A macroporous multi-nucleus structure agarose gel microsphere and a preparation method thereof

Abstract

The application discloses a macroporous multi-core structure agarose gel microsphere and a preparation method thereof. The shell layer of the microsphere is agarose gel with a pore size of 2-4 microns, and the core of the microsphere is an agarose small sphere with a particle size of not more than 50 microns, which is distributed in the interior of the macroporous gel. The preparation method is simultaneously provided. The core-shell type microsphere has the pore structure of the core and the macroporous structure of the shell layer, and the rich pore structure is beneficial to efficient mass transfer of biological molecules in the medium, and the existence of the core can reduce the structural softness caused by the macropore.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agarose gel microspheres, in particular to a macroporous multi-nuclear structure agarose gel microsphere and a preparation method thereof. BACKGROUND

[0002] Agarose is one of the natural polysaccharides obtained from agar extracted from marine red algae. Agarose chromatography medium has the advantages of good hydrophilicity, porous structure, high stability, low non-specific adsorption, and easy functionalization, and is widely used in chromatographic analysis of proteins, viruses, nucleic acids and peptides. Agarose gel microspheres are a mature gel filtration medium, which has been widely used as a chromatographic packing material for purifying biomolecules due to its inherent neutral, hydrophilic and porosity. Under the high requirements of biomolecule chromatographic separation, agarose microspheres can easily cope with proteins with smaller molecular weight, but the mass transfer rate of chromatographic separation of some proteins with larger molecular weight will be greatly reduced. The mass transfer rate is a problem that agarose microspheres need to overcome. An important influencing factor for the mass transfer of biological macromolecules inside the medium is the pore size inside the separation medium. In order to solve these problems, the performance of balanced agarose gel microspheres in the use process. Zhou first prepared super-porous agarose microspheres by using calcium carbonate particles as a porogen. The main principle is to uniformly disperse calcium carbonate particles as a solid porogen in an agarose aqueous solution, pour it into an oil phase containing a surfactant by a traditional emulsification method, and stir to form droplets. After cooling and solidification, agarose microspheres with calcium carbonate particles inside are obtained. Then, the calcium carbonate is dissolved and washed with dilute acid solution, at this time, large pores different from the agarose gel network pores are formed inside the microspheres. Zhao first prepared highly cross-linked macroporous agarose gel microspheres by combining pre-crosslinking and surfactant micelle swelling method in 2019. In 1996, Gustavsson first prepared macroporous agarose gel microspheres by using double emulsion method based on the preparation method of porous agarose gel microspheres proposed by Larsson (1992). The microspheres prepared by Gustavsson contain two kinds of pores inside, one is the diffusion pore of the original agarose gel, and the other is the super-large flow pore obtained by secondary emulsification, which greatly promotes the diffusion efficiency inside the particles, and is successfully applied to the purification of plasmid DNA.

[0003] The disadvantage of agarose as a natural material is soft and obvious compared with polymers. Therefore, in order to meet the needs of high speed chromatography, it is urgent to develop rigid agarose microspheres with enhanced mass transfer performance. Crosslinking is a commonly used technique to improve the mechanical strength of agarose microspheres. It chemically combines polysaccharide chains through crosslinking agents to stabilize the gel skeleton. Traditionally, crosslinking agents are introduced after the formation of gel particles, which means that the gel structure changes due to the addition of crosslinking agents, which increases the steric hindrance and hinders the diffusion process. Another way to alleviate the pressure caused by flow rate is to use larger particles, but at the cost of greatly increasing the mass transfer distance within the particles, which is not desirable for the separation of biological macromolecules. SUMMARY

[0004] In order to solve these problems and balance the performance of agarose gel microspheres during use, the present application proposes a preparation method of macroporous multi-core structure agarose gel microspheres. The method is characterized by using crosslinked agarose gel microspheres as the inner core and macroporous agarose microspheres prepared by the multiple emulsion method as the shell layer to synthesize macroporous multi-core core-shell structure. This core-shell type microsphere has both the fine pore structure of the inner core and the macroporous structure of the shell layer. The high-resolution chromatographic medium prepared by crosslinking treatment has a rich pore structure that is beneficial to the efficient mass transfer of biological molecules within the medium, and the existence of the inner core can reduce the structural softness caused by macropores.

[0005] The present application aims to provide a macroporous multi-core structure agarose gel microsphere.

[0006] In order to achieve the above-mentioned purpose, the present application provides a macroporous multi-core structure agarose gel microsphere, characterized in that the shell layer of the microsphere is agarose gel with a pore size of 2-4 μm, and the core of the microsphere is agarose beads with a particle size of not more than 50 μm, which are distributed inside the macroporous gel.

