Composite gel filtering medium as well as preparation method and application thereof

Abstract

The invention relates to the technical field of chromatography media, in particular to a composite gel filter medium as well as a preparation method and application thereof. The preparation method of the composite gel filtering medium comprises the following steps that S1, cross-linked agarose microspheres are swelled, activated and brominated, and the pressure resistance of cross-linked agarose is larger than or equal to 0.3 MPa; s2, in an alkaline solution, carrying out grafting reaction on the brominated agarose microspheres and glucan, so as to obtain glucan grafted agarose microspheres; and S3, carrying out a cross-linking reaction on the sepharose microspheres grafted with the glucan and a cross-linking agent to obtain the composite gel filter medium. The agarose microspheres with small particle size and high crosslinking degree are used as a skeleton of the gel filter medium, have excellent pressure resistance, are subjected to bromination treatment, then are grafted with glucan, and are further crosslinked to prepare the composite gel filter medium, so that the flow velocity of the gel filter medium in the separation and purification process of a target object is increased, and the separation and purification efficiency of the target object is improved. Further, the separation and purification efficiency of the target object is realized.

Description

Technical Field

[0001] The invention relates to the technical field of chromatography media, and in particular to a composite gel filtration medium and a preparation method and application thereof. Background Art

[0002] In separation and purification technology, chromatography technology is widely used due to its high separation efficiency, wide applicability, and easy process scale-up. The characteristics of the chromatographic medium in chromatography technology affect the effect of separation and purification. Among them, agarose has the characteristics of high hydrophilicity, high porosity, good biocompatibility, low production cost, etc., and carries a large number of hydroxyl groups that can react with other chemical groups. When used as a chromatographic medium material, it has good adsorption performance but poor mechanical strength. At present, the mechanical strength of agarose microspheres is mostly improved by increasing the agarose content or chemically cross-linking the agarose, but the improved agarose microspheres have a low loading capacity for the target.

[0003] The prior art discloses a high-load agarose chromatography medium, which uses dextran to modify agarose to increase the loading capacity of the chromatography medium for the target object. However, the chromatography medium has the problems of slow flow rate and low efficiency in separation and purification of the target object. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defect of slow flow rate of modified agarose gel filtration medium in the prior art, thereby providing a composite gel filtration medium and a preparation method and application thereof, so as to improve the separation and purification efficiency of the target object.

[0005] Another technical problem to be solved by the present invention is to overcome the defect of low resolution of modified agarose gel filtration medium in the prior art, thereby providing a composite gel filtration medium and a preparation method and application thereof to improve the resolution of the target object.

[0006] On the one hand, the present invention provides a method for preparing a composite gel filtration medium, comprising the following steps: S1, swelling, activating and brominating cross-linked agarose microspheres, wherein the pressure resistance of the cross-linked agarose is greater than or equal to 0.3 MPa; S2, in an alkaline solution, grafting the brominated agarose microspheres with dextran to obtain agarose microspheres grafted with dextran; S3, cross-linking the agarose microspheres grafted with dextran with a cross-linking agent to obtain a composite gel filtration medium.

[0007] In some embodiments, the cross-linking agent in step S3 includes a bifunctional cross-linking agent containing an epoxy group.

[0008] Preferably, the cross-linking agent includes at least one of a halogenated epoxy compound and a glycidyl ether.

[0009] More preferably, the halogenated epoxy compound includes epichlorohydrin.

[0010] Preferably, the glycidyl ether includes at least one of ethylene glycol diglycidyl ether, propylene glycol glycidyl ether, butanediol diglycidyl ether and pentaerythritol glycidyl ether, more preferably butanediol diglycidyl ether.

[0011] In some of the embodiments, the volume ratio of the agarose microspheres grafted with dextran to the cross-linking agent is (1-5):(1-5).

[0012] In some embodiments, the cross-linking reaction temperature is 40-60° C., and the reaction time is 12-24 hours.

[0013] In some of the embodiments, in step S1, the step of swelling the cross-linked agarose microspheres comprises stirring the cross-linked agarose microspheres and the alkaline solution at 180-200 rpm for 0.5-1 h.

[0014] In some embodiments, the activation step of the swollen cross-linked agarose microspheres further includes washing and drying the activated cross-linked agarose.

