Chromatography medium and chromatography apparatus

Abstract

This application relates to a chromatography medium and a chromatography apparatus including the chromatography medium. The chromatography medium is composed of a polymer material crosslinked by at least a partially crosslinkable substance containing rigid nanoparticles having at least one non-spherical symmetric shape, and is characterized in that the rigid nanoparticles form at least a locally ordered array structure in the chromatography medium. The chromatography medium and the chromatography apparatus disclosed in this application form a stationary phase by deposition of polymer microparticles having at least a locally ordered internal structure and pore distribution, or use the entire structure in which the rigid nanoparticles form at least a locally ordered array structure in the chromatography medium as the stationary phase, thereby effectively improving the separation and purification rate, shortening the elution time, improving the symmetry of the peak shape, effectively reducing the half-peak width, and improving the column efficiency.

Description

Technical Field

[0001] The present invention relates to a chromatography medium and a chromatography apparatus including the chromatography medium, and particularly to a chromatography medium and a chromatography apparatus composed of polymer fine particles having a regular internal structure and pore arrangement.

Background Art

[0002] The technique known as Gel Filtration Chromatography (GFC), also known as Molecular Exclusion Chromatography (MEC), is a field of Size Exclusion Chromatography (SEC). It is a liquid chromatography method that mainly utilizes the molecular sieve effect of a gel with a porous structure and separates based on the difference in molecular size. Its characteristics are that there is no chemical interaction between components and the sample recovery rate is high under mild separation conditions. Therefore, it is currently one of the most widely used separation methods in the field of life science.

[0003] The basic principle of GFC is demonstrated when a mixture of biopolymers such as proteins and other complex molecules with different molecular weights and sizes passes through a chromatography column containing a porous gel. Small protein molecules enter the interior of the stationary phase through small and large pores, larger molecules enter the interior of the stationary phase through large pores, but even larger protein molecules do not enter the interior of the stationary phase and remain in the gaps between the stationary phase particles and flow out with the mobile phase. In principle, large protein molecules are eluted first, and small protein molecules are eluted later, thereby achieving effective separation of the mixture.

[0004] The conventional gel dialysis chromatography method usually adopts the method of filling the stationary phase in a column. When introducing the separation target into the column, the mobile phase containing specific components is also introduced into the chromatography column simultaneously. The mobile phase is generally inert, and a solvent with a certain dissolving ability for the sample is used. Since the separation degree is not controlled, the separation result of the chromatography column does not depend on the interaction between the sample and the mobile phase. Relatively speaking, it is the characteristics of the stationary phase that determine the separation effect. The commonly used types of stationary phases include hydrophilic organic gels. In particular, polysaccharide gel beads are widely applied in ion exchange chromatography, affinity chromatography, and hydrophobic interaction chromatography columns for small molecules and bioactive substances.

[0005] In view of the wide applications and great success in the separation of biopharmaceuticals, there are a very large number of related patent documents regarding agar-based biopolymer microspheres. The early related literature includes Biochim. Biophys. Acta 1964, 79:393-398 by Hjerten, S. and Biochim. Biophys. Acta 1964, 79:399 by Bengtsson et al., S. The first patent regarding agar microspheres is US Patent No. 4,647,536. The polysaccharide microspheres used in chromatography columns have appeared in academic and patent documents even earlier.

[0006] According to research, in technologies such as affinity chromatography and ion exchange chromatography, the pore structure of the medium and the contact area of the protein are closely related and determine the separation effect of the protein (such as capacity and resolution). Therefore, the internal pore diameter structure, size, size distribution, size and distribution of the spheres, shape, and mechanical properties of the polysaccharide gel spheres used as the medium of the chromatography column have a great impact on the separation effect and separation speed. However, currently known polysaccharide gel spheres are manufactured by mechanical stirring, homogeneous emulsification, membrane emulsification method, etc., so their internal pore structure cannot be controlled. Also, usually, these polysaccharide gel spheres have inherent flexible properties and limited pressure resistance, so the corresponding chromatography columns are also limited to low-speed biological protein separation applications. Furthermore, the raw material of the agar microspheres currently most widely used is obtained through multi-step extraction from seaweed, so it is not suitable for large-scale industrial production and is a raw material with high costs.

[0007] On the other hand, some bioactive substances to be separated are less resistant to changes in temperature, shear force, and solution environment, and are easily inactivated due to structural changes. Therefore, the chromatography conditions for active substances have relatively strict requirements, and good mechanical properties, chemical stability, and high-efficiency separation performance are required. Otherwise, there is a risk of causing denaturation and deterioration of the separation target.

[0008] Therefore, there is a need to provide a chromatography medium with uniform and controllable particle size, a regular internal structure and pore distribution, and a certain strength. This can improve the separation efficiency of the chromatography column in gel permeation chromatography and also lead to time savings.

Summary of the Invention

[0009] The object of the present invention is to provide a chromatography device applicable to gel filtration chromatography. Thereby, a polymer material with at least a locally ordered array structure in the internal pores can be used as the chromatography medium to meet the above needs.

[0010] Examples using systems, tools, and methods for embodiments of the present invention and their objectives will be described below. These examples are merely illustrative and explanatory, not restrictive. In different embodiments, one or more of the above market needs may be satisfied by the present invention, or in other embodiments, other improvements may be addressed.

[0011] A main objective of the present invention is to provide a chromatography medium for use in gel filtration chromatography. At least a part of the chromatography medium is composed of a polymer material formed by cross-linking at least a partially cross-linkable substance containing rigid nanoparticles, and at least one of the above rigid nanoparticles has an asymmetric shape in solution and forms at least a locally ordered array structure within the chromatography medium.

[0012] One objective of the present invention is to provide a chromatography medium, where at least a part of the filled polymer material consists of polymer microparticles, and rigid nanoparticles are locally or entirely ordered within the polymer microparticles.

