Chelated microparticle, chromatographic medium, preparation method therefor, and use thereof

Abstract

The present application relates to a chelated microparticle suitable for use in metal-chelate affinity chromatography. The chelated microparticle comprises a chelating ligand coupled to a support, the chelating ligand immobilizing a metal ion by means of coordination, the support having an effective hydroxyl group, and at least part thereof being a polymer microparticle of which internal pore channels have an at least partially ordered arrangement structure. Moreover, the present application further relates to a chromatographic medium containing the chelated microparticle. Compared with a traditional agarose matrix medium, in the chromatographic medium for the metal-chelate affinity chromatography technique of the present invention, polymer microparticles which have an at least partially ordered internal structure and pore channel distribution are used as supports, and the chromatographic medium has both a high dynamic binding capacity and a high mass transfer speed. In addition, an epoxy activation process is optimized, and the density of the coupled chelating ligands and immobilized metal ions is further increased. The chromatographic medium can tolerate conventional reagents, exhibits little metal ion leaching, and has a long service life, making the chromatographic medium suitable for further promotion and application.

Description

Chelating microparticles, chromatography media and methods of making and using the same

[0001] This application is based on and claims priority to Chinese patent application No. CN202410671317.1, filed on May 28, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to a chelating microparticle, more particularly to an immobilized metal chelating microparticle having a regular internal structure and a chromatography medium using the same, which can ensure high yield and high purity in the purification of proteins. BACKGROUND

[0003] In recent years, the rapid development of high-end biological drugs, especially macromolecular drugs, has rapidly driven the simultaneous development of the chromatographic separation industry as an important downstream branch of biological drug products; among them, the chromatographic column is the core component of the chromatographic separation system, and the chromatographic packing (also known as "chromatography medium" when applied in the field of biological macromolecules) is the core material of the chromatographic column, which is a very important link in pharmaceutical production. Chromatography medium has been widely used in the separation and purification of fields such as vaccines, recombinant protein drugs, IVD raw materials, CGT, antibody drugs, etc., and the cost of the separation process accounts for 40%-70% of the processing cost of biological therapeutic drugs.

[0004] Affinity chromatography is a chromatographic technique that separates biological molecules by using their specific affinity. Due to its strong selectivity and binding force, and high resolution, and the characteristic of reversible binding, affinity chromatography is a commonly used method for protein and antibody purification. Metal chelate affinity chromatography (IMAC) as an important member of the affinity chromatography family has been used as a protein purification technique for several years. Its working principle is that amino acids (especially histidine) form a coordination covalent bond with transition metal ions, thereby being retained on the stationary phase, while the heteroprotein that does not form a coordination bond is eluted. Transition metal ions such as cobalt, nickel or copper are used to purify histidine-containing proteins or peptides; while iron, zinc or gallium are used to purify phosphorylated proteins or peptides. Methods for eluting target proteins include changing the pH value or adding "competitive molecules" (competitive molecules), such as imidazole. In addition, IMAC can not only separate and purify, but also realize protein recognition and identification, directional fixation of proteins and renaturation purification of proteins.

[0005] The loading of IMAC media is an important parameter for the pharmaceutical factory to choose, the higher the loading, the more feed liquid can be treated by the same column volume of media, and the higher the production efficiency. The loading is theoretically related to the density of chelating ligand and the ligand fixed phase. The chelating ligands on the fixed phase, such as iminodiacetic acid (IDA), nitrilotriacetic acid (NTA) and N,N,N-3-carboxymethyl ethylenediamine (TED), are widely reported and used, which are the most common chelating agents. The empirical order of the binding ability of metal ions to chelating ligands is TED > NTA > IDA, so the number of coordination sites available for protein binding decreases in the opposite direction. Related literatures include V. Riguero et al., Journal of Chromatography A 2020 and M. Peterka et al., Journal of Chromatography A 2006. The loading of the IMAC media in the above prior art is relatively low, which may be due to the fact that the pore structure of the media is closely related to the accessible area of the protein, which determines the protein separation effect including loading and resolution. This may be the key to the poor separation effect of traditional chromatography media microspheres (such as agarose); in addition, according to the different binding ability of metal ions to chelating ligands, the media based on TED ligand is more stable than the media based on IDA ligand, but the protein adsorption capacity is relatively weak, and it will significantly reduce the yield of target protein, and even affect the purity of the product. Therefore, the IMAC media in the prior art always seeks a balance between loading and stability.

[0006] In view of the above problems of the prior art, it is necessary to provide an affinity chromatography medium that can improve the loading and the elution speed of impurities. SUMMARY

[0007] As described in the background, the metal chelate affinity chromatography (IMAC) medium in the prior art has problems of loading and stability due to the structure of the fixed phase. In view of the above, the present application is made.

[0008] One aspect of the present application is to provide a chelating microparticle suitable for use in metal chelate affinity chromatography, which meets the needs by using a chelating microparticle with a support that has a higher number of chelating ligands coupled to the surface and an arrangement structure of at least part of the internal pores being locally ordered.

[0009] Another aspect of the present application is to provide a chromatography medium comprising the above chelating microparticle, which has a high dynamic binding capacity and a high mass transfer rate.