[0015] In some embodiments, the mass concentration of agarose in the cross-linked agarose microspheres is 4% to 8%, preferably 4% to 6%; and / or,

[0016] In some embodiments, the particle size of the cross-linked agarose microspheres is less than or equal to 60 μm, preferably 25 to 45 μm.

[0017] In some of the embodiments, the activation step of the swollen cross-linked agarose microspheres in step S1 includes adding allyl glycidyl ether to the swollen cross-linked agarose microspheres and reacting at 40 to 70° C. for 8 to 24 hours.

[0018] Preferably, the activated highly cross-linked agarose is washed with water, alcohol, and water until the conductivity is the same as that of pure water.

[0019] In some of the embodiments, the bromination step of the activated cross-linked agarose microspheres includes mixing the activated cross-linked agarose with bromine and a pH adjuster in an aqueous solvent at a speed of 220-260 rpm and reacting for 20-30 minutes.

[0020] Preferably, the pH regulator includes sodium acetate or sodium acetate trihydrate. During the bromination treatment, sodium acetate (or sodium acetate trihydrate) is added thereto. Sodium acetate mainly plays the role of adjusting the pH value of the system. It can be hydrolyzed in water to form OH - Negative ions can neutralize the acidic ions formed during the bromination process and ensure the continuous stability of the reaction process.

[0021] Preferably, the highly cross-linked agarose after bromide treatment is washed with water until the conductivity is the same as that of pure water.

[0022] In some of the embodiments, after the bromination treatment, sodium formate is added to the reaction system, and the sodium formate is oxidized and reduced with the unreacted bromine to generate salt and gas to remove excess bromine in the reaction system.

[0023] In some of the embodiments, the volume ratio of cross-linked agarose microspheres to bromine is (20-10):1.

[0024] In some embodiments, the grafting reaction temperature of step S2 is 40-70° C., and the reaction time is 8-24 hours.

[0025] Preferably, the composite microspheres grafted with dextran are washed with water until the conductivity is the same as that of pure water.

[0026] In some of the embodiments, the mass ratio of the volume of the cross-linked agarose microspheres to allyl glycidyl ether is (1-5):(1-2), preferably (1-2):1, and the unit is mL:g.

[0027] In some embodiments, the molecular weight of the dextran in step S2 is 20,000 to 400,000, preferably 40,000 to 200,000.

[0028] In some embodiments, the step S2 uses a dextran aqueous solution to provide dextran for the grafting reaction, wherein the mass concentration of dextran in the dextran aqueous solution is 10-50%.

[0029] In some embodiments, the volume ratio of the cross-linked agarose microspheres to the dextran solution is 1:(1-2).

[0030] In some embodiments, the alkaline solution for swelling the cross-linked agarose in step S2 and / or step S1 comprises a metal hydroxide solution, and the metal hydroxide solution comprises at least one of a sodium hydroxide solution and a potassium hydroxide solution. Optionally, the mass concentration of the alkaline solution is 40-60%, preferably 50%.

[0031] In another aspect, the present invention provides a composite gel filtration medium, which is prepared by the above-mentioned preparation method of the composite gel filtration medium.

[0032] In some embodiments, the composite gel filtration medium has a particle size of 25 to 45 μm.

[0033] In some embodiments, the separation range of the composite gel filtration medium is 3000-70000 Da.

[0034] In some embodiments, the separation range of the composite gel filtration medium is 10,000 to 600,000 Da.

[0035] At the same time, the present invention provides the application of the composite gel filtration medium in the separation and purification of biomolecules.

[0036] In some embodiments, the biomolecule includes at least one of a polypeptide, a recombinant protein, a nucleotide, and a small molecule.

[0037] In some embodiments, the biomolecule further comprises at least one of antibody monomers, dimers and aggregates in monoclonal antibodies.

[0038] The technical solution of the present invention has the following advantages:

[0039] 1. A method for preparing a composite gel filtration medium provided by the present invention comprises the following steps: S1, swelling, activating and brominating cross-linked agarose microspheres, wherein the pressure resistance of the cross-linked agarose is greater than or equal to 0.3 MPa; S2, in an alkaline solution, grafting the brominated agarose microspheres with dextran to obtain agarose microspheres grafted with dextran; S3, cross-linking the agarose microspheres grafted with dextran with a cross-linking agent to obtain a composite gel filtration medium. The present invention uses agarose microspheres with small particle size and high cross-linking degree as the skeleton of the gel filtration medium, which itself has excellent pressure resistance; after bromination treatment, dextran is grafted, and further cross-linked, the obtained agarose / dextran composite gel filtration medium has a uniform and stable particle size distribution, has the advantages of fast operating flow rate, good mechanical strength, low non-specific adsorption, high selectivity and high resolution, and improves the separation and purification efficiency of the target.