[0013] Another objective of the present invention is to provide a chromatography medium formed by cross-linking as a whole at least a partially cross-linkable substance containing rigid nanoparticles. In the above chromatography medium, the rigid nanoparticles form at least a locally ordered array structure.

[0014] Another objective of the present invention is to provide a chromatography apparatus using the above chromatography medium.

[0015] Based on the object of the present invention, the present invention aims to provide the above-mentioned chromatography medium composed of a polymer material formed from at least partially crosslinkable substances containing at least some rigid nanoparticles. In this chromatography medium, at least one of the above-mentioned rigid nanoparticles has an asymmetric shape in solution and forms at least a locally ordered array structure within the chromatography medium.

[0016] As a further improvement of the present invention, the above-mentioned chromatography medium is formed by laminating the above-mentioned polymer material, and at least a part of the polymer material is composed of polymer microparticles. These polymer microparticles are formed by crosslinking at least partially crosslinkable substances containing rigid nanoparticles, and at least one rigid nanoparticle has an asymmetric shape in solution and forms at least a locally ordered array structure within the polymer microparticles.

[0017] Furthermore, as a further improvement of the present invention, inside the above-mentioned polymer microparticles, there are one or more regions where the rigid nanoparticles are orderly arranged, and the molecular arrangements between these multiple regions are unrelated, related, or partially related.

[0018] Also, as an improvement of the present invention, the above-mentioned chromatography medium is formed by crosslinking as a whole from at least partially crosslinkable substances containing rigid nanoparticles, and at least one of the above-mentioned rigid nanoparticles has an asymmetric shape in solution and forms at least a locally ordered array structure within the chromatography medium.

[0019] As a further improvement of the present invention, the shape of the above-mentioned asymmetric rigid nanoparticles is rod-shaped, strip-shaped, sheet-shaped, needle-shaped, or linear, characterized by the long axis direction of the molecule.

[0020] Also, as a further improvement, the shape of the above-mentioned asymmetric rigid nanoparticles is disk-shaped with the characteristic direction perpendicular to the plane direction.

[0021] Furthermore, as an improvement of the present invention, the entire interior of the above-mentioned chromatography medium is orderly arranged, and the above-mentioned characteristic direction is distributed along the radial direction of the polymer microparticles, along the bipolar axis direction, or so as to form a plurality of concentric circles inside.

[0022] Furthermore, as an improvement of the present invention, in the orderly arranged local regions, the above-mentioned characteristic directions are arranged parallel, fan-shaped, or spiral-shaped.

[0023] Also, as a further improvement of the present invention, the above-mentioned rigid nanoparticles are selected from any one or more of polypeptides, proteins, nucleic acids, polysaccharides, and lipids.

[0024] Also, as a further improvement of the present invention, as the rigid nanoparticles having an asymmetric shape, cellulose nanocrystals or cellulose nanofibers are used.

[0025] Also, as an improvement of the present invention, the above-mentioned chromatography medium further contains a polysaccharide compound that does not take a clear asymmetric shape in solution, and these polysaccharide compounds copolymerize with the above-mentioned rigid nanoparticles to form a chromatography medium.

[0026] Furthermore, the mass ratio of the above-mentioned rigid nanoparticles to the above-mentioned polysaccharide compound is 1:10 - 50:1.

[0027] The polysaccharide compound is selected from any one or more of agar, agarose, dextran, starch, chitosan, and trehalose.

[0028] The separation range of the above-mentioned chromatography medium is 50 - 300000 kDa.

[0029] On the other hand, the present invention also discloses a chromatography apparatus as follows. This chromatography apparatus includes a support, a chromatography medium filled inside the support or coated on the surface of the support, The chromatography medium is any of the above.

[0030] As an improvement, the support is any of a cylindrical wall material, a flat substrate, or a curved hollow tube.

[0031] Also, the axial cross-section of the cylindrical wall material is any of circular, elliptical, or polygonal.

[0032] Furthermore, the material of the support is any one of plastic, glass, ceramic, or metal, or a combination thereof.

Advantages of the Invention

[0033] The chromatography medium and chromatography apparatus for gel filtration chromatography disclosed by the present invention form a stationary phase by laminating polymer microparticles having at least a locally ordered internal structure and pore distribution, or adopt a stationary phase using an overall structure in which rigid nanoparticles form at least a locally ordered structure within the chromatography medium. Thereby, it is possible to improve the separation and purification speed, shorten the peak time, improve the symmetry of the peak shape, reduce the half-width, and improve the column efficiency.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0035] To clarify the object, technical content, and advantages of the present invention, the following will detail the technical content using specific embodiments of the present invention and drawings. The embodiments described herein are only a part of the present invention, not an exhaustive list of all embodiments, and do not limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments that can be obtained by an ordinary technician without creative effort are also included in the protection scope of the present invention.

[0036] In conventional "whole columns" and "particle-packed columns", the most prominent feature of the chromatography media used in chromatography is that the pores are disordered. For example, as shown in Fig. 1(a), in a chromatography media composed of general agarose particles, the agarose-type chromatography media 100 is constituted by packing agarose particles 101. In the detailed enlarged view of the local A part (Fig. 1(b)), the deposition structure of the agarose particles is shown, and further in Fig. 1(c), it is clarified that the porous structure of the agarose particles is an unordered arrangement and cross-linked structure.

