Chromatography media 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 device including the chromatography medium, and particularly to a chromatography medium and a chromatography device composed of polymer microparticles 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 sciences.

[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] Conventional gel dialysis chromatography typically involves packing a stationary phase into a column, and simultaneously introducing a fluid phase containing specific components into the chromatography column when the samples to be separated are introduced. The fluid phase generally has inertia, and a solvent with a certain solubility for the sample is used. Since the degree of separation is not controlled, the separation results of the chromatography column do not depend on the interaction between the sample and the fluid phase. Relatively speaking, the properties of the stationary phase determine the separation effect. Commonly used stationary phases include hydrophilic organic gels, and polysaccharide gel spheres in particular are widely used in ion exchange chromatography, affinity chromatography, and hydrophobic interaction chromatography columns for small molecules and bioactive substances.

[0005] Given their wide range of applications and significant success in biopharmaceutical separation, there is a vast amount of relevant patent literature on biomolecular microspheres on agar substrates. Early relevant literature includes Biochim. Biophys. Acta 1964, 79:393-398 by Hjerten, S. and Biochim. Biophys. Acta 1964, 79:399 by Bengtsson et al., S., with U.S. Patent No. 4,647,536 being cited as the first patent relating to agar microspheres. Polysaccharide microspheres used in chromatography columns appeared even earlier in academic and patent literature.

[0006] Research indicates that in techniques such as affinity chromatography and ion exchange chromatography, the pore structure of the medium and the contact area with the protein are closely related, determining the protein separation effect (capacity, resolution, etc.). Therefore, the internal pore size, size, and size distribution of polysaccharide gel spheres used as the medium for chromatography columns, as well as the size and distribution of the spheres, their shape, and mechanical properties, significantly affect the separation effect and separation speed. However, currently known polysaccharide gel spheres are manufactured using methods such as mechanical stirring, homogeneous emulsification, and membrane emulsification, making it impossible to control their internal pore structure. Furthermore, these polysaccharide gel spheres typically possess inherent flexibility and have limited pressure tolerance, so the corresponding chromatography columns are limited to low-speed biological protein separation applications. In addition, the raw material for the most widely used agar microspheres is obtained through multi-step extraction from seaweed, making it unsuitable for large-scale industrial production and resulting in a high-cost raw material.

[0007] On the other hand, some biologically active substances, which are the target of separation, have low tolerance to changes in temperature, shear force, and solution environment, and easily lose their activity due to structural changes. Therefore, the chromatographic conditions for active substances are relatively stringent, requiring good mechanical properties, chemical stability, and high separation efficiency. Otherwise, there is a risk of denaturation or degradation of the target substance.

[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 consistent strength. This will improve the separation efficiency of chromatography columns in gel permeation chromatography and lead to time savings. [Overview of the project]

[0009] The object of the present invention is to provide a chromatography apparatus applicable to gel filtration chromatography. This allows the use of polymer materials having at least locally ordered pore structures as the chromatography medium, thereby addressing the above-mentioned needs.

[0010] The embodiments and objectives of the present invention will be described below, with examples of systems, tools, and methods. These examples are illustrative and explanatory, and not limiting. In different embodiments, one or more of the above-described market needs may be met by the present invention, or other embodiments may address different improvements.

[0011] The primary object of the present invention is to provide a chromatography medium for use in gel filtration chromatography. At least a portion of the chromatography medium is composed of a polymer material formed by crosslinking at least partially crosslinkable materials, including rigid nanoparticles, wherein at least one of the rigid nanoparticles has a non-spherically symmetric shape in solution and forms at least locally ordered arrangement structures within the chromatography medium.

[0012] One of the objectives of the present invention is to provide a chromatography medium in which the packed polymer material consists at least partly of polymer microparticles, and rigid nanoparticles are locally or entirely orderedly arranged within the polymer microparticles.

[0013] Another object of the present invention is to provide a chromatography medium formed by crosslinking a material that is at least partially crosslinkable and contains rigid nanoparticles. Within this chromatography medium, the rigid nanoparticles form an ordered arrangement structure at least locally.