[0040] 2. The preparation method of the composite gel filtration medium provided by the present invention, wherein the crosslinking agent in step S3 comprises a bifunctional crosslinking agent containing an epoxy group. The present invention adopts a bifunctional crosslinking agent containing an epoxy group to increase the crosslinking degree of agarose microspheres grafted with dextran, which can effectively enhance the mechanical strength of the composite gel filtration medium, improve the resolution of the medium for the target, and effectively shorten the time required for separation and purification.

[0041] 3. The preparation method of the composite gel filtration medium provided by the present invention, wherein the cross-linking agent in step S3 includes at least one of a halogenated epoxy compound and a glycidyl ether reagent. Due to the repulsive force between polysaccharide macromolecules, the cross-linking degree of the polysaccharide is proportional to the chain length of the cross-linking agent. The present invention can reduce the three-dimensional network pore size of the gel medium and improve the pressure resistance of the gel medium by increasing the chain length of the cross-linking agent.

[0042] 4. The method for preparing the composite gel filtration medium provided by the present invention, wherein the mass ratio of the agarose composite microspheres grafted with glucan to the cross-linking agent in step S3 is (1-5):(1-5). By limiting the mass ratio of the agarose composite microspheres grafted with glucan to the cross-linking agent to be within the range of (1-5):(1-5), the prepared composite gel filtration medium has good sphericity, no broken balls, no sticky balls, and a uniform and stable particle size distribution.

[0043] 5. The preparation method of the composite gel filtration medium provided by the present invention, wherein the molecular weight of the dextran in step S2 is 20,000 to 400,000, preferably 40,000 to 200,000. The present invention utilizes the dextran with a larger molecular weight to have more active sites, which can be effectively bound to the highly cross-linked agarose microspheres in a covalent bond manner, which is beneficial to improving the mechanical strength of the composite gel filtration medium on the one hand, and on the other hand, it is beneficial to improve the charged environment on the surface of the agarose microspheres, reduce nonspecific adsorption, and improve the recovery rate of the target.

[0044] 6. The preparation method of the composite gel filtration medium provided by the present invention, wherein in step S1, the step of swelling the highly cross-linked agarose microspheres comprises stirring the highly cross-linked agarose microspheres and the alkaline solution at 180 to 200 rpm for 0.5 to 1 hour. The present invention utilizes stirring to swell the cross-linked agarose microspheres with an alkaline solution, so that the alkaline solution fully enters the interior of the pores of the cross-linked agarose microspheres, thereby accelerating the reaction rate of the subsequent activation of the highly cross-linked agarose microspheres.

[0045] 7. The composite gel filtration medium prepared by the preparation method of the composite gel filtration medium provided by the present invention can be applied to the separation and purification of biomolecules. The gel filtration medium with a separation range of 3000 to 70000Da can quickly separate and purify polypeptides, recombinant proteins, nucleotides and other small molecules; the separation range is 10000-600000Da, and can separate antibody monomers, dimers and aggregates present in monoclonal antibodies. The composite gel filtration medium prepared by the present invention provides extremely high resolution for the separation, purification and product formulation of monoclonal antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the specific implementation methods of the present invention 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 invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0047] Figure 1 is an optical microscope image of the composite gel filtration medium obtained in Example 1 of the present invention, Figure 1a in the figure is one of the dielectric microspheres, and the particle size of the dielectric microsphere is 42.4 μm. Figure 1 b in the figure is another dielectric microsphere, the particle size of which is 27.8 μm;

[0048] Figure 2 is a particle size distribution diagram of the composite gel filtration medium prepared in Example 1 of the present invention;

[0049] Figure 3 is a particle size distribution diagram of the composite gel filtration medium prepared in Example 2 of the present invention;

[0050] Figure 4 is the pressure resistance-flow rate curve of the composite gel filtration medium prepared in Example 1 of the present invention;

[0051] Figure 5 is the selectivity curve of the composite gel filtration medium prepared in Example 1 of the present invention;

[0052] Figure 6 This is the selectivity curve of the composite gel filtration medium prepared in Example 2 of the present invention. DETAILED DESCRIPTION

[0053] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0054] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0055] The mass concentration of agarose in the examples is 4%, 6%, and the highly cross-linked agarose microspheres, and the uncross-linked and low-cross-linked agarose microspheres in the comparative examples are all from Suzhou Lanxiao Biotechnology Co., Ltd.