[0037] Actually, the specific manufacturing method of the agarose particles 101 is to disperse agarose in water, use an appropriate emulsification technique to form minute aqueous droplets containing polysaccharides in the oil phase, and as shown in the enlarged view of the internal B part of the agarose microsphere shown in Fig. 1(c), after cooling, a double helix structure is formed from the single strands of the polysaccharide compound 102, whereby pores 103 are generated between the molecular bundles, and finally polysaccharide microparticles are formed by a cross-linking agent 104. Since the polysaccharide molecules are arranged disorderly in water, the pores in the generated gel microparticles are also arranged disorderly. Also, due to the inherent flexibility of the polysaccharide molecules, the microparticles formed by cross-linking are usually relatively soft.

[0038] Based on the concept of the present invention, by using rigid nanoparticles that do not have spherical symmetry, a porous chromatography medium with an ordered molecular arrangement, controllable pores, and excellent mechanical properties can be produced. As shown in FIGS. 2(a) and (b), the chromatography medium 200 whose internal structure in FIG. 2(a) has at least local order has at least partially ordered molecular arrangement, and the formed pores also have an ordered array structure (see FIG. 2(b)).

[0039] In nature, many biopolymers take a form that does not have spherical symmetry and exhibits rigidity when alone or dispersed in water. Based on the lyotropic liquid crystal theory, such rigid nanoparticles (or biopolymers described later) may be arranged disorderly according to the concentration and characteristics of the rigid nanoparticles when dispersed in a solvent, or when the concentration increases, they may form an ordered molecular arrangement of a certain lyotropic liquid crystal such as a nematic phase (e.g., nanomaterials of tobacco mosaic virus), a smectic phase, a cholesteric phase, a columnar phase liquid crystal, etc. Since the orderly arranged liquid crystal material usually exhibits the birefringence property of light, droplets and solvents containing non-spherical biopolymers can be clearly observed under a polarized light microscope and show typical liquid crystal structures.

[0040] Based on the spirit of the present invention, FIG. 2(b) is a detailed enlarged view of part C of FIG. 2(a), showing the chromatography medium 200 with an internal structure having at least local order. This chromatography medium contains rigid nanoparticles 201. When the rigid nanoparticles 201 reach or exceed the critical concentration of the liquid crystal, the nanoparticles form an ordered array, and ordered pores, that is, ordered pores 202, are formed between the nanoparticles. On the other hand, some nanoparticles cannot form an ordered array, resulting in the formation of disordered pores 203. Furthermore, through a cross-linking process, by adding a cross-linking agent and other flexible molecules, rigid nanoparticles 201 that reach or exceed the critical concentration region of the liquid crystal after cross-linking are generated. At this time, some nanoparticles continue to maintain an ordered array, and accordingly, a chromatography medium with ordered pores 202 arranged can also be obtained.

[0041] Similar to the chromatographic medium formed by the deposition of porous agarose microparticles, based on the spirit of the present invention, one method of forming a chromatographic medium with pores arranged in an orderly manner is to utilize the deposition of polymer microparticles with pores arranged in an orderly manner. Specific explanations are shown in Figure 3.

[0042] Based on the spirit of the present invention, when the rigid nanoparticles are biopolymers, at least the molecules are locally ordered, and it is possible to produce porous biopolymer microparticles with pores arranged in an orderly manner. After depositing these microparticles with ordered pores, a structure that is at least partially ordered can be obtained in the overall structure of the above chromatographic medium.

[0043] Specifically, cellulose nanocrystals (CNC) with an appropriate aspect ratio and size distribution are biopolymers that have a certain rigidity in water, which is their solvent, and can form a lyotropic liquid crystal phase. Based on the spirit of the present invention, when the biopolymer is cellulose nanocrystals (CNC), when the concentration reaches the critical value, the CNC molecules self-organize to form an ordered arrangement and exhibit an ordered liquid crystal phase. In addition, cellulose nanocrystal CNC has chirality as a biopolymer, and the formed ordered molecular arrangement structure has the arrangement of liquid crystal molecules with a helical structure such as a cholesteric phase.

[0044] Figure 3 shows an example of polymer microparticles with a porous structure disclosed based on the present invention. Specifically, (a) in Figure 3 shows a partial cross-sectional view of the interior along the radial direction of the polymer microparticles. The rigid nanoparticles at least locally maintain an ordered arrangement after cross-linking, and the pores formed under the influence of the molecular arrangement may also be locally regularly arranged (see Figure 3(b)). The rigid nanoparticle solution shown in (a) of Figure 3 is emulsified when in a locally ordered state to form emulsion droplets, and the rigid nanoparticles 201 within the emulsion droplets also attempt to be locally ordered in the same way. When the temperature of the emulsion decreases, the rigid nanoparticles 201 maintain their arrangement and further cross-link to form polymer microparticles, and their internal structure at least partially retains the previous ordered structure. At the same time, as shown in Figure 3(b), the ordered pores 202 existing between the arrangements of the rigid nanoparticles also inherit a similar ordered structure, and the pores of the porous microparticles form at least a locally ordered arrangement structure. Here, "ordered (having)" means that there is a certain regularity in the arrangement of direction and position between at least a locally sectional area of the above-mentioned pores and at least a locally sectional area of the adjacent pores within at least a local region. Specifically, it shows that within at least a local region, at least a locally sectional area of the above-mentioned pores and at least a locally sectional area of the adjacent pores are arranged parallel, fan-shaped, or spiral-shaped. As shown in Figure 3(d), as a preferred embodiment, the diameter of the pores is 1 - 1000 nanometers.

[0045] Based on the spirit of the present invention, by controlling the concentration of rigid nanoparticles, the ordered array structure formed in the emulsion droplets can include one or more regions. At the same time, the molecular arrangements of the multiple regions may be unrelated, related, or partially related. Also, the above-mentioned ordered array structure may have order globally or locally. When having order globally, in the locally ordered regions, the characteristic directions of the rigid nanoparticles are distributed along the radial direction of the microparticles, along the bipolar axis direction of the microparticles, or are distributed to form a plurality of concentric circles inside the microparticles. When having order locally, in the locally ordered regions, the characteristic directions of the rigid nanoparticles are arranged parallel, fan-shaped, or spiral.