[0010] Another aspect of the present invention is to provide a separation method having high loading capacity and high selectivity, by using the chromatography medium, according to the method of the present invention, substantially pure expression products of various expression systems such as His-tag labeled fusion proteins expressed by bacterial, yeast, insect and mammalian cells, etc. expression systems can be obtained.

[0011] Another aspect of the present invention is to provide a chromatography column for purifying a sample of a beneficial biomolecule and an impurity using the above-described chromatography medium.

[0012] Another aspect of the present invention is to provide the use of the above-described chromatography medium in liquid chromatography technology.

[0013] According to the purpose of the present application, the present invention relates to a chelating microparticle suitable for use as a metal-chelating affinity chromatography, comprising a chelating ligand coupled to a support through an activating group, said chelating ligand immobilizing a metal ion through coordination, said support being a microparticle of a polymer having effective hydroxyl groups and at least partially ordered arrangement structure of internal pores at least locally.

[0014] As a further improvement of the present application, said microparticle of a polymer has a diameter of 10-150 μm.

[0015] As a further improvement of the present application, said microparticle of a polymer is formed by cross-linking at least partially cross-linkable substances including rigid nanoparticles, at least one of said rigid nanoparticles having a non-spherical shape in solution, said rigid nanoparticles forming an ordered arrangement structure in said microparticle of a polymer at least locally.

[0016] As a further improvement of the present application, said microparticle of a polymer further comprises a polysaccharide compound not forming a non-spherical shape in solution, said polysaccharide compound being copolymerized with said rigid nanoparticles to form said microparticle of a polymer.

[0017] As a further improvement of the present application, said polysaccharide compound is selected from at least one of agar, agarose, dextran, starch, chitosan and trehalose.

[0018] As a further improvement of the present application, said chelating ligand is a polydentate ligand.

[0019] As a further improvement of the present application, said polydentate ligand is selected from any one of iminoalkyldicarboxylic acid-based compounds, dicarboxymethylamino acid derivatives and ethylenediamine-based alkyl polycarboxylic acid compounds.

[0020] As a further improvement of the present application, said polydentate ligand is any one of N-carboxymethyl aspartic acid (CM-ASP), iminodiacetic acid (IDA), nitrilotriacetic acid (NTA) or N,N,N-3-carboxymethyl ethylenediamine (TED).

[0021] As a further improvement of this application, the activating group is provided by an activator containing an epoxy group.

[0022] As a further improvement of this application, the epoxy-containing activator is at least one selected from epichlorohydrin, methyl epichlorohydrin, epibromopropane, diglycidyl ether, trimethylolpropane triglycidyl ether, and 1,4-butanediol diglycidyl ether.

[0023] As a further improvement to this application, the metal ion is selected from Cu. 2+ Ni 2+ Zn 2+ Co 2+ Fe 3+ Cr 3+ and Ga 3+ Any one of them.

[0024] In the second aspect, this application discloses a method for preparing the above-mentioned chelating microparticles, comprising the following steps:

[0025] (a) Providing at least one support containing effective hydroxyl groups and at least a portion of the support being a polymeric microparticle with an internal pore structure that is at least locally ordered;

[0026] (b) The hydroxyl groups on the support are activated using an activator containing an epoxy group, and at least one chelating ligand with a metal ion immobilized on at least one activated hydroxyl group is provided to form the chelated microparticle.

[0027] In a third aspect, this application discloses a chromatography medium containing the aforementioned chelating microparticles.

[0028] In a fourth aspect, this application also discloses a method for purifying a sample comprising beneficial biomolecules and impurities, comprising: loading a mobile phase containing the sample onto the above-described chromatography medium; and adding an eluent to release the biomolecules from the chromatography medium.

[0029] As a further improvement to this application, the sample is a natural histidine-containing protein.

[0030] As a further improvement to this application, the method also includes the step of cleaning the medium with a cleaning solution.

[0031] As a further improvement of this application, the cleaning solution is an alkaline cleaning solution containing at least 0.1 mol / L NaOH.

[0032] In a fifth aspect, this application also discloses a chromatographic column for purifying samples containing beneficial biomolecules and impurities, the chromatographic column comprising the aforementioned chromatographic medium.

[0033] In a sixth aspect, this application also discloses the application of the above-mentioned chromatography media in liquid chromatography technology.

[0034] Beneficial effects:

[0035] Compared to traditional agarose-based media, the chromatographic medium of the metal chelate affinity chromatography technique of this invention uses high molecular weight microparticles with at least locally ordered internal structures and pore distribution as supports, which have both high dynamic binding capacity and high mass transfer rate; at the same time, the epoxy activation process is optimized to further improve the density of coupled chelate ligands and immobilized metal ions; it can withstand conventional reagents, has less metal ion shedding, and a longer service life, making it suitable for further promotion and application. Attached Figure Description

[0036] Figure 1 illustrates the different steps for epoxy activation of the support using monoepoxy-terminated halogen compounds (upper part of Figure 1) and diepoxy-terminated ether compounds (lower part of Figure 1).

[0037] Figure 2 illustrates the steps for chelating and coupling the epoxy-activated product with iminoalkyl dicarboxylic acid compounds (top of Figure 2), dicarboxymethyl amino acid derivatives (middle of Figure 2), and alkyl polycarboxylic acid ethylenediamine compounds (bottom of Figure 2); R refers to structures other than the functional groups and supports involved in the reaction.