[0007] The present application also provides a preparation method of the macroporous multi-core structure agarose gel microsphere, which comprises the following steps:

[0008] S1. Preparation of inner core: mix and stir the oil phase and agarose solution to emulsify, then cool and solidify to form spherical shape; centrifuge the solution containing the spherical shape to discard the supernatant, and repeatedly precipitate and wash with deionized water to obtain the inner core;

[0009] The oil phase is obtained by mixing a surfactant and an oily substance, and the content of the surfactant is 0.5%-2% of the volume of liquid paraffin, and the oily substance is liquid paraffin;

[0010] The concentration of the agarose solution is 4%-6% w / v;

[0011] The volume ratio of the oil phase to the agarose solution is 200:(50-80); preferably, the volume ratio is 200:60;

[0012] The emulsification is 1200-3500 rpm for 10-30 min; the solidification is 500-600 rpm for cooling to 15-25℃;

[0013] S2 Crosslinking of the inner core agarose microspheres: the obtained inner core is placed in a mixed solution containing deionized water, lye, 1,4-dioxane, and crosslinking agent for reaction. After the reaction, the obtained microspheres are repeatedly washed with deionized water to remove NaOH and organic solvent in the microsphere solution, thereby obtaining the crosslinked inner core agarose microspheres.

[0014] The crosslinking agent is at least one of halogenated epoxide compounds and diglycidyl ether reagents; preferably, the halogenated epoxide compound is epichlorohydrin, and the diglycidyl ether reagent is selected from any one of ethylene glycol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether;

[0015] The ratio of the inner core, deionized water, lye, 1,4-dioxane, and crosslinking agent is 36 g: 24 mL: 14 mL: 14 mL: 14 mL;

[0016] The reaction conditions are 200-300 rpm, 20-38℃ for 2-8 h;

[0017] S3 Obtaining of the macroporous multi-core structure agarose gel microspheres:

[0018] S3.1 Preparation of the first o / w emulsion:

[0019] Preparation of the first aqueous phase: after heating the 4%-6% w / v agarose solution to be clear and transparent to completely dissolve the agarose, the crosslinked inner core agarose microspheres are added and uniformly mixed; the addition amount of the crosslinked inner core agarose microspheres is 5wt%-20wt% of the agarose solution;

[0020] Preparation of the first oil phase: the first surfactant and the first organic solution are mixed to obtain the first oil phase at 60℃; preferably, the first surfactant is Tween, and the first organic solution is cyclohexane; the mass content of the first surfactant in the cyclohexane is 2wt%-8wt%;

[0021] The first oil phase is poured into the stirring first aqueous phase, and mixed and stirred to form the first o / w emulsion; preferably, the volume ratio of the oil phase to the aqueous phase is 1:2;

[0022] The stirring speed of the mixing and stirring is 600-1000 rpm, and the time is 3-5 min; preferably, 4 min;

[0023] S3.2 Preparation of the second w / o / w emulsion:

[0024] a second surfactant is mixed with a second organic solution to prepare a second oil phase; preferably, the second oil phase is prepared at 60°C; the second surfactant is Span 85, and the second organic solution is cyclohexane; the second surfactant is added in an amount of 6w / v% to 12w / v% of the second organic solution;

[0025] The second oil phase is poured into the first o / w emulsion and continues to be stirred to emulsify to form a second w / o / w emulsion; preferably, the volume ratio of the first emulsion to the second emulsion is 1:2;

[0026] The stirring speed is 1000-1200 rpm, and the stirring time is 1-3 min;

[0027] S3.3 The second w / o / w emulsion is solidified, and the precipitate is repeatedly washed to obtain transparent agarose gel microspheres.

[0028] Further, NaOH, 1,4-dioxane and a second crosslinking agent are added to the obtained microspheres, and the mixture is stirred at a speed of 300 rpm for 1-12 h; the supernatant is discarded after centrifugal separation and repeated washing until clean; and a crosslinked macroporous multi-nuclear structure agarose gel microsphere is obtained.

[0029] Further, an ion exchanger and 5 mol / L NaOH are added to 5 g-50 g of the crosslinked macroporous nuclear shell agarose gel microspheres, and the mixture is reacted at 20-40°C and a speed of 200-300 rpm for 2-24 h; and the ion exchange macroporous multi-nuclear structure agarose gel microspheres are obtained after washing with deionized water multiple times.