[0056] Example 1

[0057] This embodiment provides a method for preparing a composite gel filtration medium, and the specific steps and parameters are as follows:

[0058] (1) Swelling of highly cross-linked agarose: 100 mL of highly cross-linked agarose microspheres were taken, rinsed with pure water, drained, and placed in a 500 mL three-necked flask. 18 g of water and 143 g of a 50% NaOH solution were added to the three-necked flask. The highly cross-linked agarose microspheres were stirred and swollen at 180 rpm for 1 h at 25° C. The agarose content of the highly cross-linked agarose microspheres was 6% w / v, the particle size range was 25 to 45 μm, and the pressure resistance reached above 0.3 MPa.

[0059] (2) Activating highly cross-linked agarose: Add 52 g of allyl glycidyl ether to the swollen highly cross-linked agarose obtained in step (1), and react at 50° C. for 16 h. After the reaction is completed, drain the mother liquor, wash with 10 volumes of water, 5 volumes of ethanol, and 10 volumes of water until the conductivity of the activated highly cross-linked agarose is the same as that of pure water, and drain for later use.

[0060] (3) Bromination treatment: The activated highly cross-linked agarose spheres were placed in a 500 mL three-necked flask, 22.1 g of sodium acetate trihydrate and 260.5 g of water were added, and the mixture was stirred at 240 rpm for 10 min. 10 mL of bromine was added dropwise until the orange color did not fade. The mixture was reacted for 30 min. After the reaction, a 5% sodium formate solution was added until the system returned to white. The mother liquor was drained and the mixture was washed with water for 10 volumes until the conductivity of the highly cross-linked agarose spheres after bromination treatment was the same as that of pure water. The mixture was drained and set aside for use.

[0061] (4) Grafting dextran: Place the brominated agarose microspheres in a 500 mL three-necked flask, add 100 mL of 25% dextran, where the molecular weight of dextran is 40,000, stir at 25°C for 1 h, add 15 g of 50% NaOH solution, and react at 50°C for 18 h. After the reaction is completed, drain the mother liquor, wash the agarose microspheres grafted with dextran with 10 volumes of water until the conductivity is the same as that of pure water, and drain for later use.

[0062] (5) Cross-linking reaction: The agarose composite microspheres grafted with dextran were drained, placed in a 500 mL three-necked flask, 20.5 g of water was added, stirred evenly, and 20 mL of 1,4-butanediol diglycidyl ether and 46.6 mL of 2M NaOH solution were added dropwise within 2 h (using a peristaltic pump to control the dropwise acceleration rate), and the reaction was continued at 50° C. for 18 h. After the reaction was completed, it was washed with deionized water until neutral to obtain a composite gel filtration medium, which was stored in 20% ethanol.

[0063] Example 2

[0064] This embodiment provides a method for preparing a composite gel filtration medium. The specific steps and parameters are the same as those in Example 1, except that highly cross-linked agarose microspheres of equal mass with an agarose content of 4% w / v are used to replace the highly cross-linked agarose microspheres in step (1).

[0065] Example 3

[0066] This embodiment provides a method for preparing a composite gel filtration medium. The specific steps and parameters are the same as those in Embodiment 1, except that an equal volume of pentaerythritol glycidyl ether is used to replace 1,4-butanediol diglycidyl ether in step (5).

[0067] Example 4

[0068] This embodiment provides a method for preparing a composite gel filtration medium. The specific steps and parameters are the same as those in Example 1, except that an equal volume of propylene glycol triglycidyl ether is used to replace 1,4-butanediol diglycidyl ether in step (5).

[0069] Example 5

[0070] This embodiment provides a method for preparing a composite gel filtration medium. The specific steps and parameters are the same as those in Example 1, except that an equal volume of ethylene glycol diglycidyl ether is used to replace 1,4-butanediol diglycidyl ether in step (5).

[0071] Example 6

[0072] This embodiment provides a method for preparing a composite gel filtration medium. The specific steps and parameters are the same as those in Example 1, except that an equal volume of epichlorohydrin is used to replace 1,4-butanediol diglycidyl ether in step (5).