[0038] Figure 3 illustrates the different steps by which the chelating ligand coupling products chelate with metal ions;

[0039] Figure 4 shows the dynamic binding capacity (DBC) of several commercially available agarose gel-based IMAC media as a function of retention time;

[0040] Figure 5 shows the purification of the PET28a histidine tag protein, analyzed by reduction SDS-PAGE electrophoresis. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, not all of them, and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0042] definition

[0043] The term "rigid nanoparticles" refers to biomacromolecules that exhibit a non-spherical, rigid morphology when alone or dispersed in water. They may be arranged randomly in the solvent, or they may form an ordered molecular arrangement of some kind of lyotropic liquid crystal, such as nematic phase, smectic phase, cholesteric phase, columnar phase liquid crystal, etc., as the concentration of rigid nanoparticles increases.

[0044] The term "polydentate ligand" refers to a compound whose molecular formula contains an atom that provides a lone pair of electrons, such as O, S, N, etc., that is, a compound that can provide at least two coordination sites for a metal ion.

[0045] The term "spacer arm" refers to an element that separates a multidentate ligand from a support.

[0046] One of the main challenges in IMAC purification is the balance between high binding capacity, high mass transfer rate, and high purity. High mass transfer rate usually comes at the cost of high binding capacity and high purity. Therefore, the inventors sought an IMAC medium that overcomes these shortcomings, possesses high binding capacity and high mass transfer rate, and can guarantee the purity of the final product.

[0047] In a first aspect, this application relates to a chelating microparticle suitable for use in metal chelate affinity chromatography. More specifically, it employs a polymeric microparticle with at least locally ordered internal pores as a support, and further comprises a chelating ligand coupled to the support via an activating group, wherein the chelating ligand immobilizes metal ions through coordination. The support is a polymeric microparticle having effective hydroxyl groups and at least partially having an internally ordered arrangement of at least locally ordered pores.

[0048] Currently, commercially available IMAC media typically use organic polymer matrices (polysaccharide polymers are the most commonly used). Because ordinary polysaccharide molecules are randomly arranged in water, the internal pores of the resulting gel microspheres are also randomly arranged. Furthermore, given the inherent softness of ordinary polysaccharide molecules, the microspheres formed by their cross-linking are usually relatively soft. Compared to existing technologies, this application uses rigid nanoparticles with non-spherical symmetric shapes in solution. These ordered pores can significantly increase the surface area of ​​the support, achieving a high surface area without reducing the support diameter, as a reduction in support diameter usually leads to a significant decrease in mass transfer rate.

[0049] In some embodiments, the polymeric microparticles have a diameter of 10-150 μm. The polymeric microparticles are formed by crosslinking at least partially crosslinkable substances, including rigid nanoparticles, wherein at least one of the rigid nanoparticles has a non-spherical symmetric shape in solution, and the rigid nanoparticles form at least locally an ordered arrangement within the polymeric microparticles.

[0050] In this application, when rigid nanoparticles are dispersed in a solvent, they form an ordered molecular arrangement of some kind of lyotropic liquid crystal, including nematic, smectic, cholesteric, and columnar liquid crystals, as the concentration increases. Liquid crystal materials with ordered arrangements typically exhibit birefringence of light. Therefore, microdroplets or solvents containing non-spherical symmetric biomolecules forming ordered arrangements are clearly visible under a polarizing microscope and exhibit common liquid crystal conformations. Furthermore, the rigid nanoparticles can be selected from biomolecules, and even further, the biomolecules are selected from at least one of peptides, proteins, nucleic acids, polysaccharides, and lipids.

[0051] The polymeric microparticles may further include polysaccharide compounds that do not have a non-spherical symmetric shape. Although these polysaccharide compounds do not spontaneously arrange themselves in an orderly manner in solution, due to various interactions, including hydrogen bonds, between these polysaccharide compounds and biomolecules, they will align with the biomolecules and eventually copolymerize with them to form polymeric microparticles. This further improves the pressure resistance of the formed polymeric microparticles without destroying their ordered structure. These polysaccharide compounds are selected from at least one of agar, agarose, dextran, starch, chitosan, and trehalose. In a specific embodiment of this application, the polysaccharide compound is agarose.

[0052] In some embodiments, the chelating ligand is a polydentate ligand. The chelating ligand binds to the support by introducing a covalent bonding method between the support and the chelating agent, which can be performed using any conventional covalent bonding method, for example, by means of use. These methods are well known in the art and readily performed by those skilled in the art. In some embodiments, the chelating ligand binds to the solid support in a covalent manner through activated functional groups, with the spacer arms residing therebetween. Further, the polydentate ligand is selected from any of iminoalkyl dicarboxylic acid compounds, dicarboxymethyl amino acid derivatives, and alkyl polycarboxylic acid ethylenediamine compounds. In some specific embodiments, the polydentate ligand is selected from any of N-carboxymethyl aspartic acid (CM-ASP), iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), or N,N,N-3-carboxymethyl ethylenediamine (TED).

[0053] In some embodiments, the activating group is provided by an activator containing an epoxy group. In some specific embodiments, the activator containing an epoxy group is selected from at least one of epichlorohydrin, methyl epichlorohydrin, epibromopropane, diglycidyl ether, trimethylolpropane triglycidyl ether, and 1,4-butanediol diglycidyl ether.