[0030] The second crosslinking agent is at least one of a halogenated epoxy compound and a diglycidyl ether reagent; preferably, the halogenated epoxy compound is epichlorohydrin, and the diglycidyl ether reagent is any one of ethylene glycol diglycidyl ether, butanediol diglycidyl ether and hexanediol diglycidyl ether;

[0031] The volume ratio of the lye to the second crosslinking agent is 1:2;

[0032] The reaction is stirred at a speed of 200 rpm; preferably, the reaction time is 2 h;

[0033] The ion ligand of the ion exchanger is any one of diethylaminoethyl, quaternary aminoethyl, triethylamine ethyl, p-aminobenzyl, dimethyl-B-hydroxyethylamine, carboxymethyl, sulfonic acid group, sulfopropyl, and phosphoric acid group. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 is a result picture of the morphology and pore size of the macroporous multi-nuclear structure agarose gel microspheres of Example 1;

[0035] Figure 2 This is a morphological diagram of the cross-linked macroporous multinucleated agarose gel microspheres of Example 2;

[0036] Figure 3 This is a morphological diagram of the ion-exchange macroporous multinuclear agarose gel microspheres of Example 3;

[0037] Figure 4 This is a morphological diagram of the ion-exchange macroporous multinucleated agarose gel microspheres of Example 4;

[0038] Figure 5 This is a morphological diagram of the ion-exchange macroporous multinuclear agarose gel microspheres of Example 5;

[0039] Figure 6 The graph shows the ion exchange capacity results of the macroporous multinuclear agarose gel microspheres of Examples 3, 4, and 5.

[0040] Figure 7 The graph shows the dynamic protein loading results of the ion-exchange macroporous multinuclear agarose gel microspheres in Examples 3, 4, and 5.

[0041] Figure 8 This is a morphological diagram of the agarose gel microspheres in Comparative Example 1;

[0042] Figure 9 This is a morphological diagram of the agarose gel microspheres in Comparative Example 2;

[0043] Figure 10 This is a morphological diagram of the agarose gel microspheres in Comparative Example 3;

[0044] Figure 11 This is a morphological diagram of the agarose gel microspheres in Comparative Example 4;

[0045] Figure 12 The particle size distribution diagrams are shown for macroporous multinucleate agarose gel microspheres in Comparative Examples 3 and 4 and Example 1.

[0046] Figure 13 The figures are A, B, and C, showing the pore size results of the macroporous multinucleated agarose gel microspheres of Example 1 and the agarose gel microspheres of Comparative Examples 3 and 4, respectively.

[0047] Figure 14 The graph shows the ion exchange capacity and dynamic protein loading results of the ion exchange macroporous multinucleated agarose gel microspheres of Comparative Examples 5, 6 and Example 5.

[0048] Figure 15 The graph shows the mechanical strength results of macroporous multinucleated agarose gel microspheres in Comparative Examples 3 and 4 and Example 1. Detailed Implementation

[0049] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0050] Example 1: Preparation of a macroporous multinucleated agarose gel microsphere

[0051] S1. Preparation of the core: The oil phase and agarose solution are mixed and stirred to emulsify, and then cooled and solidified to form a spherical shape; the solution containing the spherical shape is centrifuged and the supernatant is discarded, and the core is obtained by repeated precipitation and washing with deionized water;

[0052] An oil phase is obtained by mixing a surfactant and an oily substance, wherein the surfactant (Span80) content is 1.5% of the volume of liquid paraffin, the oily substance is 200 mL of liquid paraffin, and the Span80 content is 3 g.

[0053] The concentration of the agarose solution is 6% w / v: 3.6g of agarose powder is dispersed in 60mL of deionized water and heated until it is clear, transparent and completely dissolved to prepare a 6% w / v agarose solution.

[0054] The oil phase is 200 mL, and the agarose solution is 60 mL.

[0055] The volume ratio of the oily substance to the agarose solution is 200:60;

[0056] The emulsification process is carried out at 1200 rpm for 15 minutes; the curing process is carried out by cooling down to 20°C at 500 rpm.

[0057] Crosslinking of S2 kernel agarose microspheres: The obtained kernel is placed in a mixed solution containing deionized water, alkaline solution, 1,4-dioxane and crosslinking agent for reaction. After the reaction is completed, the obtained microspheres are repeatedly washed with deionized water to remove NaOH and organic solvent contained in the microsphere solution, thus obtaining crosslinked kernel agarose microspheres.

[0058] The crosslinking agent is at least one of a halogenated epoxy compound and a diglycidyl ether reagent; preferably, the halogenated epoxy compound is epichlorohydrin, and the diglycidyl ether reagent is selected from any one of ethylene glycol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether.

[0059] The amount ratio of the core: deionized water: lye: crosslinking agent is 36g: 24mL: 14mL: 14mL;

[0060] The reaction condition is 200rpm, 30℃ for 6h;

[0061] S3. Preparation of the second emulsion of w / o / w:

[0062] S3.1 Preparation of the first emulsion of o / w:

[0063] Preparation of the first water phase: 50mL 6%w / v agarose solution is heated to be clear and transparent to make it completely dissolved, then the crosslinked core agarose microspheres (2.5g, 5g, 10g) are added and mixed evenly;

[0064] Preparation of the first oil phase: the first surfactant and the first organic solution are mixed to obtain the first oil phase in 60℃; the first surfactant is 2mL Tween80, and the first organic solution is 50mL cyclohexane;

[0065] The first oil phase is poured into the stirring first water phase, and mixed and stirred to form the first emulsion of o / w;