[0046] Within the range of having order globally, these ordered array structures can form a special structure in terms of structure. For example, in the radial shape shown in Fig. 4(a) (an ordered array with the characteristic direction being the radial direction), a first hole 401 regularly arranged towards the center can be formed inside. Also, in the bipolar type shown in Fig. 4(b) (an ordered array with the characteristic direction being the bipolar axis direction), a second hole 402 regularly arranged along the bipolar axis direction can be formed inside. Furthermore, in the annular shape shown in Fig. 4(c) (the characteristic direction is arranged in a plurality of concentric circles), a third hole 403 regularly arranged concentrically can also be formed inside, etc. However, this application is not limited to these, and other ordered configurations are also possible. At the same time, these special configurations form special optical phenomena under a polarized light microscope due to the optical birefringence characteristics that rigid nanoparticles usually have. For example, as shown in Fig. 4(a), since the characteristic directions of the rigid nanoparticles 201 are ordered radially in the emulsion droplets and the internal structure and holes of the formed polymer microparticles are also ordered radially, it has a radial configuration. Thereby, it can show the optical anisotropy of the Maltese black cross under an orthogonal polarized light microscope (see Fig. 4(d)).

[0047] In the range having local order, as shown in Fig. 3(a), inside the polymer microparticles, a D part shown in Fig. 3(b) and an E part shown in Fig. 3(c) are included. The D part is a local region where the characteristic directions of the rigid nanoparticles have order, and the E part is a region where the characteristic directions are randomly arranged. Inside this polymer microparticle, an ordered pore 202 and a disordered pore 203 are simultaneously formed. At this time, although the polymer microparticle does not have a specific configuration, since its characteristic directions are still regularly arranged within a narrow range, it can still show color under a cross-polarized light microscope.

[0048] Through the manufacturing method according to the present invention, at least locally ordered rigid nanoparticle droplets of different sizes can be obtained, and after crosslinking, polymer microparticles with at least locally ordered molecules and pores can be formed. As a preferred embodiment, these polymer microparticles usually have an average particle size of 1 - 500 microns in an aqueous solvent, and more preferably, the particle size is in the range of 5 - 150 microns. If the particle size of the polymer microparticle is too small, the back pressure of the chromatography column may increase, and if the particle size is too large, the column efficiency will decrease.

[0049] Based on the spirit of the present invention, the polymer microparticle 301 is formed by crosslinking with at least a partially crosslinkable substance containing rigid nanoparticles 201 (i.e., biopolymers). At this time, at least one of the above rigid nanoparticles has an asymmetric shape in solution, and the shape of the rigid nanoparticle is a rod shape with the long axis direction of the molecule as the characteristic direction. Also, it may have a banana-like shape with the long axis direction of the molecule as the characteristic direction, a disk shape with the characteristic direction perpendicular to the plane direction, or even a plate shape, needle shape, or linear shape, but the present application is not limited to these, and other non-spherical symmetric shapes that meet other requirements can also be adopted.

[0050] Rigid nanoparticles with or without an asymmetric shape are selected from at least one of polypeptides (such as insulin and growth hormone), proteins (such as chlorophyll and collagen), nucleic acids (such as DNA), polysaccharides (such as cellulose and chitin), and lipids (such as monoglycerides, phospholipids, glycolipids, and steroids). These biopolymers are widely present in living organisms and mainly exhibit rod-like or flat shapes in solution. As a preferred embodiment, biopolymers with an asymmetric shape may or may not have chirality. Furthermore, chiral biopolymers include biopolymers with left-handed and right-handed chirality, and the formed liquid crystal phase forms an array of liquid crystal molecules with a helical structure of the cholesteric phase. One specific example of the present application is cellulose nanocrystal (CNC), a biopolymer, and its structural formula is as follows. [Chemical formula]

[0051] The rod-like structure formed by biopolymers has a large aspect ratio and is likely to form a liquid crystal state. As a more preferred embodiment, the length of cellulose nanocrystals is 20 - 1000 nanometers, the width is 2 - 100 nanometers, and the aspect ratio is from 1:5 to 1:200.

[0052] As shown in Fig. 3(b), the polymer microparticles can further contain a polysaccharide compound 102 without an asymmetric shape. These polysaccharide compounds copolymerize with biopolymers to form polymer microparticles. These polysaccharide compounds are selected from at least one of agar, agarose, dextran, starch, chitosan, and trehalose. In a specific example of the present application, the polysaccharide compound is agarose, and its structural formula is as follows. [Chemical formula]

[0053] The polysaccharide compound is a gel-like dispersion with fluidity before emulsification. After emulsification to form emulsion droplets, through processes such as cooling, solidification, and aging, as shown in Fig. 3(b), the polysaccharide compound 102 forms a double helix from a single strand, further forms a bundled state, and finally forms stable solid microparticles. These polysaccharide compounds cannot spontaneously form an ordered arrangement in solution, but through various interactions including hydrogen bonds between these polysaccharide compounds and biopolymers, they follow the arrangement of biopolymers and finally form an ordered arrangement. Also, in certain situations, at a specific concentration, the biopolymer is locally ordered to form a liquid crystal state, and the polysaccharide compound is arranged by using the polymer arrangement method as a model. These polysaccharide compounds are further copolymerized with the help of biopolymers and cross-linking agents to form a stable microparticle structure. At this time, without damaging the ordered structure of the formed polymer microparticles, the pressure resistance of the polymer microparticles can be further improved. At the same time, in the manufacturing process of the polymer microparticles, the polysaccharide compound (such as agarose) solidifies the emulsion droplets emulsified by cooling and provides structural support for subsequent cross-linked polymer formation, thus simplifying the manufacturing process.

