1 for protein in situ purification are recorded as separation and purification microsphere sample 3.

[0091] Experimental Example 1

[0092] Experimental Example 1 of the present application carried out a separation and purification experiment on the separation and purification microsphere samples 1 to 3 provided in Example 3.

[0093] The experimental process includes: adding samples 1 to 3 to the protein fermentation liquid respectively, applying a constantly changing magnetic field, controlling the magnetic microspheres to circulate in the solution, adsorbing the target protein, and the adsorption time is 20 minutes to obtain samples 1 to 3 adsorbing the target protein; then adjusting the changing magnetic field to a constant magnetic field, aggregating samples 1 to 3 adsorbing the target protein, and removing the remaining protein fermentation liquid; then using HAC-NaAC buffer to wash the magnetic microspheres to remove impurities weakly bound to the medium or antibody, and then adding a strong acid buffer for elution, applying a constant magnetic field to collect and separate the magnetic microspheres, and obtaining a purified antibody protein solution; repeating the above steps three times, so that samples 1 to 3 adsorb the target protein in the protein fermentation liquid as much as possible, and obtaining a purified antibody protein solution.

[0094] At the same time, this experimental example also uses Protein A affinity chromatography column separation and purification as a comparative experiment. The experimental process includes: adding the protein fermentation broth to a Protein A gel equilibrium liquid chromatography column with a column height of 8 to 12 cm and 6 to 8 times the column bed volume, loading 60 to 80% of the maximum processing capacity of the Protein A gel, and the contact time between the supernatant and the Protein A gel is 6 to 10 minutes. The flow-through liquid is collected to obtain the bacterial supernatant after Protein A adsorption; the immunoglobulin adsorbed on the chromatography column is eluted with an eluent, and the linear flow rate of the eluent is 1.0 to 1.5 cm / min to obtain a Protein A eluent; the Protein A eluent is repeated three times in step 2 to obtain a purified antibody protein solution;

[0095] In the process of separation and purification of Protein A affinity chromatography column, the Protein A gel used is MabSelectTMPrismA; the Protein A gel is filled into the chromatography column to obtain the Protein A chromatography column, and the Protein A chromatography column is balanced with 6 to 8 times the column bed volume of Protein A gel equilibration liquid at a linear flow rate of 1 cm / min to obtain a Protein A gel equilibration liquid chromatography column; the Protein A gel equilibration liquid contains 10 to 30 mmol / L sodium dihydrogen phosphate and 0.1 to 0.3 mol / L sodium chloride in water for injection, with a pH of 7.0 to 7.5; the eluent contains 0.1 to 0.5 mol / L glycine in water for injection, with a pH of 3.0 to 4.0.

[0096] The results of the separation and purification experiments of samples 1 to 3 and the separation and purification experiments using Protein A affinity chromatography columns are shown in Table 1.

[0097] Table 1: Results of separation and purification experiments

[0098]

[0099] As can be seen from Table 1, the recovery rate of protein separated and purified by Protein A affinity chromatography column is low, only 75.2%. Compared with the protein separated and purified by Protein A affinity chromatography column, the separation and purification microspheres (samples 1-3) provided in Examples 1-2 and Comparative Example 1 can better separate and purify proteins, with a better amount of purified protein, and the protein recovery rate reaches 88.7-93.6%. This shows that compared with the use of fixed-position fillers in the process of protein separation and purification by Protein A affinity chromatography column, the separation and purification microspheres (samples 1-3) provided in Examples 1-2 and Comparative Example 1 can use a magnetic field to control the movement of the microspheres, thereby increasing the probability of collision, contact and adsorption of the microspheres with the target protein in the protein fermentation broth, thereby greatly improving the utilization rate of the separation and purification microspheres and improving the adsorption capacity of the target protein.

[0100] It can be further seen from Table 1 that compared with the separation and purification microspheres (samples 1-2) provided in Examples 1-2, the separation and purification microspheres (sample 3) provided in Comparative Example 1 have a poor effect in separating and purifying proteins. This is because the separation and purification microspheres provided in Comparative Example 1 are not swelled, the separation and purification microspheres provided in Comparative Example 1 have a small particle size, are easily agglomerated, have a small surface area, and have a low probability of colliding, contacting and adsorbing the target protein in the protein fermentation broth during the separation and purification process, resulting in a small adsorption capacity for the target protein.

[0101] Experimental Example 2

[0102] Experimental Example 2 of the present application repeated the separation and purification experiments on the separated and purified microsphere samples 1 to 3 provided in Example 3, and calculated the amount of purified protein after repeating 3 times, 10 times and 20 times.

[0103] The experimental process is as follows: Samples 1 to 3 are added to the protein fermentation broth respectively, and a constantly changing magnetic field is applied to control the circulation of the magnetic microspheres in the solution to adsorb the target protein for 20 minutes, thereby obtaining samples 1 to 3 adsorbing the target protein; then the changing magnetic field is adjusted to a constant magnetic field, and samples 1 to 3 adsorbing the target protein are aggregated, and the remaining protein fermentation broth is removed; next, the magnetic microspheres are washed with HAC-NaAC buffer to remove impurities weakly bound to the medium or antibody, and then a strong acidic buffer is added for elution, and a constant magnetic field is applied to collect and separate the magnetic microspheres to obtain a purified antibody protein solution; the above steps are repeated three times, ten times, and twenty times to obtain a purified antibody protein solution, and the amount of purified protein is calculated; the experimental results are shown in Table 2.