[0054] In some embodiments, the metal ion is selected from Cu. 2+ Ni 2+ Zn 2+Co 2+ Fe 3+ Cr 3+ and Ga 3+ Any one of them.

[0055] Secondly, this application relates to a method for preparing chelated microparticles, specifically comprising the following steps:

[0056] (a) Providing at least one support containing effective hydroxyl groups and at least a portion of the support being a polymeric microparticle with an internal pore structure that is at least locally ordered;

[0057] (b) The hydroxyl groups on the support are activated using an activator containing an epoxy group, and at least one chelating ligand with a metal ion immobilized on at least one activated hydroxyl group is provided to form the chelated microparticle.

[0058] In this application, the support containing effective hydroxyl groups can be a variety of porous materials, including at least partially polymeric microparticles with locally ordered internal pore structures, and can also include porous cross-linked polysaccharide materials, which are relatively easy to prepare by standard methods, such as membrane emulsification, high-shear emulsification, and microfluidic methods. Both types of supports contain effective active groups such as hydroxyl or amino groups on their surfaces, which can covalently bind to chelating ligands.

[0059] More specifically, it includes the following steps:

[0060] 1. Preparation of the support

[0061] In this application, the portion where the support is a polymeric microparticle can be prepared according to Chinese Patent CN116554536A or Chinese Patent CN114700055A. In an advantageous embodiment, the preparation of the polymeric microparticle is as described in Chinese Patent CN116554536A, which is incorporated herein by reference. In short, firstly, biomolecules and polysaccharide compounds are dispersed to form a dispersion, specifically, the biomolecules and polysaccharide compounds are dispersed in water to form a dispersed phase solution; secondly, the dispersion is further dispersed in a continuous phase containing an emulsifier using a method including membrane emulsification, emulsifying to form droplets; finally, the droplets are crosslinked, specifically by adding a crosslinking agent to the formed droplets to crosslink the biomolecules in the droplets, forming polymeric microparticles.

[0062] 2. Epoxy Activation

[0063] The specific operation of epoxy activation involves constructing spacer arms and epoxy functional groups on the surface of a support using an activator containing epoxy groups. Specifically, the support is chemically coupled to give it epoxidative properties. Compound molecules with mono- or poly-epoxy groups are used. Specifically, as shown in Figure 1, terminal halogen compounds containing mono-epoxy groups (upper part of Figure 1) or ether compounds with double-epoxy groups (lower part of Figure 1) can be used. However, this application is not limited to these. More specifically, in some embodiments, at least one of epichlorohydrin, methyl epichlorohydrin, epibromopropane, diglycidyl ether, trimethylolpropane triglycidyl ether, and 1,4-butanediol diglycidyl ether can be used for reaction activation. The specific procedure is as follows: Under stirring conditions, 0.5-3 times the mass of the epoxide agent is subsequently added to the support; then, a 2 mol / L sodium hydroxide solution containing 0.3% sodium borohydride is added to the reaction system, and the reaction is carried out at 20-40℃ for 6-12 hours. The product is washed until neutral, and the epoxy group density of the epoxy-activated product is determined. For more detailed information on the determination of epoxy group density, please refer to the method provided in "The Determination of Epoxide Groups" (Dobinson B et al. 1969).

[0064] 3. Chelating ligand coupling steps

[0065] The epoxy-activated product from step 2 is mixed evenly with an equal volume of sodium carbonate solution (2 mol / L) containing 0.3% sodium borohydride, and finally 10% wt of chelating agent is added. The mixture is reacted overnight at 50-70°C. The product is washed multiple times with deionized water until the system is neutral, as illustrated in Figure 2 (upper part). The chelating agent can be an iminoalkyl dicarboxylic acid compound. In a specific example, when R is CH2, the chelating agent is IDA. As illustrated in Figure 2 (middle part), the chelating agent can also be a dicarboxymethyl amino acid derivative. In a specific example, when n is 4, the chelating agent is N,N-bis(carboxymethyl)-L-lysine. As illustrated in Figure 2 (lower part), the chelating agent can also be an alkyl polycarboxylic acid ethylenediamine compound. In a specific example, when n is 2, the chelating agent is TED. However, other options are possible within the scope of this application and no specific limitation is imposed.

[0066] 4. Chelates with transition metal ions

[0067] As shown in Figure 3, the chelated ligand coupling product from step 3 is mixed with a metal salt solution to obtain the aforementioned metal chelate affinity chromatography medium. Specifically, an equal volume of 0.5 mol / L metal salt solution is added to the chelated ligand coupling product, stirred overnight at room temperature, and washed until no metal ions are detected. Those skilled in the art can easily attach metal ions to the chelated ligand coupling product. See "Immobilized metal affinity adsorption and immobilized metal affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions" (Porath J, Biochemistry, 1983, 22(7):1621-30), which is cited here for reference.

[0068] Thirdly, this application relates to a metal chelate affinity chromatography medium containing the aforementioned chelate particles.

[0069] Fourthly, this application relates to a method for purifying a sample comprising beneficial biomolecules and impurities, specifically comprising loading a mobile phase containing the sample onto the aforementioned chromatography medium; adding an eluent to release the biomolecules from the chromatography medium. Further, it also includes recovering beneficial biomolecules from the eluent.