[0054] In accordance with the spirit of the present invention, a preferred method for obtaining a chromatography medium having at least a partially pore-ordered structure is that, as shown in Fig. 5(a), the whole of the chromatography medium 200 with an at least locally ordered internal structure includes non-spherically symmetric rigid nanoparticles and is further composed of polymer microparticles 301 in which at least partially the molecules are ordered. In accordance with the spirit of the present invention, the chromatography medium 200 with an at least locally ordered internal structure having at least a partially pore order can also be formed by stacking polymer microparticles 301 with pore order and microparticles 501 having disordered pores inside, as shown in Fig. 5(b). Furthermore, it can also be formed by stacking polymer microparticles 301 with pore order and non-porous microspheres 502, as shown in Fig. 5(c). Generally, microspheres with disordered pores include polysaccharide microspheres (such as agarose microspheres), and common non-porous microspheres include inorganic silicon dioxide microspheres, hydroxyapatite, etc.

[0055] In accordance with the spirit of the present invention, the production of a chromatographic medium containing polymer microparticles involves dispersing rigid nanoparticles alone or together with an appropriate amount of amorphous monomer or oligomer to form a lyotropic liquid crystal solution, forming emulsion droplets by an emulsification method, and then forming polymer porous microspheres through procedures such as solidification and cross-linking. At this time, the rigid nanoparticles may be physically encapsulated by the surrounding polymer within the polymer microparticles and incorporated into the polymer microspheres, or may directly participate in the cross-linking reaction and chemically bond to become part of the polymer.

[0056] When the chromatographic medium is formed by cross-linking an at least partially cross-linkable substance containing rigid nanoparticles as a whole, at least one of the above-mentioned rigid nanoparticles has an asymmetric shape in solution, and the rigid nanoparticles form at least a locally ordered structure within the above-mentioned chromatographic medium.

[0057] Specifically, as shown in Fig. 5(d), the chromatographic medium 200 with an internal structure having at least local order is an overall structure formed by an overall polymer arranged in an orderly manner. However, the present invention is not limited to this, and the above-mentioned chromatographic medium can include other foreseeable ranges.

[0058] In accordance with the spirit of the present invention, when using the overall structure formed by cross-linking rigid nanoparticles as a chromatographic medium, different from the method for producing polymer porous microspheres, after forming a locally or overall ordered lyotropic liquid crystal solution, a cross-linking agent is directly added to perform in-situ polymerization. As a specific operation, a dispersion containing rigid nanoparticles is added to a stainless steel pre-packed column, the cross-linking agent is dropped while controlling the temperature, the pH is adjusted, and after the reaction, it is dried to obtain at least a structure in which the rigid nanoparticles and the rigid nanoparticles are at least locally ordered within the overall structure.

[0059] The filling methods of chromatographic media usually include high-pressure homogenization filling, wet filling, dry filling, and high-density high-viscosity filling. High-pressure homogenization filling is a method of manufacturing a uniformly filled column by suspending a filler in an appropriate homogenizing liquid and quickly pushing it into the column at a very high flow rate with a high-pressure pump before sedimentation. This is a common filling method for analytical and manufacturing chromatography and is also the method adopted in the present invention. Wet filling is usually used to obtain a uniform and dense column bed for high-efficiency analytical columns filled with small particle size fillers of 3-10 microns. Dry filling is usually used for powdery fillers with large particle sizes (widely used in the manufacture of chromatography), such as fillers with particle sizes of 50 microns or more. The application environment of high-density high-viscosity filling is when the density of the chromatographic filler is very large and there is a high requirement for the uniformity of the column bed. Usually, a high-density or high-viscosity homogenizing liquid is prepared to suspend the filler in the homogenizing liquid, and the filler is deposited in layers by applying an external pressure.

[0060] The present invention also discloses a chromatographic apparatus. FIG. 6 is a schematic diagram of the structure of a column-type chromatographic apparatus according to the present invention. As shown in FIG. 6(a), the chromatographic apparatus includes a support 600 and a chromatographic medium 200 filled inside the support or coated on the surface of the support, the internal structure of which has at least local order. The above-mentioned chromatographic apparatus also further includes a sample inlet 601 to be filtered and a sample outlet 602 to be filtered, which are located at both ends of the support.

[0061] The separation principle of the chromatography apparatus of the present invention is as shown in Fig. 6(b). When porous polymer microparticles 301 separate molecules of different sizes (molecular weight sizes), such as proteins, small protein molecules 603 can enter the stationary phase through small and large pores, large protein molecules 604 can enter the stationary phase only through large pores, and ultra-large protein molecules 605 cannot enter the stationary phase and remain in the gaps between the particles of the stationary phase and later flow out with the mobile phase. In principle, ultra-large protein molecules are eluted first, large protein molecules are eluted next, and small protein molecules are eluted last, thus achieving the purpose of effectively separating the mixture.

[0062] Furthermore, since the polymer microparticles in the present invention have at least locally ordered characteristic direction arrays and pore arrays, the paths for molecules of the same size to diffuse into and out of the polymer microparticles are regular and clear. Compared with a stationary phase with an irregular internal arrangement, the peak appearance time of the sample molecules to be separated in the chromatography apparatus of the present invention is shortened, that is, its retention time is further reduced.

[0063] As a preferred technical solution, as shown in Fig. 7, the support can be selected from a cylindrical wall material 701 (shown in Fig. 7(a)), a flat substrate 702 (shown in Fig. 7(b)) or a curved hollow tube. Specifically, as shown in Fig. 6, the cross-sectional shape of the support 600 in the A-A' direction can be any of a circle (shown in Fig. 8(a)), an ellipse (shown in Fig. 8(b)), a rectangle (shown in Fig. 8(c)) or a polygon such as a hexagon (shown in Fig. 8(d)). When the support is a flat substrate, the chromatography medium is coated on the surface of the flat substrate with a certain thickness. When the support is a cylindrical wall material or a curved hollow tube, the chromatography medium is filled inside the support. As a preferred technical solution, the material of the support is selected from any of plastics, glass, ceramics, metals or their composite materials.