[0014] Another object of the present invention is to provide a chromatography apparatus that uses the above-mentioned chromatography medium.

[0015] Based on the objectives of the present invention, the present invention provides a chromatography medium comprising a polymer material formed from a substance that is at least partially crosslinkable and comprises at least a portion of rigid nanoparticles. In this chromatography medium, at least one of the rigid nanoparticles has a non-spherically symmetric shape in solution and forms at least locally ordered arrangement structures within the chromatography medium.

[0016] As a further improvement of the present invention, the above-mentioned chromatography medium is made up of stacked polymer materials, and at least a portion of the polymer material is composed of polymer microparticles. These polymer microparticles are formed by crosslinking of at least partially crosslinkable materials, including rigid nanoparticles, and at least one rigid nanoparticle has a non-spherically symmetric shape in solution, forming at least locally ordered arrangement structures within the polymer microparticles.

[0017] Furthermore, as a further improvement of the present invention, the polymer microparticles contain one or more regions in which rigid nanoparticles are arranged in an ordered manner, and the molecular arrangements between these multiple regions are unrelated, related, or partially related.

[0018] Furthermore, as an improvement to the present invention, the above-mentioned chromatography medium is formed by crosslinking as a whole from a material that is at least partially crosslinkable and contains rigid nanoparticles, wherein at least one of the rigid nanoparticles has a non-spherically symmetric shape in solution and forms at least locally ordered arrangement structures within the chromatography medium.

[0019] As a further improvement of the present invention, the shape of the above-mentioned non-spherically symmetric rigid nanoparticles may be rod-shaped, strip-shaped, sheet-shaped, needle-shaped, or linear, characterized in the direction of the molecular long axis.

[0020] Furthermore, as a further improvement, the shape of the non-spherically symmetric rigid nanoparticles described above is disc-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 chromatographic medium is orderly arranged, and the above 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 region, the above characteristic direction is arranged in parallel, fan-shaped, or spiral.

[0023] Also, as a further improvement of the present invention, the above 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 chromatographic medium further contains polysaccharide compounds that do not take a clear asymmetric shape in solution, and these polysaccharide compounds copolymerize with the above rigid nanoparticles to form a chromatographic medium.

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

[0027] The polysaccharide compounds are selected from any one or more of agar, agarose, dextran, starch, chitosan, and trehalose. <0000**088> The separation range of the above chromatographic medium is 50 - 300000 kDa. <00000**90>

[0029] On the other hand, the present invention also discloses the following chromatographic apparatus. This chromatographic apparatus comprises a support, and a chromatographic 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. As a result, 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

[0034] [Figure 1] FIG. 1 is a schematic structural view of a porous chromatography medium in the prior art. [Figure 2] FIG. 2 is a schematic structural view of a porous chromatography medium in the present invention. [Figure 3] FIG. 3 includes four figures. (a) is a cross-sectional view of the polymer microparticles disclosed in the present invention, (b) is an enlarged view of part D of (a) in FIG. 3, (c) is an enlarged view of part E of (a) in FIG. 3, and (d) shows a scanning electron microscope image of the polymer microparticles in some embodiments. [Figure 4]Figure 4 includes four images: (a) shows the radial structure of polymer nanoparticles, (b) shows the bipolar structure of polymer nanoparticles, (c) shows the cyclic structure of polymer nanoparticles, and (d) shows the orthogonal polarized light microscope image of the radial structure of polymer nanoparticles. [Figure 5] Figure 5 shows schematic diagrams of the structures of various chromatography media used in the present invention. [Figure 6] Figure 6 includes two diagrams: (a) is a schematic diagram of the structure of a chromatography apparatus, and (b) is a diagram illustrating the separation principle of the chromatography apparatus. [Figure 7] Figure 7 shows schematic diagrams of the structures of two types of chromatography apparatus. [Figure 8] Figure 8 shows the axial cross-sectional shape in the A-A' direction of the chromatography apparatus shown in Figure 6(a). [Figure 9] Figure 9 shows orthogonal polarized microscope images of polymer nanoparticles prepared in Preliminary Example 1 of the present invention. [Figure 10] Figure 10 shows orthogonal polarized microscope images of polymer nanoparticles prepared in Preliminary Example 2 of the present invention. [Figure 11] Figure 11 shows orthogonal polarized light microscope images of polymer nanoparticles prepared in Preliminary Example 3 of the present invention. [Figure 12] Figure 12 shows orthogonal polarized microscope images of polymer nanoparticles prepared in Preliminary Example 4 of the present invention. [Figure 13] Figure 13 includes three images showing the gel filtration chromatography (GFC) separation results of each standard separated by chromatography apparatus prepared in the 1-1 ratio of Examples 1-4 shown. (a) is acetone, (b) is cytochrome C, and (c) is bovine serum albumin. [Figure 14] Figure 14 shows the gel filtration chromatography (GFC) separation results of each standard separated using the chromatography apparatus prepared in Example 5 and Proportional 2. [Modes for carrying out the invention]