[0070] This method can be used to purify natural histidine-containing protein samples. It is applicable to the purification of macromolecular bioactive substances such as viruses, proteins, and enzymes, and can be specifically applied in the fields of biomedicine, blood products, vaccines, cosmetics, and health products.

[0071] Fifthly, this application relates to a chromatographic column for purifying samples of beneficial biomolecules and impurities, the column comprising a packing material or operating in the form of an expanded bed; in some embodiments, the column may be made of conventional materials, such as plastics, glass, ceramics, and metals.

[0072] Sixthly, this application also discloses the application of the above-mentioned chromatography media in liquid chromatography technology.

[0073] The structure and preparation of the chromatography medium of the present invention, as well as the separation effect of the chromatography apparatus and its preparation method, will be described in detail below with reference to specific embodiments. In the embodiments of the present invention, unless otherwise specified, all ratios are mass ratios.

[0074] experiment

[0075] The following examples are provided for illustrative purposes only and are not intended to limit the invention as defined in the appended claims. This invention relates to a two-step method for preparing IMAC chromatography media from ordered, porous polymeric microparticles based on biomacromolecules. The two steps are an epoxy activation step and a chelating ligand coupling step.

[0076] Preliminary Example 1: Preparation of Polymer Microparticles

[0077] 0.9 g of cellulose nanocrystals and 0.9 g of agarose were dispersed in 28.2 g of water and stirred at 90 °C for 3 h to form a suspension. This suspension was then added to 270 g of liquid paraffin containing SPAN 80 (mass percentage concentration: 10%) and emulsified at 110 °C for 20 min. The dispersed phase and continuous phase were mixed at a ratio of 1:10 and emulsified at 80 °C and 650 rpm for 4 min. After cooling and solidification, the continuous phase solution was removed, and the gel was washed and weighed. An equal volume of deionized water and 2,3-dibromopropanol were added, followed by 10 mL of 40 wt% sodium hydroxide solution. The mixture was stirred at 30 °C for 15 h to obtain the final product.

[0078] Example 1:

[0079] 10g of washed and dried polymer microparticles were placed in a 100mL round-bottom flask. An equal volume of acetone was added under stirring. After stirring for 15 minutes, 20mL of epichlorohydrin was added. During stirring, 40mL of a 2mol / L sodium hydroxide solution containing 0.3wt% sodium borohydride was added. The reaction was carried out at 30℃ for 6 hours. The product was washed multiple times with ethanol and deionized water until neutral, yielding epoxy-activated microspheres. The microspheres were then added to a flask along with an equal volume of a 2mol / L sodium carbonate solution containing 0.3wt% sodium borohydride. 10wt% iminodiacetic acid (IDA), a chelating agent, was added. The reaction was carried out overnight at 60℃. The product was washed with deionized water until neutral, and then an equal volume of a 0.5mol / L nickel sulfate solution was added. The mixture was stirred overnight at room temperature. The product was washed multiple times with deionized water until no Ni ions were detected in the washings.

[0080] Example 2

[0081] 10g of washed and dried polymer microparticles were placed in a 100mL round-bottom flask. An equal volume of sodium hydroxide solution (1.5 mol / L) was added with stirring. After stirring for 15 min, 1,4-butanediol-diglycidyl ether (twice the mass of the polymer microparticles) was added. The mixture was reacted at 25℃ for 18 h. The resulting product was washed multiple times with ethanol and deionized water until neutral, yielding epoxy-activated microspheres. The microspheres were then added to a flask along with an equal volume of sodium carbonate solution (2 mol / L) containing 0.3 wt% sodium borohydride. 10 wt% iminodiacetic acid (IDA) was added as a chelating agent. The mixture was reacted overnight at 60℃. The product was washed with deionized water until neutral, and then an equal volume of 0.1 mol / L nickel sulfate solution was added. The mixture was stirred overnight at room temperature. The product was washed multiple times with deionized water until no Ni ions were detected in the washings.

[0082] Example 3

[0083] 10g of washed and dried polymer microparticles were placed in a 100mL round-bottom flask. An equal volume of acetone was added under stirring. After stirring for 15 minutes, 20mL of epichlorohydrin was added. During stirring, 40mL of a 2mol / L sodium hydroxide solution containing 0.3wt% sodium borohydride was added. The reaction was carried out at 30℃ for 16 hours. The product was washed multiple times with ethanol and deionized water until neutral, yielding epoxy-activated microspheres. The microspheres were then added to a flask along with an equal volume of a 2mol / L sodium carbonate solution containing 0.3wt% sodium borohydride. 10wt% iminodiacetic acid (IDA), a chelating agent, was added. The reaction was carried out overnight at 60℃. The product was washed with deionized water until neutral, and then an equal volume of a 0.5mol / L nickel chloride solution was added. The mixture was stirred overnight at room temperature. The product was washed multiple times with deionized water until no Ni ions were detected in the washings.

