A core-shell composite mode filler and a preparation method thereof

Abstract

This invention discloses a core-shell composite packing material and its preparation method. The matrix is ​​activated using two types of activating compounds with different properties. The first activation involves grafting short-length functional groups, which should be inert during the subsequent second activation and the coupling reaction with the packing shell. The second activation involves grafting longer, network-like or dendritic functional groups, which are used to couple one or more layers of polymer compounds, acting as the shell in the core-shell structure to achieve size exclusion. Furthermore, the size exclusion molecular weight can be controlled by adjusting the coverage from small to large. After achieving satisfactory size exclusion through shell coupling, the groups grafted to the core during the first activation are fully activated, allowing for further coupling of functional groups with different mechanisms of action. The preparation method of the core-shell composite packing material disclosed in this invention is stable. The core-shell composite packing material prepared by the method disclosed in this invention has high loading capacity and stable functional group activity.

Description

Technical Field

[0001] This invention relates to the field of composite packing synthesis, and more particularly to a core-shell composite packing and its preparation method. Background Technology

[0002] The booming development of the biopharmaceutical industry has placed higher demands on the production capacity of upstream and downstream biopharmaceutical products. How to purify the required components from a wide variety of complex expression systems has become a primary challenge for major manufacturers. As a separation and purification method with mild conditions and minimal loss of product bioactivity, chromatographic chromatography has been widely used in the separation and purification steps of various biopharmaceuticals.

[0003] The core of chromatographic chromatography technology is the chromatographic medium, also known as the packing material, which plays a crucial role in separating different components. Its performance is a key factor affecting the effectiveness and efficiency of the entire purification process. With the continuous development of chromatographic chromatography technology, there are now hundreds of different types of chromatographic media. However, based on their separation mechanisms, they are mainly classified into four categories: size exclusion, ion interaction, hydrophobic interaction, and affinity interaction. In recent years, based on this, chromatographic packing materials with dual or multiple functions, such as ion-plus-hydrophobic and size exclusion-plus-ion-plus-hydrophobic, have also been developed—i.e., composite mode chromatographic media.

[0004] Core-shell composite chromatography media are a type of composite packing material that couples size exclusion and other separation mechanisms. They are mainly used for the separation and purification of biomacromolecules such as viruses, exosomes, and proteins. Taking viruses as an example, traditional methods involve purification via gel filtration chromatography or ion exchange chromatography. While the former can greatly ensure viral activity and purity, its production efficiency is low, with a theoretical loading capacity of only 10% CV. The latter, although possessing a higher loading capacity, makes it difficult to simultaneously achieve both virus recovery and protein removal rates.

[0005] Against this backdrop, Cytiva proposed a core-shell composite chromatography medium—Capto TM The Core 700 consists of an inert, size-exclusion-resistant outer shell and a core with anionic and hydrophobic ligand functional groups. During separation and purification, impurity protein molecules smaller than a certain volume in the viral stock solution enter the microsphere through the pores of the outer shell and are captured by the functional groups, while the virus flows through the chromatographic column in a flow-through mode, thus achieving a win-win situation of high loading capacity and high recovery rate.

[0006] Existing methods for synthesizing core-shell composite chromatography packing materials mostly employ partial bromination. However, bromine, a potentially dangerous precursor for toxic and explosive substances, requires registration, and its purchase and use are cumbersome, demanding high compliance from operators and stringent safety requirements for the production workshop. Furthermore, the process is difficult to control; due to its high volatility, the amount of brominating reagent involved in the chemical reaction is difficult to quantify accurately during large-scale production, leading to batch-to-batch variations in the final product. Moreover, the exclusion effect is achieved through an inert surface region, but the pore structure and porosity of the matrix necessitate a trade-off between exclusion efficiency and accessibility of active regions. Generally, to achieve satisfactory exclusion, the inert layer on the substrate surface needs to be at least 5 μm thick, wasting a significant amount of the substrate's surface region, which is readily bound to proteins, thus reducing its loading capacity.

[0007] To address the aforementioned issues, recent patents such as CN109647361 have reported new synthetic approaches. Unlike the traditional inward approach of partial bromination, the matrix described in this patent is first functionalized with a water-soluble polyamine, and then coupled to the matrix with a neutral polymer compound through nucleophilic substitution reactions of aldehyde and amino groups. Although this method has the advantage of using different materials for the shell and matrix, this "outward expansion" approach still presents new challenges. Firstly, the functionalization of the polymers used is complex; commonly used allylation and aldehyde products typically require dialysis for purification. Obtaining large quantities of batch-to-batch stable, relatively homogeneous functionalized polymers is a problem this method must solve. Secondly, when using these activated molecules to modify already coupled functionalized spheres, there is a probability of structural changes in the functional groups. For example, the reaction between epoxy groups and primary or secondary amines may cause them to lose their inherent properties, such as salt tolerance. Furthermore, the subsequent reaction between the functional groups and the activated polymer may clog the pores of the coupled functional groups, further reducing the effective ligand density of the core and affecting its fundamental loading capacity. Summary of the Invention

[0008] Purpose of the invention: The purpose of this invention is to provide a core-shell composite packing with high loading capacity and its preparation method.