[0035] To clarify the purpose, technical content, and advantages of the present invention, the technical content will be described in detail below with reference to specific embodiments and drawings of the present invention. The embodiments described herein represent only a part of the present invention and do not encompass all embodiments, nor do they limit the scope of the present invention. All other embodiments that can be obtained by an ordinary person of the art without creative effort based on embodiments of the present invention are also included within the scope of protection of the present invention.

[0036] In conventional "whole columns" and "microparticle-packed columns," the most prominent characteristic of the chromatography media used in chromatography is the disordered nature of its pores. For example, as shown in Figure 1(a), in a typical chromatography media consisting of agarose microparticles, the agarose-type chromatography media 100 is composed of packed agarose microparticles 101. A detailed magnified view of localized area A (Figure 1(b)) shows the depositional structure of the agarose microparticles, and further in Figure 1(c), it is clearly shown that the porous structure of the agarose microparticles is disorderly arranged and cross-linked.

[0037] In fact, the specific method for producing agarose microparticles 101 involves dispersing agarose in water, forming tiny aqueous droplets containing polysaccharides in the oil phase using appropriate emulsification techniques, and, as shown in the magnified view of portion B inside the agarose microsphere in Figure 1(c), after cooling, forming a double helix structure from a single chain of polysaccharide compound 102, thereby generating pores 103 between molecular bundles, and finally, forming polysaccharide microparticles with the crosslinking agent 104. Because the polysaccharide molecules are arranged disorderly in water, the pores in the resulting gel microparticles are also arranged disorderly. Furthermore, due to the inherent flexibility of polysaccharide molecules, the microparticles produced by crosslinking are usually relatively soft.

[0038] Based on the concept of the present invention, by using rigid nanoparticles that do not possess spherical symmetry, it is possible to create porous chromatography media with ordered molecular arrangements, controllable pores, and excellent mechanical properties. As shown in Figures 2(a) and (b), the chromatography media 200 in Figure 2(a), whose internal structure is at least locally ordered, has molecules arranged in an ordered manner at least partially, and the pores that are formed also have an ordered arrangement structure (see Figure 2(b)).

[0039] In nature, many biomolecules, when isolated or dispersed in water, exhibit rigid forms that lack spherical symmetry. Based on lyotropic liquid crystal theory, such rigid nanoparticles (or biomolecules, described later) may be arranged randomly depending on their concentration and characteristics when dispersed in a solvent, or, at higher concentrations, may form ordered molecular arrangements of certain lyotropic liquid crystals, such as nematic phases (e.g., nanomaterials of tobacco mosaic virus), smectic phases, cholesteric phases, or columnar phase liquid crystals. Orderly arranged liquid crystal materials usually exhibit birefringence properties, so droplets or solvents containing non-spherical biomolecules can be clearly observed under a polarizing microscope and exhibit typical liquid crystal structures.