[0084] Example 4

[0085] 10g of washed and dried polymer microparticles were placed in a 100mL round-bottom flask. An equal volume of acetone was added under stirring. After stirring for 15 minutes, 20mL of epichlorohydrin was added. During stirring, 40mL of a 2mol / L sodium hydroxide solution containing 0.3wt% sodium borohydride was added. The reaction was carried out at 30℃ for 16 hours. The product was washed multiple times with ethanol and deionized water until neutral, yielding epoxy-activated microspheres. The microspheres were then added to a flask along with an equal volume of a 2mol / L sodium carbonate solution containing 0.3wt% sodium borohydride. 10wt% of the chelating agent N,N-bis(carboxymethyl)-L-lysine was added. The reaction was carried out overnight at 60℃. The product was washed with deionized water until neutral, and then an equal volume of a 0.5mol / L nickel chloride solution was added. The mixture was stirred overnight at room temperature. The product was washed multiple times with deionized water until no Ni ions were detected in the washings.

[0086] Example 5

[0087] 10g of washed and dried polymer microparticles were placed in a 100mL round-bottom flask. An equal volume of acetone was added under stirring, and the mixture was stirred for 15 min. Then, 20mL of epichlorohydrin was added. During stirring, 40mL of a 2mol / L sodium hydroxide solution containing 0.3wt% sodium borohydride was added. The reaction was carried out at 30℃ for 16 h. The product was washed multiple times with ethanol and deionized water until neutral, yielding epoxy-activated microspheres. Then, 30mL of a 0.2mol / L sodium bicarbonate solution and 20mL of ethylenediamine were added, and the mixture was stirred at low speed overnight at 50℃ for amino activation. After washing the amino-activated microspheres, 2g of bromoacetic acid, 6.5mL of a 2mol / L sodium hydroxide solution, and 6.5mL of a 1mol / L sodium bicarbonate solution were added sequentially, and the mixture was stirred at room temperature for 15 h. After the reaction was complete, the microspheres were washed thoroughly with plenty of water until neutral, and an equal volume of a 0.5mol / L nickel sulfate solution was added. The mixture was stirred overnight at room temperature. After the reaction is complete, wash with deionized water until no Ni ions are detected in the filtrate.

[0088] Example 6

[0089] 10g of washed and dried polymer microparticles were placed in a 100mL round-bottom flask. An equal volume of acetone was added under stirring. After stirring for 15 minutes, 20mL of epichlorohydrin was added. During stirring, 40mL of a 2mol / L sodium hydroxide solution containing 0.3wt% sodium borohydride was added. The reaction was carried out at 30℃ for 16 hours. The product was washed multiple times with ethanol and deionized water until neutral, yielding epoxy-activated microspheres. The microspheres were then added to a flask along with an equal volume of a 2mol / L sodium carbonate solution containing 0.3wt% sodium borohydride. 10wt% iminodiacetic acid (IDA), a chelating agent, was added. The reaction was carried out overnight at 60℃. The product was washed with deionized water until neutral, and then an equal volume of a 0.5mol / L chromium nitrate solution was added. The mixture was stirred overnight at room temperature. The product was washed multiple times with deionized water until no Cr ions were detected in the washings.

[0090] Example 7

[0091] 10g of washed and dried polymer microparticles were placed in a 100mL round-bottom flask. An equal volume of acetone was added under stirring. After stirring for 15 minutes, 20mL of epichlorohydrin was added. During stirring, 40mL of a 2mol / L sodium hydroxide solution containing 0.3% sodium borohydride was added. The reaction was carried out at 30℃ for 16 hours. The product was washed multiple times with ethanol and deionized water until neutral, yielding epoxy-activated microspheres. The microspheres were then added to a flask along with an equal volume of a 2mol / L sodium carbonate solution containing 0.3wt% sodium borohydride. 10wt% iminodiacetic acid (IDA), a chelating agent, was added. The reaction was carried out overnight at 60℃. The product was washed with deionized water until neutral, and then an equal volume of a 0.5mol / L ferric nitrate solution was added. The mixture was stirred overnight at room temperature. The product was washed multiple times with deionized water until no Fe ions were detected in the washings.

[0092] Comparative Example 1

[0093] 10g of washed and dried commercial 6FF microspheres were placed in a 100mL round-bottom flask. An equal volume of acetone was added with stirring, and the mixture was stirred for 15 min. Then, 20mL of epichlorohydrin was added. During stirring, 40mL of a 2mol / L sodium hydroxide solution containing 0.3wt% sodium borohydride was added. The mixture was reacted at 30℃ for 16 h. The resulting product was washed multiple times with ethanol and deionized water until neutral, yielding epoxy-activated microspheres. The microspheres were then added to a flask along with an equal volume of a 2mol / L sodium carbonate solution containing 0.3wt% sodium borohydride. 10wt% iminodiacetic acid (IDA), a chelating agent, was added. The mixture was reacted overnight at 60℃. The product was washed with deionized water until neutral, and then an equal volume of a 0.5mol / L nickel chloride solution was added. The mixture was stirred overnight at room temperature. The product was washed multiple times with deionized water until no Ni ions were detected in the washings.

[0094] Comparative Example 2

[0095] 10g of washed and dried commercial 6FF microspheres were placed in a 100mL round-bottom flask. An equal volume of acetone was added under stirring, and after stirring for 15min, 20mL of epichlorohydrin was added. During stirring, 40mL of a 2mol / L sodium hydroxide solution containing 0.3wt% sodium borohydride was added. The reaction was carried out at 30℃ for 16h. The product was washed multiple times with ethanol and deionized water until neutral, yielding epoxy-activated microspheres. The microspheres were then added to a flask along with an equal volume of a 2mol / L sodium carbonate solution containing 0.3wt% sodium borohydride. 10wt% N,N-bis(carboxymethyl)-L-lysine, a chelating agent, was added. The reaction was carried out overnight at 60℃. The product was washed with deionized water until neutral, and then an equal volume of a 0.5mol / L nickel chloride solution was added. The mixture was stirred overnight at room temperature. The product was washed multiple times with deionized water until no Ni ions were detected in the washings.