[0066] The rotation speed of the mixing and stirring is 600rpm, and the time is 4min;

[0067] S3.2 Preparation of the second emulsion of w / o / w:

[0068] The second surfactant and the second organic solution are mixed to prepare the second oil phase in 60℃; the second surfactant is 13.5mL Span85, and the second organic solution is 225mL cyclohexane; the addition amount of the second surfactant is 6%-12%w / v of the second organic solution;

[0069] The second oil phase is poured into the first emulsion of o / w, and the stirring is continued to emulsify to form the second emulsion of w / o / w; the volume ratio of the first emulsion: second emulsion is 1:2;

[0070] The rotation speed of the continuous stirring is 1000-1200rpm, and the time is 1min;

[0071] S3.3 The temperature of the second emulsion of w / o / w is reduced to 25℃, and the stirring is continued to solidify, and then the precipitate is repeatedly washed with ethanol and deionized water to obtain transparent agarose gel microspheres; the sample in the receiving solvent is filtered by nylon gauze, and the particle size of the macroporous multi-nuclear structure agarose gel microspheres obtained in this embodiment is 45μm-180μm, and the morphology is as shown in Figure 1 A, B and C in the figure correspond to the addition amount of 2.5g core, 5g core and 20g core respectively, and the pore size distribution results are as shown inFigure 1 The cross-section of the D portion microspheres clearly shows the existence of the pores and the core.

[0072] Example 2: Preparation of cross-linked macroporous multi-core structure agarose gel microspheres

[0073] S1. Preparation of the core: same as Example 1

[0074] S2. Cross-linking of the core agarose microspheres: same as Example 1.

[0075] S3. Obtaining of the macroporous multi-core structure agarose gel microspheres: same as Example 1, wherein the amount of the core added is 5 g.

[0076] S4. Obtaining of the cross-linked macroporous multi-core structure agarose gel microspheres:

[0077] 1% v / v NaOH, 2% v / v 1,4-dioxane and 2% v / v epichlorohydrin were added to the microspheres, and the reaction was stirred at a speed of 300 rpm for 4 h; the above-mentioned mixed system was then centrifuged to separate the layers, and the supernatant was discarded and repeatedly washed until clean. The cross-linked macroporous multi-core structure agarose gel microspheres were obtained.

[0078] The sample was filtered using nylon mesh, and the particle size of the cross-linked macroporous multi-core structure agarose gel microspheres obtained in this example was 45 μm-180 μm, and the morphology results are shown in Figure 2 .

[0079] Example 3: Preparation of ion-exchange macroporous multi-core structure agarose gel microspheres

[0080] S1. Preparation of the core agarose microspheres: same as Example 1.

[0081] S2. Cross-linking and activation of the core agarose microspheres: same as Example 1.

[0082] S3. Preparation of the macroporous multi-core structure agarose gel microspheres: same as Example 1, wherein the amount of the core added is 5 g.

[0083] S4. Preparation of the cross-linked macroporous multi-core structure agarose gel microspheres: same as Example 2.

[0084] S5. Obtaining of the ion-exchange macroporous multi-core structure agarose gel microspheres:

[0085] 5 g of the cross-linked and activated macroporous core-shell structure microspheres were taken, and 4 mL of 6 mol / L bromoacetic acid and 4 mL of 5 mol / L NaOH were added thereto, and the reaction was carried out at 30 C and a speed of 200 rpm for 24 h. After the reaction, the product was washed with deionized water for multiple times to obtain an ion-exchange macroporous multi-core structure agarose gel microsphere.

[0086] The sample is filtered by nylon mesh, and the ion exchange macroporous multi-nucleus structure agarose gel microspheres obtained have a particle size of 45 μm-180 μm, and the ion exchange capacity and dynamic protein loading capacity results are as follows Figure 3 、 6 、7.

[0087] Example 4: Preparation of ion exchange macroporous multi-nucleus structure agarose gel microspheres

[0088] S1. Preparation of inner core agarose microspheres: same as Example 1.

[0089] S2. Crosslinking and activation of inner core agarose microspheres: same as Example 1.

[0090] S3. Preparation of macroporous multi-nucleus structure agarose gel microspheres: same as Example 1, wherein the amount of inner core added is 5 g.

[0091] S4. Preparation of crosslinked macroporous multi-nucleus structure agarose gel microspheres: same as Example 2.

[0092] S5. Obtaining of ion exchange macroporous multi-nucleus structure agarose gel microspheres: the difference from Example 3 is that the ion exchanger is changed to hydroxypropyl sulfonic acid.

[0093] Take 5 g of crosslinked and activated macroporous core-shell structure microspheres, add 4 mL of 6 mol / L hydroxypropyl sulfonic acid and 2 mL of 5 mol / L NaOH, and react at 30°C and 200 rpm for 24 h. After the reaction is completed, the crosslinked and activated macroporous core-shell structure microspheres are washed with deionized water several times to obtain ion exchange macroporous core-shell structure microspheres.