[0040] In line with the spirit of the present invention, Figure 2(b) is a detailed enlarged view of portion C of Figure 2(a), showing a chromatography medium 200 having an internal structure that is at least locally ordered. This chromatography medium contains rigid nanoparticles 201. When the rigid nanoparticles 201 reach or exceed the critical concentration of liquid crystal, the nanoparticles form an ordered arrangement, and ordered pores, i.e., ordered pores 202, are formed between the nanoparticles. On the other hand, some nanoparticles fail to form an ordered arrangement, resulting in the formation of disordered pores 203. Furthermore, by adding a crosslinking agent or other flexible molecules through a crosslinking process, rigid nanoparticles 201 are produced that reach or exceed the critical concentration region of liquid crystal after crosslinking. In this case, some nanoparticles continue to maintain an ordered arrangement, and a chromatography medium with ordered pores 202 arranged accordingly is also obtained.

[0041] Similar to chromatographic media formed by the deposition of porous agarose microparticles, one method for forming a chromatographic media with orderedly arranged pores, in line with the spirit of the present invention, is to utilize the deposition of polymer microparticles with orderedly arranged pores. A specific explanation is shown in Figure 3.

[0042] In line with the spirit of the present invention, if the rigid nanoparticles are biopolymers, it is possible to produce porous biopolymer polymer microparticles in which at least the molecules have local order and the pores are arranged in an ordered manner. After depositing these microparticles with ordered pores, it is possible to obtain a structure in which the overall structure of the above-mentioned chromatography medium is at least partially ordered.

[0043] Specifically, cellulose nanocrystals (CNCs) with an appropriate major-to-minor ratio and size distribution are biopolymers that possess a certain rigidity in their solvent, water, and can form a dissolved liquid crystal phase. In line with the spirit of this invention, when the biopolymer is cellulose nanocrystal (CNC), when the concentration reaches a critical value, the CNC molecules self-assemble to form an ordered arrangement, exhibiting an ordered liquid crystal phase. Furthermore, cellulose nanocrystal CNCs possess chirality as biopolymers, and the ordered arrangement structure of the formed molecules has an arrangement of liquid crystal molecules with a helical structure, such as the cholesterol phase.

[0044] Figure 3 shows an example of polymer nanoparticles having a porous structure disclosed according to the present invention. Specifically, Figure 3(a) shows a partial cross-sectional view of the interior of the polymer nanoparticles along the radial direction, where the rigid nanoparticles maintain an ordered arrangement at least locally after crosslinking, and the pores formed under the influence of the molecular arrangement may also be arranged regularly locally (see Figure 3(b)). The rigid nanoparticle solution shown in Figure 3(a) emulsifies to form emulsion droplets when it is in a locally ordered state, and the rigid nanoparticles 201 within the emulsion droplets also attempt to arrange themselves locally in an ordered manner. As the temperature of the emulsion decreases, the rigid nanoparticles 201 maintain their arrangement and further crosslink to form polymer nanoparticles, whose internal structure retains at least partially the previous ordered structure. At the same time, as shown in Figure 3(b), the ordered pores 202 present between the arrangements of rigid nanoparticles also inherit a similar ordered structure, and the pores of the porous nanoparticles form an ordered arrangement structure at least locally. Here, "ordered" means that, at least within a local area, there is a certain regularity in the arrangement of direction and position between the local section of the hole and at least the local section of an adjacent hole. Specifically, this means that, at least within a local area, the local section of the hole and the local section of an adjacent hole are arranged in parallel, sector-shaped, or spiral. As shown in Figure 3(d), in a preferred embodiment, the diameter of the hole is 1–1000 nanometers.

[0045] In line with the spirit of the present invention, by controlling the concentration of rigid nanoparticles, the ordered arrangement structure formed in the emulsion droplet may include one or more regions. Simultaneously, the molecular arrangements of multiple regions may be unrelated, related, or partially related. Furthermore, the above ordered arrangement structure may be ordered overall or locally. When ordered overall, in the ordered local region, the characteristic directions of the rigid nanoparticles are distributed along the radial direction of the particle, along the bipolar axis direction of the particle, or distributed to form multiple concentric circles within the particle. When ordered locally, in the ordered local region, the characteristic directions of the rigid nanoparticles are arranged in parallel, sector-shaped, or helical patterns.