[0064] In accordance with the spirit of the present invention, the present invention also provides a method for manufacturing the above-mentioned chromatography medium. When the chromatography medium is formed by at least partial deposition of polymer microparticles, the manufacturing method includes the following steps.

[0065] First step: Manufacture the above-mentioned polymer microparticles. The embodiments of the present invention include methods for manufacturing polymer microparticles. First, a biopolymer and a polysaccharide compound are dispersed to form a dispersion. Specifically, a biopolymer and a polysaccharide compound are dispersed in water to form a dispersed phase solution. Next, the dispersion is emulsified to form emulsion droplets. There are many emulsification methods, including the membrane emulsification method. The membrane emulsification method refers to a method in which the dispersed phase directly enters the continuous phase through the pores of a microporous membrane during the emulsification process. Emulsion droplets are formed at the ends of the pores and are sequentially extruded. Finally, a cross-linking agent is added to the emulsion droplets to cross-link the biopolymer in the emulsion droplets to form polymer microparticles.

[0066] Second step: Weigh a certain amount of the manufactured polymer microparticles. Preferably, after washing 2-3 times by the suction filtration method, the washed polymer microparticles are sufficiently swollen in a homogeneous liquid to form a suspension. The homogeneous liquid is ultrapure water or a phosphate buffer solution of different concentrations, and there is no particular limitation in the present invention. However, in the following examples, all the homogeneous liquids used are ultrapure water.

[0067] Third step: Fill the suspension obtained in the second step into a column and pack the column by the homogenization method. The mobile phase is selected as ultrapure water. Specifically, pour the suspension into a homogenization tank, wait for the stationary phase to sediment freely, and after the sedimentation is complete, observe the corresponding relationship between the flow rate and the pressure in real time while increasing the flow rate of the mobile phase at a rate of 0.5 milliliters per minute every 10 minutes. When the pressure reaches 0.2-0.25 megapascals, stop increasing the flow rate and observe whether there is any change in the pressure. If there is no obvious increase, it is considered that the packing is firm and there is no damage to the packing material, and finally a chromatography device containing polymer microparticles can be formed.

[0068] When the chromatographic medium is formed by being cross-linked throughout with at least a partially cross-linkable substance containing rigid nanoparticles, unlike the method for producing polymer microparticles, after dispersing a biopolymer and a polysaccharide compound to form a dispersion, a cross-linking agent is directly added to perform in-situ polymerization. The specific operations are as follows. A dispersion of cellulose nanocrystals and agarose is added to a stainless-steel pre-packed column, and the temperature is controlled at 80 °C with a heating jacket to prevent the dispersion from solidifying. Next, a cross-linking agent (1,4-butanediol diglyceryl ether) is dropped in, and then a 0.5 M sodium hydroxide solution is added. After the dropping is completed, the reaction is carried out at 38 °C for 12 hours. Finally, this entire column is freeze-dried or critical-point dried to obtain a porous entire column bed. A large amount of ethanol and water are added to wash the unreacted substances, and then freeze-drying or critical-point drying is carried out again to obtain a porous entire gel chromatography device.

[0069] Hereinafter, based on specific examples, the structure, production of the chromatographic medium of the present invention, the separation effect of the chromatographic device, and further its production method will be described in detail. In the embodiments of the present invention, unless otherwise specified, all ratios indicate mass ratios.

[0070] (Preliminary Example 1) Polymeric microparticles containing 5% cellulose nanocrystals and 1% agarose were prepared. 0.5 grams of cellulose nanocrystals and 0.1 grams of agarose were dispersed in 9.4 grams of water and stirred at 90 °C to form a suspension. The above suspension was added to liquid paraffin containing 100 grams of SPAN 80 (mass percentage concentration: 10%), emulsified at 80 °C for 2 minutes, cooled, and a dispersion containing solidified emulsion droplets was formed. After washing to remove the emulsifier and liquid paraffin, the weight of the obtained gel was measured and then added to 10 milliliters of an aqueous solution in which 500 microliters of the crosslinking agent 1,4-butanediol diglyceryl ether was dissolved, and stirred for 12 hours for a reaction. Next, 500 microliters of an aqueous solution containing 40 wt% sodium hydroxide and 5 wt% sodium borohydride was added to the above reaction solution, and the reaction was carried out with stirring for 8 hours. Then, 500 microliters of epichloropropane and 500 microliters of an aqueous solution containing 40 wt% sodium hydroxide and 5 wt% sodium borohydride were mixed, added to the reaction solution, and further stirred for 12 hours for a reaction. After the reaction was completed, it was washed thoroughly. As shown in Figure 9, under an orthogonal polarizing microscope, the polymeric microparticles exhibit radial optical anisotropy (the black cross of a chiral malt), indicating that their internal structure and pore distribution are radial.

[0071] (Preparative Example 2) Different from the method of Example 1, polymeric microparticles containing 4% cellulose nanocrystals and 2% agarose were prepared. Other procedures and experimental conditions were the same as those in Example 1. As shown in Figure 10, under an orthogonal polarizing microscope, the polymeric microparticles exhibit radial optical anisotropy (the black cross of a malt), indicating their radial internal structure and pore distribution.