[0009] Technical solution: The core-shell composite packing of the present invention comprises a core-shell structure with a microsphere matrix as the base sphere, wherein short-chain activating compounds and long-chain activating compounds are connected to the base sphere, and the short-chain activating compounds are coupled with functional groups with different functions; one or more layers of polymeric compounds are coupled to the long-chain activating compounds as the outer shell of the core-shell structure, wherein the short-chain activating compounds are shown in general formula I, and the long-chain activating compounds are shown in general formula II.

[0010]

[0011] In the formula, R1 is selected from C1 to C10 straight-chain alkyl, repeating polymeric units, or alkyl chains containing ester groups; R2 is selected from epoxy groups or halogen substituents; R3 is selected from C1 to C10 straight-chain alkyl or repeating polymeric units; and R4 is selected from epoxy groups, olefins, or halogen substituents.

[0012] The core-shell composite packing material of the present invention is constructed from the inside out, and the coverage is adjustable. That is, by adjusting the pore size of the core and shell, the exclusion effect on proteins of different volumes can be achieved.

[0013] The preparation method of the core-shell composite packing of the present invention includes the following steps:

[0014] (1) First activation reaction of the nucleus: The microsphere matrix, activation compound 1 and inorganic alkaline solution are mixed, and the microspheres after the first activation are obtained after the reaction is completed; the structure of the activation compound 1 is shown in general formula I:

[0015]

[0016] Wherein, R1 is selected from C1 to C10 straight-chain alkyl, repeating polymeric unit or alkyl chain containing ester group, and R2 is selected from epoxy group or halogen substituent;

[0017] (2) Second activation reaction of the nucleus: Microspheres after the first activation, activated compound 2 and inorganic alkaline solution are added to the solvent. After the reaction is completed, microspheres after the second activation are obtained.

[0018] The structure of the activated compound 2 is shown in general formula II:

[0019]

[0020] Wherein, R3 is selected from C1 to C10 straight-chain alkyl or repeating polymeric units, and R4 is selected from epoxy, olefin or halogen substituents;

[0021] (3) Coupling shell: Add the microspheres after the second activation, the polymer compound and the inorganic alkaline solution, and the core-shell spheres are obtained after the reaction is completed;

[0022] (4) Ligand coupling: Activator is used to activate the pre-reserved activation group in the core of the core-shell sphere, and then one or more functional groups with different functions are coupled. The functional groups have ion exchange function, hydrophobic function, hydrophobic ion recombination function or affinity function; thus, the core-shell composite packing is obtained.

[0023] The preparation method of this invention involves activating the matrix with two types of activating compounds with different properties. The first activation involves grafting shorter functional groups, which should be inert during the subsequent second activation and the coupling reaction with the packing shell. The second activation involves grafting longer functional groups that exhibit a network or dendritic structure, used to couple one or more layers of polymer compounds as the outer shell in the core-shell structure, achieving size exclusion. Furthermore, by adjusting the coverage amount from small to large, the size exclusion molecular weight can be controlled from large to small. After achieving satisfactory size exclusion through shell coupling, the groups grafted to the core during the first activation are fully activated, allowing for further coupling of functional groups with different mechanisms of action, such as ion exchange, hydrophobic interactions, hydrophobic ion recombination modes, and affinity interactions. A schematic diagram of the composite chromatography packing material prepared by this invention is shown below. Figure 1 As shown, the core base sphere is grafted with activated compounds 1 and 2, the former for coupling functional groups and the latter for grafting the shell.

[0024] Further, the microsphere matrix in step (1) includes, but is not limited to, one or more of agarose microspheres, dextran microspheres, konjac microspheres, cellulose microspheres, polymethacrylate microspheres, and polystyrene-divinylbenzene microspheres, and other similar microsphere matrices may also be used; the activated compound 1 includes one or more of allyl glycidyl ether, allyl iodine, 3,4-epoxy-1-butene, 1,2-epoxy-9-decene, and glycidyl methacrylate; the inorganic alkaline solution includes NaOH solution or KOH solution.

[0025] Further, in step (1), the mass ratio of activated compound 1 to the microsphere matrix is ​​0.1-3:1; the temperature of the first activation reaction is 25-65℃, the reaction time is 10-24h, the reaction conditions are 50-300rpm, and the concentration of the inorganic alkaline solution is 0-18M.