[0104] Table 2: Results of repeated separation and purification experiments

[0105]

[0106] As can be seen from Table 2, the amount of purified protein in the separation and purification microsphere sample 3 decreased rapidly during the separation and purification experiment repeated ten times and twenty times. Only 90% of the purified protein was retained after ten repetitions, and only 75% of the purified protein was retained after twenty repetitions. This shows that during the separation and purification of the separation and purification microsphere sample 3, some magnetic particles precipitated and fell off, resulting in a decrease in the magnetic properties of the separation and purification microsphere sample 3, which is not conducive to adsorption concentration and protein purification; while the amount of purified protein in the separation and purification microsphere samples 1-2 decreased very slowly during the separation and purification experiment repeated ten times and twenty times, and more than 92% of the purified protein was retained after twenty repetitions. This shows that the magnetic polysaccharide separation and purification microspheres provided in the present application are cross-linked after swelling and incubation, so that the pores of the polysaccharide microspheres are tightened, and the magnetic particles are stably loaded inside the microspheres. In the subsequent protein adsorption separation and purification process, the magnetic nanoparticles are not easy to precipitate, and the magnetic properties can be maintained for a long time, thereby realizing the reuse of the separation and purification microspheres.

[0107] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing magnetic polysaccharide separation and purification microspheres, characterized in that: Includes steps: Step S1, adding polysaccharide into water and heating to dissolve, to obtain a polysaccharide aqueous solution; Step S2, adding the magnetic particle suspension to the polysaccharide aqueous solution and stirring evenly to obtain a magnetic polysaccharide aqueous solution; Step S3, emulsifying the magnetic polysaccharide aqueous solution into the oil phase component containing the emulsifier to obtain a water-in-oil emulsion in which the magnetic polysaccharide aqueous droplets are dispersed; Step S4, solidifying the dispersed phase magnetic polysaccharide water droplets in the water-in-oil emulsion into gel to obtain a suspension of dispersed magnetic polysaccharide gel microspheres; Step S5, sedimenting and stratifying the suspension containing dispersed magnetic polysaccharide gel microspheres to obtain magnetic polysaccharide gel microspheres; Step S6, swelling the magnetic polysaccharide gel microspheres in an aqueous solution to obtain magnetic polysaccharide gel microspheres with modified surface area; Step S7, mixing and incubating the surface area modified magnetic polysaccharide gel microspheres and the magnetic nanoparticle suspension to obtain magnetically modified magnetic polysaccharide gel microspheres; Step S8, mixing the magnetically modified magnetic polysaccharide gel microspheres and the cross-linking agent and performing a cross-linking reaction to obtain magnetic polysaccharide separation and purification microspheres.

2. The method for preparing magnetic polysaccharide separation and purification microspheres according to claim 1, characterized in that: In step S4, the curing to form gel is performed by lowering the temperature to form gel or adding a cross-linking agent to form gel.

3. The method for preparing magnetic polysaccharide separation and purification microspheres according to claim 1, characterized in that: In step S2 and step S7, the particle size of the magnetic nanoparticles in the magnetic nanoparticle suspension is 30-100 nm; The particle size of the surface area modified magnetic polysaccharide gel microspheres is 30-200 μm.

4. The method for preparing magnetic polysaccharide separation and purification microspheres according to claim 1, characterized in that: In step S1, the polysaccharide is selected from at least one of agarose, dextran and cellulose.

5. The method for preparing magnetic polysaccharide separation and purification microspheres according to claim 1, characterized in that: In step S6, the swelling time is 8 to 24 hours.

6. The method for preparing magnetic polysaccharide separation and purification microspheres according to claim 1, characterized in that: In step S8, the cross-linking agent is selected from at least one of epichlorohydrin, epibromohydrin, 2,3-dibromopropanol, 1,4-butanediol diglycidyl ether, and glutaraldehyde.

7. A protein in situ purification microsphere, characterized in that: It comprises the magnetic polysaccharide separation and purification microspheres according to any one of claims 1 to 6 and recombinant protein A; The recombinant protein A is coupled to the surface of the magnetic polysaccharide separation and purification microspheres.

8. A method for preparing protein in situ purification microspheres, characterized in that: Includes steps: Step S9, subjecting the magnetic polysaccharide separation and purification microspheres according to any one of claims 1 to 6 to an activation reaction to obtain activated polysaccharide magnetic microspheres with epoxy groups grafted on the surface; Step S10, coupling reaction is performed on the activated polysaccharide magnetic microspheres with epoxy groups grafted on the surface and the recombinant protein A to obtain protein in situ purified microspheres.

9. Use of the protein in situ purification microsphere according to any one of claims 1 to 6 in the field of protein purification or detection.

10. A method for in situ protein purification, characterized in that: Includes steps: Step A, adding the protein in situ purification microspheres and protein fermentation liquid according to claim 7 or 8 into a purification container; Step B, applying a moving magnetic field around the purification container to control the cyclic movement of the protein in situ purification microspheres in the purification container to obtain the protein in situ purification microspheres that adsorb the target protein; Step C: applying a constant magnetic field to separate and collect the protein in situ purification microspheres adsorbing the target protein, and eluting the protein in situ purification microspheres adsorbing the target protein to obtain the purified target protein.

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