[0072] (Preparative Example 3) Different from the method of Example 1, polymeric microparticles containing 2% cellulose nanocrystals and 4% agarose were prepared. Other procedures and experimental conditions were the same as those in Example 1. As shown in Figure 11, under an orthogonal polarizing microscope, no obvious radial optical anisotropy (the black cross of a malt) is seen in the polymeric microparticles, indicating that the tendency of their radial internal pore structure is weakened.

[0073] (Preparative Example 4) Different from the method of Example 1, polymer microparticles containing 1% cellulose nanocrystals and 5% agarose were produced. Other procedures and experimental conditions were the same as those in Example 1. As shown in Figure 12, under an orthogonal polarized light microscope, no obvious radial optical anisotropy (the black cross of Maltese) was observed in the polymer microparticles, indicating that the tendency of the radial internal pore structure was weakened.

[0074] (Examples 1-4) The polymer microparticles prepared in Preparative Examples 1-4 were sieved, and the polymer microparticles with a particle size of 60 to 90 microns were left, and then washed three times by the suction filtration method. The washed polymer microparticles were dispersed in ultrapure water and sufficiently swollen, and the formed suspension was injected into a 5*100 mm hollow column by the above homogenization method, and the corresponding chromatography apparatuses were manufactured respectively.

[0075] (Example 5) The polymer microparticles prepared in Preparative Example 2 were sieved, and the polymer microparticles with a particle size of 60 to 90 microns were left, and then washed three times by the suction filtration method. The washed polymer microparticles were dispersed in ultrapure water and sufficiently swollen, and the formed suspension was injected into a 16*400 mm hollow column by the above homogenization method, and a chromatography apparatus was manufactured.

[0076] (Comparative Example 1) Different from the method of Preparative Example 1, agarose balls with a solid content of 6% were produced. Other procedures and experimental conditions were the same as those in Preparative Example 1. The agarose balls with a solid content of 6% were sieved, and the polymer microparticles with a particle size of 60 to 90 microns were used as the stationary phase, and other procedures were the same as those in Examples 1-4.

[0077] (Comparative Example 2) Different from the method of Preliminary Example 1, agarose balls with a solid content of 6% were produced. Other procedures and experimental conditions were the same as those in Preliminary Example 1. The agarose balls with a solid content of 6% were sieved, and polymer microparticles with a particle size of 60 to 90 microns were used as the stationary phase. Other procedures were the same as those in Example 5.

[0078] To verify that the chromatography apparatus provided by the present invention has an excellent separation effect, sample separation was performed with a plurality of standard products in the chromatography apparatuses of each example and the comparative example. The protein purification apparatus used in the chromatography column was AKTA PURE. Ultra-pure water was used as the mobile phase for the separation of acetone, and a mixed buffer solution of PBS solution and sodium chloride solution was used as the mobile phase for the separation of bovine serum albumin and cytochrome C.

[0079] (Experimental Example) The chromatography apparatuses obtained in Examples 1-4 and Comparative Example 1 were respectively connected to the protein purification apparatus, and the flow rate was controlled to 0.1 milliliter / minute to inject the sample. The sample was a 25 microliter 0.5% acetone solution. The ultra-pure water in the chromatography apparatus was replaced with a 0.2 mmol / liter PBS solution and a 0.15 mol / liter sodium chloride mixed buffer solution, and then the flow rate was controlled to 0.1 milliliter / minute to inject the sample. The samples were respectively 25 microliter 0.8 milligram / milliliter bovine serum albumin (BSA) solution and 25 microliter 0.5 milligram / milliliter cytochrome C (Cytochrome C) solution to test the separation effect. The results are shown in Table 1 and Figure 13.

[0080] The chromatography apparatuses of Example 5 and Comparative Example 2 were each connected to a protein purification apparatus, and the flow rate was controlled to 1 milliliter per minute and the sample was injected. The samples were 300 microliters of 1% acetone solution and 300 microliters of 1 milligram / milliliter Blue Dextran 2000 solution, and the separation effect was tested. The ultrapure water in the chromatography apparatus was replaced with a 0.2 millimolar / liter PBS solution and a 0.15 molar / liter sodium chloride mixed buffer solution, and then the flow rate was controlled to 1 milliliter per minute and the sample was injected. The samples were 300 microliters of 0.8 milligram / milliliter bovine serum albumin solution, 300 microliters of 0.5 milligram / milliliter Cytochrome C solution, and 300 microliters of 1 milligram / milliliter IgG solution, respectively, and the separation effect was tested. The results are shown in Table 2 and Figure 14.

[0081] Table 1 Separation effect data by the chromatography apparatuses of Examples 1-4 and Comparative Example 1

Table 1

[0082] Table 2 Separation effect data by the chromatography apparatuses of Example 5 and Comparative Example 2

Table 2

[0083] Since acetone molecules can pass through all the spaces (including the gaps between fine particles and the pores inside the fine particles) in the chromatography medium of the chromatography apparatus, generally, the number of theoretical plates and symmetry are calculated using the retention volume value of acetone, and it is possible to evaluate whether the packing of the chromatography apparatus meets the application criteria. The normal evaluation criteria are that the number of theoretical plates is ≥ 3000 and the asymmetry is in the range of 0.8 - 1.5. Based on this, from the data in Table 1 and Figure 13, it was shown that all the samples of other examples met the packing criteria except for the data of Example 4. By analyzing the symmetry of the sample peaks, it was confirmed that the asymmetry of the sample peaks in Example 3 was close to or exceeded 1.5, and the tailing phenomenon was significant, indicating that the pore sizes of these packings were the smallest and not suitable for separating samples with relatively large molecular weights. Overall, when compared comprehensively, the polymer fine particles obtained in Preliminary Example 1 and Preliminary Example 2 are replaceable compared with the conventional chromatography packings with the same solid content.