[0026] Furthermore, the microsphere matrix is ​​preferably agarose microspheres or polymethacrylate microspheres. Activating compound 1 is preferably allyl glycidyl ether. The concentration of the NaOH solution is preferably 10M.

[0027] Further, the solvent mentioned in step (2) includes one or more of water, DMSO, dioxane, and DMF; the activated compound 2 includes one or more of 1,2,7,8-diepoxyoctane, 1,4-butanediol diglycidyl ether, pentanediol diglycidyl ether, poly(ethylene glycol) diglycidyl ether, poly(propylene glycol) diglycidyl ether, and trimethylolpropane triglycidyl ether.

[0028] Further, in step (2), the mass ratio of activated compound 2 to the microspheres after the first activation is 0.1-5:1; the temperature of the second activation reaction is 25-65℃, the reaction time is 10-24h, the reaction conditions are 50-300rpm, and the concentration of the inorganic alkaline solution is 0-5M.

[0029] Furthermore, the activating compound 2 is preferably poly(ethylene glycol) diglycidyl ether.

[0030] Furthermore, the mass ratio of the solvent to the microspheres after the first activation is 1 to 3:1.

[0031] Furthermore, the polymeric compound mentioned in step (3) can be a natural or synthetic hydroxyl-rich compound with an average molecular weight ≥10,000 Da, preferably 10,000-500,000 Da, including but not limited to polysaccharides (cellulose, dextran, molten agarose, etc.) and polymers (polyethylene glycol, polyvinyl alcohol, epoxy-hydrolyzed polyglycidyl methacrylate). The polymeric compound should minimize the number of groups containing ion exchange or hydrophobic interactions as branches or main chains in its structure to avoid unwanted interactions between the separated components and the filler during application.

[0032] Further, in step (3), the mass ratio of the polymer compound to the microspheres after the second activation is 0.1-3:1, the concentration of the inorganic alkaline solution is 0-5M, the reaction temperature is 25-65℃, the reaction time is 10-24h, and the reaction conditions are 50-300rpm.

[0033] Further, the activator mentioned in step (4) is an alkyl halide; the reaction temperature of the activation reaction is 0-50℃, the reaction time is 0-10h, and the reaction conditions are 50-300rpm.

[0034] Furthermore, the reagent used for coupling in step (4) can be a functional group with ionic hydrophobic complexation effect, including one or more of n-octylamine, polyethyleneimine, and polyacrylamine, preferably n-octylamine or polyethyleneimine.

[0035] Furthermore, the reagent used for coupling in step (4) can be an affinity group, including one or more of protein A, protein G, protein L, and 3-aminophenylboronic acid.

[0036] Further, the alkyl halide may be N-bromosuccinimide (NBS), N-bromophthalimide, N-bromoacetamide, tetrabromobenzoquinone, 1,3-dibromo-5,5-dimethylhydantoin (DBH), PyHBr3, carbon tetrabromide, 1,3,5-tribromo-1,3,5-thiazine-2,4,6-trione (TBCA), 1,3-dibromo-1,3,5-triazine-2,4,6-trione (DBI), or N-bromo-o-sulfonylbenzoimide (NBSac), preferably NBS or TBCA.

[0037] Furthermore, when the outer shell pore size of the core-shell spheres obtained in step (3) is larger than the diameter of the exclusion protein, the effect is that when the exclusion protein binds to the filler, steps (2) and (3) need to be repeated.

[0038] Furthermore, the activating compound used in repeating step (2) includes epichlorohydrin, 1,4-butanediol diglycidyl ether, 1,2,7,8-diepoxyoctane, preferably epichlorohydrin.

[0039] The core-shell composite packing material prepared by the method described in this invention achieves its size exclusion effect without relying on the inerting of the surface region of the microspheres themselves. While partially resolving the instability of the bromination process, it also retains the original surface spheres that can be used to couple ligands, further increasing the packing capacity. Secondly, the size exclusion effect of the polymer compound can be easily controlled by adjusting its molecular weight or density, and its spatial thickness is thinner, reducing the difficulty for molecules that do not require size exclusion to enter or exit the shell, allowing for faster binding and elution of proteins. Finally, the preparation method of this invention reserves semi-activated groups in the core that do not participate in subsequent reactions, which can be selectively activated after the shell is coated, grafting the required ligands and avoiding functional group deactivation or loss that may occur during the shell coating process.

[0040] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: The preparation method of the core-shell composite packing material described in this invention is stable. The prepared core-shell composite packing material has a high loading capacity, reaching over 31 mg / mL, and stable functional group activity. Depending on purification requirements, different functional groups can be activated and coupled at the core, and the pore size of the shell can be adjusted to exclude target analytes of different particle sizes, thus achieving excellent separation and purification of multiple target analytes. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the composite chromatography packing material structure described in this invention;

[0042] Figure 2 This is a scanning electron microscope image of the packing material prepared in Example 1 of the present invention;

[0043] Figure 3 The chromatographic result of the adsorption capacity of small-particle OVA by the packing material prepared in Example 1 of the present invention is shown (where UV: ultraviolet (UV, 280nm) absorption peak; Cond: conductivity curve; Conc: buffer solution ratio (manual liquid change, so this concentration does not change)).