[0094] The sample is filtered by nylon mesh, and the ion exchange macroporous multi-nucleus structure agarose gel microspheres obtained have a particle size of 45 μm-180 μm, and the ion exchange capacity and dynamic protein loading capacity results are as follows Figure 4 、 6 、7.

[0095] Example 5: Preparation of ion exchange macroporous multi-nucleus structure agarose gel microspheres

[0096] S1. Preparation of inner core agarose microspheres: same as Example 1.

[0097] S2. Crosslinking and activation of inner core agarose microspheres: same as Example 1.

[0098] S3. Preparation of macroporous multi-nucleus structure agarose gel microspheres: same as Example 1, wherein the amount of inner core added is 5 g.

[0099] S4. Preparation of crosslinked macroporous multi-nucleus structure agarose gel microspheres: same as Example 2.

[0100] S5. Preparation of ion-exchange macroporous multi-nucleus structure agarose gel microspheres: the difference from Example 3 is that the ion exchanger is changed to 2,3-epoxypropyl trimethyl ammonium chloride.

[0101] Take 5 g of the cross-linked activated macroporous multi-nucleus structure microspheres, and add 2 g of 2,3-epoxypropyl trimethyl ammonium chloride (ETA) and 4 mL of 5 mol / L NaOH, and react at 30°C and 200 rpm for 24 h. After the reaction is completed, wash the product with deionized water multiple times to obtain ion-exchange macroporous multi-nucleus structure agarose gel microspheres.

[0102] Filter the sample using nylon mesh, and the ion-exchange macroporous multi-nucleus structure agarose gel microspheres obtained have a particle size of 45 μm-180 μm, and the ion exchange capacity and dynamic protein loading capacity results are as follows: Figure 5 、 6 、7.

[0103] Preparation of macroporous multi-nucleus structure agarose microspheres

[0104] S1. Preparation of the inner core: same as Example 1.

[0105] S2. Cross-linking of the inner core agarose microspheres: same as Example 1.

[0106] S3. Preparation of macroporous multi-nucleus structure agarose gel microspheres: same as Example 1, except that the amount of the inner core added is 2 g. The results are shown in Figure 8 .

[0107] Preparation of macroporous multi-nucleus structure agarose microspheres

[0108] S1. Preparation of the inner core: same as Example 1.

[0109] S2. Cross-linking of the inner core agarose microspheres: same as Example 1.

[0110] S3. Preparation of macroporous multi-nucleus structure agarose gel microspheres: same as Example 1, except that the amount of the inner core added is 25 g. The results are shown in Figure 9 .

[0111] Preparation of macroporous agarose gel microspheres

[0112] No S1, S2 steps;

[0113] S3 step:

[0114] Preparation of the first emulsion of o / w: the difference from Example 1 is that the first aqueous phase is an agarose solution containing 6% w / v agarose, which is heated to be clear and transparent so as to be completely dissolved; the others are the same as Example 1.

[0115] Preparation of the second emulsion of w / o / w: same as Example 1;

[0116] S3.3 The procedure for transparent agarose gel microspheres is the same as in Example 1;

[0117] The average particle size and particle size distribution of the macroporous agarose gel microspheres obtained by filtering the sample with nylon mesh were measured using a BT-9300ST laser particle size analyzer. The average particle size of the microspheres in water was 122 μm and the particle size distribution coefficient Rpan was 0.57. The results of the morphology and cross-sectional pore size are shown in Figure 8 , 12 , 13(B).

[0118] Preparation of multi-core agarose gel microspheres

[0119] S1. Preparation of the inner core: The same as in Example 1.

[0120] S2. Crosslinking of the inner core agarose microspheres: The same as in Example 1

[0121] S3. Obtaining of the multi-core agarose gel microspheres: The same as in Example 1, except that no macropores are formed in the shell layer

[0122] Preparation of the oil phase: Mix the surfactant and the oily substance and stir at 80°C; add 3 mL of surfactant (Span 80) and 200 mL of liquid paraffin as the oily substance;

[0123] Disperse the agarose powder in deionized water, heat to a clear and transparent complete dissolution, and prepare a solution containing 6% w / v agarose, and add 6 g of crosslinked inner core and mix uniformly;

[0124] Mix and stir the above two solutions to emulsify, and then cool and solidify to form spherical shapes;

[0125] Centrifuge the solution containing the spherical shapes at high speed, discard the supernatant, and repeatedly precipitate and wash with deionized water to obtain multi-core agarose gel microspheres.

[0126] The average particle size and particle size distribution of the multi-core agarose gel microspheres obtained by filtering the sample with nylon mesh were measured using a BT-9300ST laser particle size analyzer. The average particle size of the microspheres in water was 132 μm and the particle size distribution coefficient Rpan was 0.50. The results of the morphology and cross-sectional pore size are shown in Figure 9 , 12 , 13(C).