[0073] Example 7

[0074] This embodiment provides a method for preparing a composite gel filtration medium. The specific steps and parameters are the same as those in Example 1, except that the molecular weight of the grafted dextran in step (4) is 20,000.

[0075] Example 8

[0076] This embodiment provides a method for preparing a composite gel filtration medium. The specific steps and parameters are the same as those in Example 1, except that the molecular weight of the grafted dextran in step (4) is 400,000.

[0077] Example 9

[0078] This embodiment provides a method for preparing a composite gel filtration medium. The specific steps and parameters are the same as those in Example 1, except that in step (5), the amount of the cross-linking agent 1,4-butanediol diglycidyl ether is 35 mL added dropwise within 2 hours.

[0079] Example 10

[0080] This embodiment provides a method for preparing a composite gel filtration medium. The specific steps and parameters are the same as those in Example 1, except that the cross-linking temperature in step (5) is 60°C.

[0081] Embodiment 11

[0082] This embodiment provides a method for preparing a composite gel filtration medium. The specific steps and parameters are the same as those in Example 1, except that the cross-linking temperature in step (5) is 40°C.

[0083] Example 12

[0084] This embodiment provides a method for preparing a composite gel filtration medium, and the specific steps and parameters are as follows:

[0085] (1) Swelling of highly cross-linked agarose: 100 mL of highly cross-linked agarose microspheres were taken, rinsed with pure water, drained, and placed in a 500 mL three-necked flask. 18 g of water and 143 g of a 40% KOH solution were added to the three-necked flask. The highly cross-linked agarose microspheres were stirred and swollen at 200 rpm for 0.5 h at 25° C. The agarose content of the highly cross-linked agarose microspheres was 8% w / v, the particle size range was 25 to 45 μm, and the pressure resistance reached above 0.3 MPa.

[0086] (2) Activating highly cross-linked agarose: Add 100 g of allyl glycidyl ether to the swollen highly cross-linked agarose obtained in step (1), and react at 70° C. for 8 h. After the reaction is completed, drain the mother liquor, wash with 10 volumes of water, 5 volumes of ethanol, and 10 volumes of water until the conductivity of the activated highly cross-linked agarose is the same as that of pure water, and drain for later use.

[0087] (3) Bromination treatment: The activated highly cross-linked agarose spheres were placed in a 500 mL three-necked flask, and 22.1 g of sodium acetate trihydrate and 260.5 g of water were added. The mixture was stirred at 220 rpm for 30 min, and 5 mL of bromine was added dropwise until the orange color did not fade. The mixture was reacted for 30 min. After the reaction, a 5% sodium formate solution was added until the system returned to white. The mother liquor was drained and the mixture was washed with water for 10 volumes until the conductivity of the highly cross-linked agarose spheres after bromination was the same as that of pure water. The mixture was drained and set aside for use.

[0088] (4) Grafting dextran: Place the brominated agarose microspheres in a 500 mL three-necked flask, add 200 mL of 10% dextran, where the molecular weight of dextran is 40,000, stir at 25°C for 1 h, add 15 g of 50% NaOH solution, and react at 40°C for 24 h. After the reaction is completed, drain the mother liquor, wash the agarose microspheres grafted with dextran with 10 volumes of water until the conductivity is the same as that of pure water, and drain for later use.

[0089] (5) Cross-linking reaction: The agarose composite microspheres grafted with dextran were drained, placed in a 500 mL three-necked flask, 20.5 g of water was added, stirred evenly, and 20 mL of 1,4-butanediol diglycidyl ether and 46.6 mL of 2M NaOH solution were added dropwise within 2 h (using a peristaltic pump to control the dropwise acceleration rate), and the reaction was continued at 50° C. for 18 h. After the reaction was completed, it was washed with deionized water until neutral to obtain a composite gel filtration medium, which was stored in 20% ethanol.

[0090] Embodiment 13

[0091] This embodiment provides a method for preparing a composite gel filtration medium, and the specific steps and parameters are as follows:

[0092] (1) Swelling of highly cross-linked agarose: 100 mL of highly cross-linked agarose microspheres were taken, rinsed with pure water, drained, and placed in a 500 mL three-necked flask. 18 g of water and 143 g of a 60% NaOH solution were added to the three-necked flask. The highly cross-linked agarose microspheres were stirred and swollen at 200 rpm for 0.5 h at 25° C. The agarose content of the highly cross-linked agarose microspheres was 8% w / v, the particle size range was 25 to 45 μm, and the pressure resistance reached above 0.3 MPa.