[0044] Figure 4 The chromatography diagram of the Tg+OVA mixed sample prepared in Example 2 of the present invention (where UV: ultraviolet (UV, 280nm) absorption peak; Cond: conductivity curve; Conc: buffer solution ratio (manual solution change, so this concentration does not change));

[0045] Figure 5 The HPLC chromatogram of the elution peak of the packing material prepared in Example 2 of this invention (red line: sample Tg+OVA; blue line: elution peak OVA);

[0046] Figure 6 The chromatography diagram of the HSF+OVA mixed sample prepared by coupling the shell only once in Example 3 of the present invention is shown (where UV: ultraviolet (UV, 280nm) absorption peak; Cond: conductivity curve; Conc: buffer solution ratio (manual solution change, so this concentration does not change)).

[0047] Figure 7 The chromatogram of the HSF+OVA mixed sample prepared by coupling the shell only once in Example 3 of this invention is shown (red line: sample HSF+OVA; blue line: elution peak HSF+OVA).

[0048] Figure 8 The image shows the chromatography of the HSF+OVA mixed sample prepared in Example 3 of this invention (where UV: ultraviolet (UV, 280nm) absorption peak; Cond: conductivity curve; Conc: buffer solution ratio (manual solution change, so this concentration does not change)).

[0049] Figure 9 The HPLC chromatogram of the elution peak of the packing material prepared in Example 3 of this invention is shown (red line: sample HSF+OVA; blue line: flow-through peak HSF). Detailed Implementation

[0050] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0051] Example 1

[0052] 1) The first activation of the nucleus

[0053] 100g of Agarosix microspheres (average particle size 90μm, 6% cross-linked agarose matrix, catalog number 250290990) purchased from Saifen Technology Co., Ltd. was weighed and added to 100g of 10M NaOH solution. After stirring evenly, allyl glycidyl ether (activating compound 1) was added. The mass ratio of activating compound 1 to microspheres was 1:1. The reaction was carried out at 45℃ and 150rpm for 18h. After the reaction was completed, the microspheres were filtered and washed repeatedly with anhydrous ethanol and deionized water until the pH of the microspheres was neutral, thus obtaining the microspheres after the first activation.

[0054] 2) Secondary activation of the nucleus

[0055] Take the microspheres after the first activation, add 100g of DMSO and 50g of 1.5M NaOH solution, stir evenly, and then slowly add poly(ethylene glycol) diglycidyl ether (activating compound 2) dropwise at 37℃ and 150rpm. The mass ratio of activating compound 2 to microspheres is 1.4:1. The addition takes about 3 hours. After the addition is complete, react under the same conditions for another 4 hours. After the reaction is complete, filter and wash to obtain the microspheres after the second activation.

[0056] 3) Coupling shell

[0057] The microspheres after the second activation were mixed evenly with a 25% dextran (MW.70000) solution, and an equal mass of 3M NaOH solution was added. The reaction was carried out at 50℃ and 150 rpm for 18 h. After the reaction was completed, the microspheres were filtered and washed to obtain core-shell microspheres.

[0058] 4) Ligand coupling

[0059] Take 50g of core-shell spheres, add 50mL of deionized water and 3.5g of sodium acetate, stir well, then add 2g of N-bromosuccinimide (NBS), stir at room temperature for 2h, then add sodium formate until the color of the reaction solution disappears, indicating that the unreacted bromine has been completely quenched; after filtration and washing, add 100mL of deionized water and 25g of n-octylamine, stir well, and place in a shaker at 45℃ and 100rpm for 18h. After the reaction is completed, filter and wash to obtain the composite mode chromatography packing material.

[0060] The adsorption loading of the packing material was evaluated using small-particle-size ovalbumin (OVA), and the test conditions were as follows:

[0061] Loading buffer: 20 mM Tris, 0.15 M NaCl, pH 7.5;

[0062] Sample: 3 mg / mL OVA dissolved in binding buffer;

[0063] Elution buffer: 20 mM Tris, 1.5 M NaCl, pH 7.5;

[0064] Regeneration (Cleaning-in-place, CIP): Dissolve 30% isopropanol (2-propanol) in 1M NaOH;

[0065] Flow velocities: 60 cm / h (combined);

[0066] After loading the sample to 10% flow-through and equilibrating for elution, CIP was performed, followed by two repeatability tests.