[0127] Preparation of ion exchange macroporous agarose gel microspheres

[0128] Without S1 and S2 steps;

[0129] S3 step:

[0130] Preparation of the first emulsion of o / w: The difference from Example 4 is that the first aqueous phase is an agarose solution containing 6% w / v agarose heated to clear transparency so as to be completely dissolved; the others are the same as Example 4.

[0131] Preparation of the second emulsion of w / o / w: the same as Example 1;

[0132] S3.3 The steps of preparing the transparent agarose gel microspheres are the same as Example 1;

[0133] S4. 1% v / v NaOH, 2% v / v 1,4-dioxane and 2% v / v epichlorohydrin are added to the microspheres, and the reaction is stirred at a speed of 300 rpm for 2 h; then the mixed system is centrifuged to separate the layers, the supernatant is discarded, and repeated washing is performed until the washing is clean. Thus, the cross-linked macroporous structure agarose gel microspheres are obtained;

[0134] S5. 5 g of the cross-linked activated macroporous core-shell structure microspheres are taken, 4 mL of 6 mol / L hydroxypropyl sulfonic acid and 2 mL of 5 mol / L NaOH are added thereto, and the reaction is carried out at 30 C and a speed of 200 rpm for 24 h. After the reaction is completed, the product is washed with deionized water for multiple times to obtain an ion-exchange macroporous structure agarose gel microsphere.

[0135] Preparation of an ion-exchange multi-core agarose gel microsphere

[0136] S1. Preparation of the inner core: the same as Example 1,

[0137] S2. Cross-linking of the inner core agarose microspheres: the same as Example 1,

[0138] S3. Obtaining of the multi-core agarose gel microspheres: the difference from Example 5 is that the shell layer is non-porous.

[0139] Preparation of the oil phase: the surfactant and the oily substance are mixed and stirred at 80 C;

[0140] The agarose powder is dispersed in deionized water, heated to clear transparency and completely dissolved to prepare a 6% w / v agarose solution, and the cross-linked inner core is added and uniformly mixed;

[0141] The above two solutions are mixed and emulsified, and then cooled and solidified to form a spherical shape;

[0142] The solution containing the spherical shape is centrifuged at high speed, the supernatant is discarded, and deionized water is repeatedly precipitated and washed to obtain a multi-core structure agarose gel microsphere.

[0143] The microspheres were added with 1% v / v NaOH, 2% v / v 1,4-dioxane and 2% v / v epichlorohydrin, and stirred at 300 rpm for 2 h; the mixed system was then centrifuged to separate layers, and the supernatant was discarded and repeatedly washed until clean. The cross-linked multi-nuclear structure agarose gel microspheres were obtained;

[0144] S4. 5 g of the cross-linked activated core-shell structure microspheres were taken, and 4 mL of 6 mol / L hydroxypropyl sulfonic acid and 2 mL of 5 mol / L NaOH were added thereto, and reacted at 30 C and 200 rpm for 24 h. After the reaction, the microspheres were washed with deionized water for multiple times to obtain ion-exchange multi-nuclear structure agarose gel microspheres.

[0145] As Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , all the macroporous multi-nuclear structure agarose gel microspheres obtained by the method of the present application have a spherical structure and are complete in shape. Figure 1 In the figure, A is 2.5 g of the inner core, and the addition amount does not affect the shape of the microspheres. B is 5 g of the inner core, and the addition amount of the shell wraps the inner core, and the spherical shape is complete without extrusion damage. C is 20 g of the inner core, and the addition amount of the microspheres is uneven in size, but the wrapping is within the limit. D is a cross-sectional view of the microspheres with 5 g of the inner core, and the inner core and the pore structure of the shell layer can be clearly seen, and the pore size is about 4 μm.

[0146] Figure 8 Preparation of macroporous core-shell microspheres with 2 g of the inner core (Comparative Example 1), the decrease of the addition amount of the inner core makes the particle size distribution of the microspheres uneven, and the inner core wrapped in the macroporous microspheres is limited, and even the macroporous microspheres do not have the inner core. Figure 9 Preparation of macroporous core-shell microspheres with 25 g of the inner core (Comparative Example 2), the increase of the addition amount of the inner core makes the particle size distribution of the microspheres uneven, and the inner core wrapped in the macroporous microspheres is limited, and even the macroporous microspheres do not have the inner core. Too much addition amount is not conducive to the stability of the system. The microscope view shows that the macroporous microspheres wrap a lot of inner cores, causing some microspheres to be distorted in shape, and some to burst, and some inner cores to be scattered in the solution. These phenomena show that too much or too little addition of the inner core will affect the shape of the microspheres, and the shape of the microspheres will affect the purification effect. It is necessary to adjust the appropriate addition amount of the inner core in Comparative Examples 1 and 2 compared with Example 1.