[0084] The large-volume column method was adopted to measure the pore volume. Among them, blue dextran 2000 (2000 kDa) is a standard product with the largest molecular weight. When this standard product passes through the column, it does not enter the pores of the polymer fine particles but passes through the gaps between the fine particles. The retention volume value of this standard product becomes the volume value of the interparticle gaps. Acetone (58 kDa) is a standard product with the smallest molecular weight, and this standard product can enter all the spaces in the stationary phase of the chromatography apparatus, including the gaps between the fine particles and the pores inside the fine particles. The retention volume value of this standard product is the sum of the volume of the interparticle gaps and the pore volume of the fine particles. Therefore, the difference in the retention volumes of acetone and blue dextran 2000 is the pore volume of the fine particles.

[0085] Calculated from the data in Table 2, the pore volume of the polymer fine particles used in Example 5 was 17.301 mL. The pore volume of the disordered pore agarose beads used in Comparative Example 2 was 18.237 mL. Therefore, the disordered pore volume of the agarose beads was 0.936 mL more than the ordered pore volume of the polymer fine particles.

[0086] Furthermore, from the data in Table 2, it was found that the retention volume value of IgG in the ordered pore stationary phase in Example 5 was 1.5 mL faster than that of the disordered pore agarose beads in Comparative Example 2. Accordingly, the retention times of bovine serum albumin and cytochrome C in Example 5 were 2.24 mL and 2.58 mL faster than those in Comparative Example 2, respectively. At the same time, from Figure 14, it was also proven that the sample had a faster outflow rate in the more ordered pore filling material, the half-width of the protein standard was smaller, the peak shape was sharper, and the column efficiency was higher.

[0087] Although this specification is described according to examples, each example does not only include an independent technical solution. Such a description method is for clarity, and those skilled in the art should understand the entire specification as a whole. The technical solutions of each example can be appropriately combined to form other implementation manners understandable to those skilled in the art.

[0088] The series of detailed descriptions shown above are only specific descriptions of the feasibility of the present invention and do not limit the protection scope of the present invention. Equivalent implementations and modifications made without departing from the technical spirit of the present invention shall be included in the protection scope of the present invention.

Claims

**Claim 1** A chromatography medium, wherein at least a part of the chromatography medium is composed of a polymer material formed by crosslinking of at least a partially crosslinkable substance containing rigid nanoparticles, at least one of the rigid nanoparticles has an asymmetric shape in solution, and the rigid nanoparticles form at least a locally ordered array structure in the chromatography medium. **Claim 2** The chromatography medium is one on which the polymer material is deposited, at least a part of the polymer material is polymer microparticles, the polymer microparticles are formed by crosslinking of at least a partially crosslinkable substance containing rigid nanoparticles, at least one of the rigid nanoparticles has an asymmetric shape in solution, and the rigid nanoparticles form at least a locally ordered array structure in the polymer microparticles. The chromatography medium according to claim 1. **Claim 3** There is one or more regions in the polymer microparticles where the rigid nanoparticles are ordered, and the molecular arrangement between the plurality of regions is unrelated, related or partially related. The chromatography medium according to claim 2. **Claim 4** The chromatography medium is formed by crosslinking of the entire at least partially crosslinkable substance containing rigid nanoparticles, at least one of the rigid nanoparticles has an asymmetric shape in solution, and the rigid nanoparticles form at least a locally ordered array structure in the chromatography medium. The chromatography medium according to claim 1. **Claim 5** The shape of the asymmetric rigid nanoparticles is rod-shaped, strip-shaped, sheet-shaped, needle-shaped or linear, with the characteristic direction being the long axis direction of the molecule. The chromatography medium according to claim 1. **Claim 6** The shape of the asymmetric rigid nanoparticles is disk-shaped, with the characteristic direction being perpendicular to the plane direction. The chromatography medium according to claim 1. **Claim 7** The entire inside of the chromatography medium is ordered, and the characteristic direction is distributed along the radial direction of the polymer microparticles, along the bipolar axis direction of the polymer microparticles, or distributed so as to form a plurality of concentric circles inside the polymer microparticles. The chromatography medium according to claim 5 or 6. **Claim 8** The chromatography medium according to claim 5 or 6, wherein in the orderly arranged local regions, the characteristic directions are arranged in parallel, fan-shaped or spiral shapes.

9. The chromatography medium according to any one of claims 1 to 4, wherein the rigid nanoparticles are at least one selected from polypeptides, proteins, nucleic acids, polysaccharides, and lipids.

10. The chromatography medium according to claim 9, wherein the rigid nanoparticles having an asymmetric shape are cellulose nanocrystals or cellulose nanofibers.

11. The chromatography medium according to claim 1, further comprising a polysaccharide compound having no asymmetric shape in solution in the chromatography medium, and the polysaccharide compound copolymerizes with the rigid nanoparticles to form the chromatography medium.

12. The chromatography medium according to claim 11, wherein the mass ratio of the rigid nanoparticles to the polysaccharide compound is 1:10 - 50:

1.

13. The chromatography medium according to claim 11, wherein the polysaccharide compound is at least one selected from agar, agarose, dextran, starch, chitosan, or trehalose.

14. The chromatography medium according to claim 1, wherein the separation range of the chromatography medium is 50 - 300000 kDa.

15. A chromatography device, comprising a support, and a chromatography medium filled inside the support or coated on the surface of the support, wherein the chromatography medium comprises the chromatography medium according to any one of claims 1 to 14.

16. The chromatography device according to claim 15, wherein the support is any one of a cylindrical wall material, a flat substrate, or a curved hollow tube.

17. The chromatography device according to claim 16, wherein the axial cross-sectional shape of the cylindrical wall material is any one of circular, elliptical, or polygonal.

18. The chromatography device according to any one of claims 16 to 17, wherein the material of the support is selected from any one or more of plastics, glass, ceramics, and metals.

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