[0093] (2) Activating highly cross-linked agarose: Add 40 g of allyl glycidyl ether to the swollen highly cross-linked agarose obtained in step (1), and react at 40° C. for 24 h. After the reaction is completed, drain the mother liquor, wash with 10 volumes of water, 5 volumes of ethanol, and 10 volumes of water until the conductivity of the activated highly cross-linked agarose is the same as that of pure water, and drain for later use.

[0094] (3) Bromination treatment: The activated highly cross-linked agarose spheres were placed in a 500 mL three-necked flask, and 22.1 g of sodium acetate trihydrate and 260.5 g of water were added. The mixture was stirred at 260 rpm for 20 min, and 5 mL of bromine was added dropwise until the orange color did not fade. The mixture was reacted for 20 min. After the reaction, a 5% sodium formate solution was added until the system returned to white. The mother liquor was drained and the mixture was washed with water for 10 volumes until the conductivity of the highly cross-linked agarose spheres after bromination treatment was the same as that of pure water. The mixture was drained and set aside for use.

[0095] (4) Grafting dextran: Place the brominated agarose microspheres in a 500 mL three-necked flask, add 100 mL of 50% dextran, where the molecular weight of dextran is 40,000, stir at 25°C for 1 h, add 15 g of 50% NaOH solution, and react at 70°C for 8 h. After the reaction, drain the mother liquor, wash the agarose microspheres grafted with dextran with 10 volumes of water until the conductivity is the same as that of pure water, and drain for later use.

[0096] (5) Cross-linking reaction: The agarose composite microspheres grafted with dextran were drained, placed in a 500 mL three-necked flask, 20.5 g of water was added, stirred evenly, and 500 mL of 1,4-butanediol diglycidyl ether and 46.6 mL of 2M NaOH solution were added dropwise within 2 h (using a peristaltic pump to control the dropwise acceleration rate), and the reaction was continued at 60° C. for 12 h. After the reaction was completed, it was washed with deionized water until neutral to obtain a composite gel filtration medium, which was stored in 20% ethanol.

[0097] Comparative Example 1

[0098] This comparative example provides a composite gel filtration medium and a preparation method thereof. The specific steps and parameters are the same as those in Example 1, except that an equal mass of uncrosslinked agarose microspheres (agarose content of 6% w / v, pressure resistance <0.1 MPa) are used to replace the highly crosslinked agarose microspheres in step (1).

[0099] Comparative Example 2

[0100] This comparative example provides a composite gel filtration medium and a preparation method thereof. The specific steps and parameters are the same as those in Example 1, except that low-crosslinked agarose microspheres of equal mass (agarose content of 6% w / v, pressure resistance of 0.1 MPa) are used to replace the highly crosslinked agarose microspheres in step (1).

[0101] Experimental Example 1

[0102] The composite gel filtration medium prepared in Example 1-2 was measured for morphology and particle size distribution using an optical microscope (objective lens magnification of 10 times) and a BT-1600 image particle analysis system. The results are shown in Figure 1-3 It can be seen that the media of Example 1 and Example 2 have good sphericity and uniform particle size distribution, and the average particle sizes are 38.2 and 33.5 μm, respectively.

[0103] Experimental Example 2

[0104] The pressure resistance flow rate of the composite gel filtration media prepared in Example 1-11 and Comparative Example 1-2 was measured. The measurement results are shown in Table 1.

[0105] The measurement conditions are as follows:

[0106] A 1.60cm×22.50cm chromatography column was selected, the medium bed height was controlled to be 22.50cm±2.00cm, and after balancing 3 column volumes with pure water at a flow rate of 6mL / min, the chromatography column was connected to the AKTA Avant medium and low pressure chromatography system. During the measurement, the column head pressure and column pressure difference at different flow rates were set from zero, and the maximum column pressure difference was 0.3MPa, and the corresponding flow rate at this time was the maximum flow rate.