[0147] In which Example 1 and Comparative Examples 3 and 4 are compared, and the existence of the inner core, the existence of the macroporous, and the transparency and inner core wrapping state of the multi-nuclear microspheres are obviously seen. The cross-sectional pore size distribution can better reflect the structure of the internal microspheres, Figure 13The macroporous structure of the microspheres in A is the main structure of the internal package of the core, and the mesh of the microspheres of the core is dense and is in sharp contrast with the macropore; the pores in B are the same as the main structure in A, which proves that this method does not violate the principle of the method even if the core is added; the C figure also shows the existence of the microsphere core, and the whole core-shell does not have the existence of macropores and is relatively dense, and the core is more dense. The whole set of figures reflect the differences of the three kinds of microspheres.

[0148] Figure 14 The ion exchange capacity and dynamic protein loading results of the agarose gel microspheres of Comparative Examples 5 and 6 and Example 5 are the actual reaction of the differences in chromatographic performance of the macroporous, macroporous multi-core structure and multi-core structure agarose gel microspheres. The macroporous agarose microspheres provide faster and more efficient separation channels, and the multi-core provides more effective binding sites for the agarose gel microspheres. The macroporous multi-core structure agarose gel microspheres combined with channels and cores solve the problems of large steric hindrance and small loading capacity, and balance the performance of agarose gel microspheres as chromatographic media in separation and purification.

[0149] Figure 15 The mechanical strength results of the agarose gel microspheres of Comparative Examples 3 and 4 and Example 1 are shown in the figure. The flow rate-pressure test: different types of agarose gel microspheres were taken and loaded into a chromatographic column with a size of 0.66*25 cm. A certain flow rate was passed, and after the column bed was stabilized, a constant pressure was used to measure the highest flow rate. By gradually increasing the pressure at intervals, after 5 minutes of stabilization, until the flow rate changes tend to be stable under a certain range of pressure, the highest flow rate of the microsphere medium can be obtained, and the flow rate-pressure curve can be drawn. It is reflected that the macroporous core-shell agarose gel microspheres prepared by the application provide support with the core agarose microspheres, and compared with the mechanical strength of the microspheres without the core, the mechanical properties are improved to some extent, which reflects that the mechanical properties of the macroporous core-shell agarose gel microspheres prepared by the application are effectively improved.

[0150] According to the embodiments of the application, the macroporous multi-core structure agarose gel microspheres are prepared by combining the double emulsion method and the emulsification solidification method. The characteristics of this structure are that the macroporous core-shell structure shortens the distance of substances entering the interior of the microspheres, accelerates the time, and improves the mass transfer rate. The smaller particle size of the core increases the specific surface area and has more active binding sites. It conforms to the development trend of the field of chromatographic media.

[0151] Although the embodiments of the application have been shown and described above, it should be understood that the above embodiments are exemplary and cannot be understood as limiting the application. Those skilled in the art can make changes, modifications, replacements and variations to the above embodiments without departing from the principles and purposes of the application within the scope of the application.

Claims

1. A macroporous multinucleated agarose gel microsphere, characterized in that, The shell of the microspheres is an agarose gel with a pore size of 2-4 μm, and the core of the microspheres is agarose microspheres with a particle size of no more than 50 μm, distributed inside the macroporous gel. Its preparation method includes the following steps: S1. Preparation of the core: The oil phase and agarose solution are mixed and stirred to emulsify, and then cooled and solidified to form a spherical shape; the solution containing the spherical shape is centrifuged and the supernatant is discarded, and the core is obtained by repeated precipitation and washing with deionized water; S2. Crosslinking of kernel agarose microspheres: The obtained kernels are placed in a mixed solution containing deionized water, alkaline solution, 1,4-dioxane and crosslinking agent for reaction. After the reaction is completed, the obtained microspheres are repeatedly washed with deionized water to remove NaOH and organic solvents contained in the microsphere solution, thus obtaining crosslinked kernel agarose microspheres. S3. Obtaining macroporous multinucleated agarose gel microspheres: Preparation of the first emulsion, S3.1 o / w: Preparation of the first aqueous phase: Heat a 4%-6% w / v agarose solution until it is clear and transparent and completely dissolved, then add the cross-linked core agarose microspheres and mix evenly; the amount of cross-linked core agarose microspheres added is 5wt%-20wt% of the agarose solution; Preparation of the first oil phase: The first surfactant is mixed with the first organic solution and placed at 60°C to obtain the first oil phase; Pour the first oil phase into the first aqueous phase that is being stirred, and mix and stir to form an o / w first emulsion; S3.2 Preparation of the w / o / w second emulsion: A second oil phase is prepared by mixing a second surfactant with a second organic solution. The second oil phase is poured into the o / w first emulsion and stirred and emulsified to form a w / o / w second emulsion. S3.3 The w / o / w second emulsion is solidified, and the precipitate is repeatedly washed to obtain transparent macroporous multinucleated agarose gel microspheres.