[0067] Its three 10% flow throughputs were 32.6, 31.2, and 31.5 mg / mL, respectively, which are significantly higher than those of Cytiva Capto. TM The Core700 is listed with a loading of 13 mg / mL. The chromatographic results are shown below. Figure 3 As shown, the loading capacity was calculated using the time to 10% flow-through and the concentration of the loading solution.

[0068] Example 2

[0069] 1) The first activation of the nucleus

[0070] Weigh 100g of Agarosix microspheres purchased from Saifen Technology, add 100g of 10M NaOH solution, stir well, and then add allyl glycidyl ether (activating compound 1). The mass ratio of activating compound 1 to microspheres is 1:1. The reaction is carried out at 45℃ and 150rpm for 18h. After the reaction is completed, filter and wash to obtain the microspheres after the first activation.

[0071] 2) Secondary activation of the nucleus

[0072] Take the microspheres after the first activation, add 100g of DMSO and 50g of 1.5M NaOH solution, stir evenly, and slowly add poly(ethylene glycol) diglycidyl ether (activating compound 2) dropwise at 37℃ and 150rpm. The mass ratio of activating compound 2 to microspheres is 0.95:1. The addition takes about 3 hours. After the addition is complete, react under the same conditions for another 4 hours. After the reaction is complete, filter and wash to obtain the secondary activated microspheres.

[0073] 3) Coupling shell

[0074] The microspheres after the second activation were mixed evenly with 25% dextran (MW.500000), and an equal mass of 3M NaOH solution was added. The reaction was carried out at 50℃ and 150 rpm for 18 h. After the reaction was completed, the microspheres were filtered and washed to obtain core-shell microspheres.

[0075] 4) Ligand coupling

[0076] Take 50g of core-shell spheres, add 50mL of deionized water and 3.5g of sodium acetate, stir well, then add 2g of N-bromosuccinimide (NBS), stir at room temperature for 2h, then add sodium formate until the color of the reaction solution disappears, indicating that the unreacted bromine has been completely quenched; after filtration and washing, add 100mL of deionized water and 25g of polyethyleneimine, stir well, and place in a shaker at 45℃ and 100rpm for 18h. After the reaction is completed, filter and wash to obtain the composite mode chromatography packing material;

[0077] The packing material was evaluated for exclusion of specific molecular weights using large-particle thyroglobulin (Tg) and small-particle ovalbumin (OVA) under the following test conditions:

[0078] Loading Buffer: 20mM Tris, 0.15M NaCl, pH 7.5;

[0079] Sample: 1mg / mL Tg and 3mg / mL OVA in loading buffer;

[0080] Elution Buffer: 20mM Tris, 1.5M NaCl, pH 7.5;

[0081] Cleaning-in-place (CIP): 30% 2-propanol in 1M NaOH;

[0082] Flow velocities: 100 cm / h;

[0083] Chromatographic results as follows Figure 4 and 5 As shown, Figure 4 Peak 1 is the flow-through peak, peak 2 is the elution peak, and peak 3 is the CIP peak. Figure 5 HPLC data analysis showed that the elution peak mainly contained OVA protein with a purity of 97.3%, indicating that Tg protein was almost completely eliminated by size exclusion flow. The results show that the packing material achieved size exclusion of Tg and adsorption of OVA.

[0084] Example 3

[0085] 1) The first activation of the nucleus

[0086] 100g of Monomix microspheres (60μm particle size, polymethyl methacrylate matrix, catalog number 280160950) purchased from Saifen Technology Co., Ltd. was weighed and added to 100g of 3M NaOH solution. After stirring evenly, allyl glycidyl ether (activating compound 1) was added. The mass ratio of activating compound 1 to microspheres was 1:1.5. The reaction was carried out at 45℃ and 150rpm for 18h. After the reaction was completed, the microspheres were filtered and washed to obtain the microspheres after the first activation.

[0087] 2) Secondary activation of the nucleus

[0088] Take the microspheres after the first activation, add 100g of dioxane and 50g of 1.5M NaOH solution, stir evenly, and then slowly add poly(ethylene glycol) diglycidyl ether (activating compound 2) dropwise at 37℃ and 150rpm. The mass ratio of activating compound 2 to microspheres is 1.4:1. The addition takes about 3 hours. After the addition is complete, react under the same conditions for another 4 hours. After the reaction is complete, filter and wash to obtain the microspheres after the second activation.

[0089] 3) Primary coupling shell

[0090] The microspheres after the second activation were mixed evenly with 25% dextran (MW.220000) solution, and an equal mass of 3M NaOH solution was added. The reaction was carried out at 50℃ and 150 rpm for 18 h. After the reaction was completed, the microspheres were filtered and washed to obtain core-shell microspheres.