[0107] The pressure-resistant flow rate reflects the mechanical strength of the microspheres, and high-rigidity gel filtration media can effectively shorten the operating time required for separation and purification. Figure 4 It can be seen from the data in Table 1 that the media prepared in Examples 1-11 have good mechanical strength, wherein the linear flow rates of Examples 1 and 2 can both reach 180 cm / h at a pressure of 0.3 MPa.

[0108] Experimental Example 3

[0109] The chromatographic performance of the composite gel filtration media prepared in Examples 1-11 and Comparative Examples 1-2 was tested, and the distribution coefficient K, which can reflect the density of the network structure and the porosity of the microspheres, was used. av Make an evaluation.

[0110] The test conditions are as follows:

[0111] A 1.60 cm × 30.00 cm chromatography column was selected, the medium bed height was controlled to be 30.00 cm ± 2.00 cm, and after balancing 3 column volumes with pure water at a flow rate of 6 mL / min, the AKTA Avant chromatography system was balanced with 0.15 M NaCl at a flow rate of 2.0 mL / min to a baseline equilibrium.

[0112] Loading relative molecular weight Mr = 243 cytidine elution volume V i As a standard, a sample of dextran with a relative molecular mass of Mr = 2000000 was loaded to determine the elution volume V0. Then, standard proteins with different relative molecular masses were loaded separately to determine the elution volume Vt; the concentration of all standard protein samples was 5.0 mg / mL, and the loading volume was 500 μL. The relative molecular masses of the standard proteins were: Mr (核糖核酸酶) =13700,Mr. (糜蛋白酶) =28000,Mr. (卵清蛋白) =45000, Mr. (BSA) =66446,Mr. (IgG) =150000,Mr. (醛缩酶) =158000,Mr. (马蹄铁蛋白) =440000.

[0113] Result calculation: Partition coefficient K av =(V t -V0) / (V i -V0), the absolute difference between three independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean.

[0114] Test results are shown in Figure 5 From Table 1, it can be seen that the medium K prepared in Example 1 av(核糖核酸酶) is 0.5548, K av(糜蛋白酶) is 0.2926, Kav(卵清蛋白) is 0.1920, K av(BSA) The steep slope of the selectivity curve indicates that the composite gel filtration medium prepared in Example 1 has a high resolution for the target substances with a relative molecular weight in the range of 3000 to 70000 Da.

[0115] Selectivity curve Figure 6 From Table 1, it can be seen that the medium K prepared in Example 2 av(核糖核酸酶) is 0.6668, K av(糜蛋白酶) is 0.5306, K av(卵清蛋白) is 0.4920, K av(BSA) is 0.4704, K av(IgG) is 0.3443, K av(醛缩酶) is 0.3739, K av(马蹄铁蛋白) The steep slope of the selectivity curve indicates that the composite gel filtration medium prepared in Example 2 has a high resolution for the target substances with a relative molecular weight in the range of 10000 to 600000 Da.

[0116] Experimental Example 4

[0117] The nonspecific adsorption amount of the composite gel filtration media prepared in Example 1-11 and Comparative Example 1-2 was tested. The test results are shown in Table 1.

[0118] The test conditions are as follows:

[0119] Select a 1.60cm×20.00cm chromatography column, control the medium bed height to 22.50cm±2.00cm, balance 3 column volumes at a flow rate of 1.0mL / min, and test after the AKTA Avant chromatography system bed is stable. Record the sample column volume V gel (mL). Balance with phosphate buffer A, flow rate of 2.0mL / min, and load 5mL of protein. Elute with buffer A until no protein appears in the eluent, then elute with phosphate buffer B, and collect the corresponding elution peak. Record the elution peak volume V E (mL). Use a UV spectrophotometer to detect the absorbance A of the cytochrome C standard working solution with different concentration gradients and the elution peak at 280nm 280 The standard working solution absorbance and protein concentration are used as the standard curve, and the elution peak protein concentration C is quantified by the standard curve. E (mg / mL).

[0120] Calculation of results: Nonspecific adsorption amount = C E V E / V gelThe absolute difference between three independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean.

[0121] The test results are shown in Table 1. The nonspecific adsorption amounts of the composite gel filtration media prepared in the embodiments of the present invention are all relatively low, wherein the nonspecific adsorption amounts of Embodiment 1 and Embodiment 2 are 16.12 mg / mL and 31.40 mg / mL, respectively.