[0096] Test Example 1: Determination of Epoxy Group Density

[0097] The epoxy-activated microsphere gel was loaded into a sintered glass funnel and repeatedly soaked, dried, and washed with deionized water (at least three times). The gel was then dried under high pressure for a period of time until no water droplets fell. 1 g of the dried gel was accurately weighed and added to 7 ml of 1.3 mol / L sodium thiosulfate solution. The reaction was allowed to proceed at room temperature for 1 h. Phenolphthalein indicator was added, and the solution was titrated to the endpoint with 0.001 mol / L hydrochloric acid. The amount of hydrochloric acid used was recorded.

[0098] Formula for determining epoxy density:

[0099]

[0100] Table 1 Results of epoxy group density determination

[0101]

[0102] As can be seen from Table 1, the epoxy group density of Comparative Examples 1-2 is significantly lower than that of Examples 1-5, indicating that epoxy treatment of the ordered chromatographic medium can provide a higher epoxy group density.

[0103] Test Example 2: ICP Test for Metal Ion Content

[0104] The chelated microparticles prepared in Examples 1-5 and Comparative Examples 1-2 were loaded into a sand core funnel and repeatedly soaked, dried, and washed with deionized water (at least three times). The gel was then dried (no water droplets fell under high pressure for a period of time). A precise amount of the dried gel was weighed and the metal ion content was determined by ICP spectrometry.

[0105] Table 2 Results of ICP test for metal ion content

[0106]

[0107] The data in Table 2 show that the chelating agent IDA can complex a larger amount of metal ions than the chelating agent NTA; however, in terms of the types of metal ions, there is no significant difference in the complexation effect between nickel chloride and nickel sulfate; under the same reaction conditions, the metal loading of chelating particles with ordered internal pores is significantly higher than that of chelating particles with disordered internal pores, and the high metal loading and high epoxy group density show the same trend.

[0108] Test Example 3: Column Packing and Dynamic Bonding Capacity (DBC) Test

[0109] Chromatography column: 1 mL specification (column length: 2 cm; diameter: 0.8 cm)

[0110] Instrument: AKTA Pure

[0111] Equilibrium solution: Tris-HCl (pH=8)

[0112] Eluent: 500mM imidazole

[0113] Protein sample: pET28a-6His_ppase-HMGR_13-433 (molecular weight: 46.35kDa, isoelectric point: 6.41)

[0114] Sample concentration: 4.745 mg / mL

[0115] Sample loading flow rate: 120 cm / h

[0116] The chelated microparticles of Example 4 and Comparative Example 2 were packed into the above chromatography column by connecting the empty column tube through the homogenization tube, and ultrapure water was used as the packing mobile phase.

[0117] DBC test conditions: Pass the eluent through the AKTA Pure system.

[0118] As described above, the dynamic binding capacity (DBC) was determined at the 10% flow-through point (Qb10%), and the results are shown in Table 3.

[0119] Table 3. Dynamic binding capacity (Qb10%) of chelated microparticles in Example 4 and Comparative Example 2

[0120]

[0121] As shown in Table 3 and Figure 4, for the above protein samples, the ordered chelating microparticles exhibited improved performance and could provide a greater protein loading capacity. The results showed that the protein loading capacity at 80% flow-through was approximately 1.24 times that of the disordered pore chromatography medium.

[0122] Test Example 4: Purification of Histidine-Tagged Proteins

[0123] Tag protein sample: pET28a-6his-tev-kras 1-169 G12D (protein molecular weight: 21.659kDa, isoelectric point: 5.98)

[0124] Buffer1: 50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1.0 mM TCEP, 5% glycerol, 10 mM imidazole, 10 μg / ml DNase I

[0125] Buffer 2: 50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1.0 mM TCEP; 5% glycerol, 10 mM imidazole

[0126] Purity and resolution were tested by gradient purification of pET28a-6his-tev-kras 1-169 G12D. The specific steps are as follows:

[0127] (1) The recombinant strain pET28a-6his-tev-kras 1-169 G12D was cultured in a medium and induced overnight for 16 h;

[0128] (2) Centrifuge to collect bacterial culture, resuspend in 300 mL of buffer 1, break up cells, and collect supernatant;

[0129] (3) Divide the supernatant into 6 portions, each 50 ml, and add 3 ml of the chelated microparticles prepared in Examples 3-4 and Comparative Examples 1-2 respectively, and incubate for 60 min;

[0130] (4) Wash the material after incubation in step (3) with 50ml of buffer Buffer2, then elute the protein with different concentrations of imidazole and collect the eluent.

[0131] The results of histidine-labeled protein isolation and purification in Examples 3-4 and Comparative Example 2 were detected by SDS-PAGE electrophoresis, and the results are shown in Figure 5. In the figure, L represents cell lysis; FT represents flow through; and 10i, 30i, 60i, 300i, and 500i represent the protein separation bands (unit: mmol / L) of the corresponding concentrations of imidazole elution buffer.