[0091] 4) Secondary coupling shell

[0092] The microspheres obtained in the previous step were mixed with equal masses of DMSO and epichlorohydrin, stirred evenly, and 10 g of 3M NaOH solution was added. The mixture was reacted at 50℃ and 100 rpm for 4 h, and then filtered and washed. The washed microspheres were mixed evenly with 25% dextran (MW.220000) solution, and 3M NaOH solution of equal mass to the microspheres was added. The mixture was reacted at 50℃ and 150 rpm for 18 h. After the reaction was completed, the mixture was filtered and washed to obtain secondary core-shell spheres.

[0093] 5) Ligand coupling

[0094] Take 50g of secondary core-shell spheres, add 50mL of deionized water and 3.5g of sodium acetate, stir well, then add 2g of N-bromosuccinimide (NBS), stir at room temperature for 2h, then add sodium formate until the color of the reaction solution disappears, indicating that the unreacted bromine has been completely quenched; after filtration and washing, add 100mL of deionized water and 25g of n-octylamine, stir well, and place in a shaker at 45℃ and 100rpm for 18h. After the reaction is completed, filter and wash to obtain the composite mode chromatography packing material;

[0095] The packing material was evaluated for exclusion of specific molecular weights using medium-sized horse spleen ferritin (HSF) and small-sized ovalbumin (OVA) under the following test conditions:

[0096] Loading Buffer: 20mM Tris, 0.15M NaCl, pH 7.5;

[0097] Sample: 0.1mg / mL HSF and 3mg / mL OVA in loading buffer;

[0098] Elution Buffer: 20mM Tris, 1.5M NaCl, pH 7.5;

[0099] Cleaning-in-place (CIP): 30% 2-propanol in 1M NaOH;

[0100] Flow velocities: 100 cm / h;

[0101] Chromatographic results as follows Figure 6 , 7 As shown in 8 and 9, Figure 6 and 8 Peak 1 is the flow-through peak, peak 2 is the elution peak, and peak 3 is the CIP peak. Figure 6 and 7 Analysis of the core-shell spheres after initial coupling showed a small flow-through peak, and the elution peak contained both HSF and OVA proteins, indicating that the shell failed to achieve the expected HSF exclusion effect; combined with Figure 8 and 9 Analysis of the core-shell spheres after secondary coupling showed a significant increase in the flow-through peak, and the peak contained only HSF protein, indicating that the packing material after secondary coupling had an exclusion effect on HSF protein. These results show that by repeatedly coupling the shell, the exclusion limit of the packing material can be further reduced, increasing the removal rate of HSF in the sample from <5% to 73%.

[0102] Example 4

[0103] 1) The first activation of the nucleus

[0104] 100g of Agarosix microspheres (average particle size 90μm, 6% cross-linked agarose matrix) purchased from Saifen Technology Co., Ltd. was weighed and 100g of 3M NaOH solution was added. After stirring evenly, allyl iodine (activating compound 1) was added. The mass ratio of activating compound 1 to microspheres was 0.1:1. The reaction was carried out at 35℃ and 150rpm for 18h. After the reaction was completed, the microspheres were filtered and washed repeatedly with anhydrous ethanol and deionized water until the pH of the microspheres was neutral, thus obtaining the microspheres after the first activation.

[0105] 2) Secondary activation of the nucleus

[0106] Take the microspheres after the first activation, add 100g of DMF and 50g of 1.5M NaOH solution, stir well, and then slowly add 1,4-butanediol diglycidyl ether (activating compound 2) dropwise at 37℃ and 150rpm. The mass ratio of activating compound 2 to microspheres is 0.8:1. The addition takes about 3 hours. After the addition is complete, react under the same conditions for another hour. After the reaction is complete, filter and wash to obtain the microspheres after the second activation.

[0107] 3) Coupling shell

[0108] The microspheres after the second activation were mixed evenly with a 5% polyvinyl alcohol (MW.200000) solution, and an equal mass of 3M NaOH solution was added. The reaction was carried out at 50℃ and 150 rpm for 18 h. After the reaction was completed, the microspheres were filtered and washed to obtain core-shell microspheres.

[0109] 4) Ligand coupling

[0110] Take 50g of core-shell spheres, add 50mL of deionized water and 3.5g of sodium acetate, stir well, then add 2g of N-bromosuccinimide (NBS), stir at room temperature for 2h, then add sodium formate until the color of the reaction solution disappears, indicating that the unreacted bromine has been completely quenched; after filtration and washing, add 100mL of deionized water and 25g of n-octylamine, stir well, and place in a shaker at 45℃ and 100rpm for 18h. After the reaction is completed, filter and wash to obtain the composite mode chromatography packing material.