[0122] Table 1 Properties of composite gel filtration media

[0123]

[0124]

[0125] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing a composite gel filtration medium, characterized in that: The following steps are included: S1, swelling, activating and brominating the cross-linked agarose microspheres, wherein the pressure resistance of the cross-linked agarose is greater than or equal to 0.3 MPa; S2, in an alkaline solution, grafting the brominated agarose microspheres with dextran to obtain dextran-grafted agarose microspheres; S3, cross-linking the agarose microspheres grafted with dextran with a cross-linking agent to obtain a composite gel filtration medium.

2. The method for preparing the composite gel filtration medium according to claim 1, characterized in that: In step S3, the crosslinking agent includes a bifunctional crosslinking agent containing an epoxy group; and / or, The cross-linking reaction temperature is 40-60°C and the reaction time is 12-24h; and / or, The volume ratio of the agarose microspheres grafted with dextran to the cross-linking agent is (1-5):(1-5).

3. The method for preparing the composite gel filtration medium according to claim 2, characterized in that: The cross-linking agent comprises at least one of a halogenated epoxy compound and a glycidyl ether; and / or, In step S1, the step of swelling the cross-linked agarose microspheres comprises stirring the cross-linked agarose microspheres and the alkaline solution at 180 to 200 rpm for 0.5 to 1 h; and / or, The activation step of the swollen cross-linked agarose microspheres also includes washing and drying the activated cross-linked agarose.

4. The method for preparing the composite gel filtration medium according to claim 3, characterized in that: The halogenated epoxy compound includes epichlorohydrin; and / or, The glycidyl ether includes at least one of ethylene glycol diglycidyl ether, propylene glycol glycidyl ether, butanediol diglycidyl ether and pentaerythritol glycidyl ether, preferably butanediol diglycidyl ether; and / or, The mass concentration of agarose in the cross-linked agarose microspheres is 4% to 8%, preferably 4% to 6%; and / or, The particle size of the cross-linked agarose microspheres is less than or equal to 60 μm, preferably 25 to 45 μm.

5. The method for preparing the composite gel filtration medium according to claim 4, characterized in that: The activation step of the swollen cross-linked agarose microspheres in step S1 comprises adding allyl glycidyl ether to the swollen cross-linked agarose microspheres, reacting at 40 to 70° C. for 8 to 24 hours; and / or, The bromination step of the activated cross-linked agarose microspheres comprises mixing the activated cross-linked agarose with bromine and a pH regulator at a speed of 220-260 rpm in an aqueous solvent, and reacting for 20-30 minutes; and / or, The volume ratio of cross-linked agarose microspheres to bromine is (20-10):1; and / or, The grafting reaction temperature in step S2 is 40-70° C. and the reaction time is 8-24 hours; and / or, The mass ratio of the volume of the cross-linked agarose microspheres to allyl glycidyl ether is (1-5):(1-2), preferably (1-2):1, and the unit is mL:g.

6. The method for preparing the composite gel filtration medium according to claim 5, characterized in that: In step S2, the molecular weight of the dextran is 20,000 to 400,000, preferably 40,000 to 200,000; and / or, In step S2, a dextran aqueous solution is used to provide dextran for the grafting reaction, wherein the mass concentration of dextran in the dextran aqueous solution is 10-50%; and / or, The volume ratio of cross-linked agarose microspheres to dextran solution is 1:(1-2); and / or, The alkaline solution for swelling the cross-linked agarose in step S2 and / or step S1 comprises a metal hydroxide solution, The metal hydroxide solution includes at least one of a sodium hydroxide solution and a potassium hydroxide solution. Optionally, the mass concentration of the alkaline solution is 40-60%, preferably 50%.

7. A composite gel filtration medium, characterized in that: The composite gel filtration medium is prepared by the preparation method of the composite gel filtration medium according to any one of claims 1 to 6.

8. The composite gel filtration medium according to claim 7, characterized in that: The particle size of the composite gel filtration medium is 25 to 45 μm; and / or, The separation range of the composite gel filtration medium is 3000 to 70000 Da; and / or, The separation range of the composite gel filtration medium is 10000-600000Da.

9. Use of the composite gel filtration medium according to claim 8 in separation and purification of biomolecules.

10. The use according to claim 9, characterized in that: The biomolecules include at least one of polypeptides, recombinant proteins, nucleotides, and small molecules. The biomolecules also include at least one of antibody monomers, dimers and aggregates in monoclonal antibodies.

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