2. The macroporous multinucleated agarose gel microspheres as described in claim 1, characterized in that, In step S1, the oil phase is obtained by mixing a surfactant and an oily substance, wherein the content of the surfactant is 0.5%-2% of the volume of the liquid paraffin, and the oily substance is liquid paraffin. The concentration of the agarose solution is 4%-6% w / v; The volume ratio of the oil phase to the agarose solution is 200:(50-80); The emulsification is carried out at 1200-3500 rpm for 10-30 min; the curing is carried out by cooling down to 15-25℃ at 500-600 rpm.

3. The macroporous multinucleated agarose gel microspheres as described in claim 1, characterized in that, In step S2 The crosslinking agent is at least one of a halogenated epoxy compound and a diglycidyl ether reagent; The ratio of the core component (deionized water, alkaline solution, 1,4-dioxane, crosslinking agent) is 36g:24mL:14mL:14mL:14mL. The reaction conditions are 200-300 rpm, 20-38℃, and 2-8 h.

4. The macroporous multinucleated agarose gel microspheres as described in claim 3, characterized in that, In step S2, the halogenated epoxy compound is epichlorohydrin, and the diglycidyl ether reagent is selected from any one of ethylene glycol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether.

5. The macroporous multinucleated agarose gel microspheres as described in claim 1, characterized in that, In the preparation of the first emulsion in S3.1 o / w, in the preparation step of the first oil phase, the first surfactant is Tween, and the first organic solution is cyclohexane; the mass content of the first surfactant is 2wt%-8wt% of cyclohexane.

6. The macroporous multinucleated agarose gel microspheres as described in claim 1, characterized in that, In the preparation step of the first emulsion S3.1 o / w, the volume ratio of the oil phase to the water phase is 1:

2.

7. The macroporous multinucleated agarose gel microspheres as described in claim 1, characterized in that, In the preparation of the first emulsion S3.1 o / w, the mixing and stirring speed is 600-1000 rpm and the time is 3-5 min.

8. The macroporous multinucleated agarose gel microspheres as described in claim 1, characterized in that, In the preparation step of the first emulsion S3.1 o / w, the mixing and stirring time is 4 min.

9. The macroporous multinucleated agarose gel microspheres as described in claim 1, characterized in that, In step S3.2, the second emulsion is prepared by mixing the second surfactant and the second organic solution at 60°C to form the second oil phase; the second surfactant is Span85, and the second organic solution is cyclohexane; the amount of the second surfactant added is 6 w / v%-12 w / v of the second organic solution.

10. The macroporous multinucleated agarose gel microspheres as described in claim 1, characterized in that, In step S3.2, the volume ratio of the first emulsion to the second emulsion is 1:

2.

11. The macroporous multinucleated agarose gel microspheres as described in claim 1, characterized in that, In the preparation of the second emulsion in S3.2 w / o / w, the stirring speed is 1000-1200 rpm and the time is 1-3 min.

12. The macroporous multinucleated agarose gel microspheres as described in claim 1, characterized in that, S1. In the core preparation step, the volume ratio of the oil phase to the agarose solution is 200:

60.

13. The macroporous multinucleated agarose gel microspheres according to any one of claims 1-12, characterized in that, Add NaOH, 1,4-dioxane, and a second crosslinking agent to the macroporous multinuclear agarose gel microspheres, and then stir the mixture at 300 rpm for 1-12 hours. Centrifuge to separate the layers, discard the supernatant, and wash repeatedly until clean to obtain crosslinked macroporous multinuclear agarose gel microspheres.

14. The macroporous multinucleated agarose gel microspheres as described in claim 13, characterized in that, Add ion exchanger and 5 mol / L NaOH to 5 g-50 g of cross-linked macroporous core-shell agarose gel microspheres and react at 20-40℃ and 200-300 rpm for 2-24 h. After the reaction, wash with deionized water several times to obtain ion-exchange macroporous multinucleated agarose gel microspheres.

15. The macroporous multinucleated agarose gel microspheres as described in claim 13, characterized in that, The second crosslinking agent is at least one of a halogenated epoxy compound and a diglycidyl ether reagent.

16. The macroporous multinucleated agarose gel microspheres as described in claim 15, characterized in that, The halogenated epoxy compound is epichlorohydrin, and the diglycidyl ether reagent is selected from any one of ethylene glycol diglycidyl ether, butylene glycol diglycidyl ether, and hexanediol diglycidyl ether.

17. The macroporous multinucleated agarose gel microspheres as described in claim 13, characterized in that, The reaction time was 2 hours with stirring at 300 rpm.

18. The macroporous multinucleated agarose gel microspheres as described in claim 14, characterized in that, The ionic ligand of the ion exchanger is any one of diethylaminoethyl, quaternary aminoethyl, triethylaminoethyl, p-aminobenzyl, carboxymethyl, sulfonic acid group, sulfopropyl group, or phosphate group.

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