[0111] The loading capacity of the obtained composite mode chromatography packing material was tested using the same method as in Example 1, with a sample loading of 20.5 mg / mL. Example 5

[0112] 1) The first activation of the nucleus

[0113] 100g of Agarosix microspheres (average particle size 90μm, 6% cross-linked agarose matrix, catalog number 250290990) purchased from Saifen Technology Co., Ltd. was weighed and added to 100g of 10M KOH solution. After stirring evenly, 1,2-epoxy-9-decene (activating compound 1) was added. The mass ratio of activating compound 1 to microspheres was 1.5:1. The reaction was carried out at 55℃ and 200rpm for 24h. After the reaction was completed, the microspheres were filtered and washed repeatedly with anhydrous ethanol and deionized water until the pH of the microspheres was neutral, thus obtaining the microspheres after the first activation.

[0114] 2) Secondary activation of the nucleus

[0115] Take the microspheres after the first activation, add 100g of DMSO and 50g of 1.5M KOH solution, stir evenly, and then slowly add poly(ethylene glycol) diglycidyl ether (activating compound 2) dropwise at 37℃ and 150rpm. The mass ratio of activating compound 2 to microspheres is 1.4:1. The addition takes about 3 hours. After the addition is complete, react under the same conditions for another 4 hours. After the reaction is complete, filter and wash to obtain the microspheres after the second activation.

[0116] 3) Coupling shell

[0117] The microspheres after the second activation were mixed evenly with polyvinyl alcohol (MW.170000) solution, and an equal mass of 3M NaOH solution was added. The reaction was carried out at 50℃ and 150 rpm for 18 h. After the reaction was completed, the microspheres were filtered and washed to obtain core-shell microspheres.

[0118] 4) Ligand coupling

[0119] Take 50g of core-shell spheres, add 50mL of deionized water and 3.5g of sodium acetate, stir well, then add 2.54g of N-bromophthalimide. Stir at room temperature for 2 hours, then add sodium formate until the color of the reaction solution disappears, indicating that the unreacted bromine has been completely quenched. After filtration and washing, add 100mL of deionized water and 25g of n-octylamine, stir well, and place in a shaker at 45℃ and 100rpm for 18 hours. After the reaction is completed, filter and wash to obtain the composite mode chromatography packing material.

[0120] The loading capacity of the obtained composite mode chromatography packing was tested using the same method as in Example 1, and the sample loading capacity was 28.4 mg / mL.

[0121] Example 6

[0122] 1) The first activation of the nucleus

[0123] 100g of Agarosix microspheres (average particle size 90μm, 6% cross-linked agarose matrix, catalog number 250290990) purchased from Saifen Technology Co., Ltd. was weighed and 100g of 10M KOH solution was added. After stirring evenly, 3,4-epoxy-1-butene (activating compound 1) was added. The mass ratio of activating compound 1 to microspheres was 1.2:1. The reaction was carried out at 45℃ and 200rpm for 18h. After the reaction was completed, the microspheres were filtered and washed repeatedly with anhydrous ethanol and deionized water until the pH of the microspheres was neutral, thus obtaining the microspheres after the first activation.

[0124] 2) Secondary activation of the nucleus

[0125] Take the microspheres after the first activation, add 100g of DMSO and 50g of 1.5M KOH solution, stir well, and then slowly add pentylene glycol diglycidyl ether (activating compound 2) dropwise at 37℃ and 150rpm. The mass ratio of activating compound 2 to microspheres is 0.8:1. The addition takes about 3 hours. After the addition is complete, react under the same conditions for another hour. After the reaction is complete, filter and wash to obtain the microspheres after the second activation.

[0126] 3) Coupling shell

[0127] The microspheres after the second activation were mixed evenly with a cellulose (MW.50000) solution, and an equal mass of 3M NaOH solution was added. The reaction was carried out at 50℃ and 150 rpm for 18 h. After the reaction was completed, the microspheres were filtered and washed to obtain core-shell microspheres.

[0128] 4) Ligand coupling

[0129] Take 50g of core-shell spheres, add 50mL of deionized water and 3.5g of sodium acetate, stir well, then add 2g of TBCA, stir at room temperature for 2h, then add sodium formate until the color of the reaction solution disappears, indicating that the unreacted bromine has been completely quenched; after filtration and washing, add 100mL of 20mM PB buffer (pre-dissolved protein A in this buffer at a concentration of 20mg / mL), pH 8.5, stir well, and place in a shaker at 25℃ for 100rpm for 18h. After the reaction, filter and wash to obtain the composite affinity chromatography packing material.

[0130] The exclusion effect of the obtained composite affinity chromatography packing shell was tested using the same method as in Example 3, with OVA replaced by hIgG at the same concentration. The results showed that the packing maintained exclusion effect on large protein molecules and captured hIgG, proving that the composite affinity chromatography packing also has practical application value.