[0132] Meanwhile, the total amount of eluted protein in Examples 3-4 and Comparative Example 2 under different imidazole elution buffer concentrations was tested (Table 4). This allowed for the determination of the corresponding IMAC medium loading capacity, further confirming that the ordered pore IMAC medium in this application has a high loading capacity.

[0133] Table 4. Changes in total protein content during purification of IMAC media in Examples 3-4 and Comparative Example 2 under different concentrations of imidazole elution buffer.

[0134]

[0135] As shown in Table 4 and Figure 3, 10-60 mM imidazole solution can elute most of the impurity proteins, while at 300 mM the target protein (the color patch near the 22 kDa mark) begins to be eluted in large quantities. When the eluent concentration reaches 500 mM, almost all proteins will be eluted. By comparing the data from Examples 3 and 4, the A280 value corresponds to the amount of protein eluted at a certain concentration. It can be found that the chromatography medium obtained by IDA modification can provide a higher protein loading capacity than the chelated microparticles obtained by NTA modification. At the same time, the proteins bound by IDA-Ni are also easier to elute, resulting in better purification effect.

[0136] The data from Example 4 and Comparative Example 2 show that the protein loading of chelating microparticles with ordered channels is higher than that of chelating microparticles with disordered channels, thus verifying the reliability of the dynamic adsorption loading test results.

[0137] Although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0138] The detailed descriptions listed above are merely specific descriptions of feasible embodiments of the present invention, and are not intended to limit the scope of protection of the present invention. All equivalent embodiments or modifications made without departing from the spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. Chelated microparticles suitable for use in metal chelate affinity chromatography, characterized in that, which comprises a chelating ligand coupled to a support via an activating group, the chelating ligand immobilizing a metal ion via coordination, the support having effective hydroxyl groups and at least partially being a microparticle of a polymer having an ordered arrangement structure of internal pores at least locally.

2. The chelating microparticles of claim 1, wherein, The microparticle of the polymer has a diameter of 10-150 μm.

3. The chelating microparticle of claim 1, wherein, The microparticle of the polymer is formed by cross-linking at least partially cross-linkable substances including rigid nanoparticles, at least one of the rigid nanoparticles having a non-spherical shape in solution, the rigid nanoparticles forming an ordered arrangement structure in the microparticle of the polymer at least locally.

4. The chelating microparticles of claim 3, wherein, The microparticle of the polymer further includes a polysaccharide compound not forming a non-spherical shape in solution, the polysaccharide compound being copolymerized with the rigid nanoparticles to form the microparticle of the polymer.

5. The chelating microparticle of claim 4, wherein, The polysaccharide compound is selected from at least one of agar, agarose, dextran, starch, chitosan and trehalose.

6. The chelating microparticle of claim 1, wherein, The chelating ligand is a polydentate ligand.

7. The chelating microparticle of claim 6, wherein, The polydentate ligand is selected from any one of iminoalkyldicarboxylic acid compounds, dicarboxymethylamino acid derivatives and ethylenediamine alkyl polycarboxylic acid compounds.

8. The chelating microparticle of claim 7, wherein, The polydentate ligand is selected from any one of N-carboxymethyl aspartic acid (CM-ASP), iminodiacetic acid (IDA), nitrilotriacetic acid (NTA) or N,N,N-3-carboxymethyl ethylenediamine (TED).

9. The chelating microparticle of claim 1, wherein, The activating group is provided by an activating agent containing an epoxy group.

10. The chelating microparticle of claim 9, wherein, The activating agent containing an epoxy group is at least one of epichlorohydrin, methyl epichlorohydrin, epibromohydrin, diglycidyl ether, trimethylolpropane triglycidyl ether and 1,4-butanediol diglycidyl ether.

11. The chelating microparticle of claim 1, wherein, The metal ion is selected from any one of Cu 2+ , Ni 2+ , Zn 2+ , Co 2+ , Fe 3+ , Cr 3+ , and Ga 3+ .

12. The method of claim 1-11, wherein the chelating microparticles are prepared by the process comprising: comprising the steps of: (a) providing at least one microparticle of a polymer having effective hydroxyl groups and at least partially being an ordered arrangement structure of internal pores at least locally; (b) activating the hydroxyl groups on the support using an activating agent containing an epoxy group to provide at least one chelating ligand immobilizing a metal ion on at least one of the activated hydroxyl groups to form the chelating microparticle.

13. A metal chelate affinity chromatography medium, characterized in that, The chromatographic medium comprises the chelating microparticle according to any one of claims 1 to 11.

14. A method of purifying a sample comprising a beneficial biomolecule and an impurity, comprising: A mobile phase comprising the sample is loaded onto the chromatographic medium according to claim 13; an eluent is added to release the biomolecule from the chromatographic medium.

15. The method of claim 14, wherein, The sample is a natural histidine-containing protein.

16. The method of claim 14, wherein, The step of washing the medium with a washing solution is further included.

17. The method of claim 14, wherein, The washing solution is an alkaline washing solution comprising at least 0.1 mol / L NaOH.

18. A chromatographic column for purifying a sample of a beneficial biomolecule and an impurity, the chromatographic column comprising the chromatographic medium according to claim 13.

19. Use of the chromatographic medium according to claim 13 in liquid chromatography.

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