Claims

1. A method for preparing a core-shell composite packing material, characterized in that, The filler comprises a core-shell structure with a microsphere matrix as the base sphere, on which short-chain activating compounds and long-chain activating compounds are connected, and the short-chain activating compounds are coupled with functional groups with different functions. The long-chain activated compound is coupled with one or more polymeric compounds as the outer shell of the core-shell structure, the short-chain activated compound is shown in general formula I, and the long-chain activated compound is activated compound 2. The preparation method includes the following steps: (1) First activation reaction of the nucleus: The microsphere matrix, activation compound 1 and inorganic alkaline solution are mixed, and the microspheres after the first activation are obtained after the reaction is completed; the structure of the activation compound 1 is shown in general formula I: ； Wherein, R1 is selected from C1~C10 straight-chain alkyl, repeating polymeric unit or alkyl chain containing ester group, and R2 is selected from epoxy or halogen substituent. (2) Second activation reaction of the nucleus: Microspheres after the first activation, activation compound 2 and inorganic alkaline solution are added to the solvent. After the reaction is completed, microspheres after the second activation are obtained. The solvent includes one or more of water, DMSO, dioxane, and DMF. The activation compound 2 is poly(ethylene glycol) diglycidyl ether. (3) Coupling shell: Add the microspheres after the second activation, the polymer compound and the inorganic alkaline solution, and the core-shell spheres are obtained after the reaction is completed; (4) When the outer shell pore size of the core-shell sphere obtained in step (3) is larger than the diameter of the exclusion protein, repeat steps (2) and (3); the activating compound used in repeating step (2) is selected from one or more of epichlorohydrin, 1,4-butanediol diglycidyl ether, and 1,2,7,8-diepoxyoctane. (5) Ligand coupling: The activation group reserved in the core of the core-shell sphere is activated by using an activator, and then one or more functional groups with different functions are coupled. The functional groups have ion exchange function, hydrophobic function, hydrophobic ion recombination function or affinity function, thus obtaining the core-shell composite packing. The activator is an alkyl halide.

2. The method for preparing the core-shell composite packing according to claim 1, characterized in that, The microsphere matrix in step (1) includes one or more of agarose microspheres, dextran microspheres, konjac microspheres, cellulose microspheres, polymethacrylate microspheres, and polystyrene-divinylbenzene microspheres; the activated compound 1 includes one or more of allyl glycidyl ether, allyl iodide, 3,4-epoxy-1-butene, 1,2-epoxy-9-decene, and glycidyl methacrylate; the inorganic alkaline solution includes NaOH solution or KOH solution.

3. The method for preparing the core-shell composite packing according to claim 1, characterized in that, In step (1), the mass ratio of activated compound 1 to the microsphere matrix is ​​0.1-3:1; the temperature of the first activation reaction is 25-65℃, the reaction time is 10-24 h, the reaction conditions are 50-300 rpm, and the concentration of the inorganic alkaline solution is 0-18 M.

4. The method for preparing the core-shell composite packing according to claim 1, characterized in that, In step (2), the mass ratio of activated compound 2 to the microspheres after the first activation is 0.1-5:1; the temperature of the second activation reaction is 25-65℃, the reaction time is 10-24 h, the reaction conditions are 50-300 rpm, and the concentration of the inorganic alkaline solution is 0-5 M.

5. The method for preparing the core-shell composite packing according to claim 1, characterized in that, The polymeric compound mentioned in step (3) is a hydroxyl-rich compound with an average molecular weight ≥10000 Da; the hydroxyl-rich compound includes one of cellulose, dextran, molten agarose, polyethylene glycol, polyvinyl alcohol, or epoxy-hydrolyzed glycidyl methacrylate; the mass ratio of the polymeric compound to the microspheres after the second activation is 0.1-3:1, the concentration of the inorganic alkaline solution is 0-5 M; the reaction temperature is 25-65℃, the reaction time is 10-24 h, and the reaction conditions are 50-300 rpm.

6. The method for preparing the core-shell composite packing according to claim 1, characterized in that, The activation reaction in step (5) is carried out at a temperature of 0-50℃, for a reaction time of 0-10 h, and under reaction conditions of 50-300 rpm. The reagents used for coupling include one or more of n-octylamine, polyethyleneimine, polyacrylamine, protein A, protein G, protein L, and 3-aminophenylboronic acid.

7. The method for preparing the core-shell composite packing according to claim 1, characterized in that, The alkyl halide in step (5) is selected from one or more of N-bromosuccinimide, N-bromophthalimide, N-bromoacetamide, tetrabromobenzoquinone, 1,3-dibromo-5,5-dimethylhydantoin, PyHBr3, carbon tetrabromide, 1,3,5-tribromo-1,3,5-thiazine-2,4,6-trione, 1,3-dibromo-5,5-dimethylhydantoin, or N-bromo-o-sulfonylbenzoimide.

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