[0046] Within the range of overall order, these ordered arrangements can form certain special structural configurations. For example, in the radial arrangement shown in Figure 4(a) (ordered arrangement where the characteristic direction is radial), first pores 401 can be formed inside that are regularly arranged toward the center. In the bipolar arrangement shown in Figure 4(b) (ordered arrangement where the characteristic direction is the bipolar axis direction), second pores 402 can be formed inside that are regularly arranged along the bipolar axis direction. Furthermore, in the annular arrangement shown in Figure 4(c) (where the characteristic direction is arranged in multiple concentric circles), third pores 403 and other pores can be formed inside that are regularly arranged in concentric circles. 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 polarizing microscope due to the optical birefringence properties that rigid nanoparticles normally possess. For example, as shown in Figure 4(a), the characteristic orientations of the rigid nanoparticles 201 are radially ordered within the emulsion droplet, and the internal structure and pores of the formed polymer microparticles are also radially ordered, resulting in a radial configuration. This allows for the observation of Malta's black cross optical anisotropy under a cross-polarizing microscope (see Figure 4(d)).

[0047] In the locally ordered regions, as shown in Figure 3(a), the polymer nanoparticles contain regions D (shown in Figure 3(b)) and E (shown in Figure 3(c)). Region D is a localized region where the characteristic directions of rigid nanoparticles are ordered, while region E is a region where the characteristic directions are arranged disorderly. Within these polymer nanoparticles, ordered pores 202 and disordered pores 203 are formed simultaneously. At this time, although the polymer nanoparticles do not have a specific structure, their characteristic directions are still regularly arranged within a narrow range, so they can still exhibit color under a cross-polarizing microscope.

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

[0049] In accordance with the spirit of the present invention, polymer nanoparticles 301 are formed by crosslinking with at least partially crosslinkable material containing rigid nanoparticles 201 (i.e., biopolymers). In this case, at least one of the rigid nanoparticles has a non-spherical symmetric shape in solution, and the shape of the rigid nanoparticle is rod-shaped with the long axis of the molecule as the characteristic direction. Alternatively, it may be bow-shaped (banana-shaped) with the long axis of the molecule as the characteristic direction, or disc-shaped with the characteristic direction perpendicular to the plane, or even plate-shaped, needle-shaped, or linear. However, this application is not limited to these, and other non-spherical symmetric shapes that satisfy other requirements can also be adopted.

[0050] Rigid nanoparticles having or not having a non-spherically symmetric shape are selected from at least one of the following: polypeptides (insulin, growth hormone, etc.), proteins (chlorophyll, collagen, etc.), nucleic acids (DNA, etc.), polysaccharides (cellulose, chitin, etc.), and lipids (monoglycerides, phospholipids, glycolipids, steroids, etc.). These biopolymers are widely present in living organisms and mainly exhibit rod-shaped or flat shapes in solution. In preferred examples, biopolymers having a non-spherically symmetric shape may or may not have chirality. Furthermore, chiral biopolymers include levorotatory and dextrorotatory biopolymers, and the resulting liquid crystal phase forms an arrangement of liquid crystal molecules with a helical structure of the cholesterol phase. One specific example of this application is cellulose nanocrystal (CNC), a biopolymer, whose structural formula is as follows. [ka]

[0051] The rod-shaped structures formed by biopolymers have a large aspect ratio and readily form a liquid crystal state. In a more preferred example, the cellulose nanocrystals have a length of 20-1000 nanometers, a width of 2-100 nanometers, and an aspect ratio of 1:5 to 1:200.

[0052] As shown in Figure 3(b), the polymer microparticles may further contain polysaccharide compounds 102 that do not have a non-spherically symmetric 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 the specific examples of this application, the polysaccharide compound is agarose, and its structural formula is as follows. [ka]

[0053] Before emulsification, the polysaccharide compound is a fluid gel-like dispersion. After emulsification to form emulsion droplets, it undergoes processes such as cooling, solidification, and aging. As shown in Figure 3(b), the polysaccharide compound 102 forms a double helix from a single chain, then 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 bonding, between these polysaccharide compounds and biomacromolecules, they follow the arrangement of the biomacromolecules and ultimately form an ordered arrangement. Furthermore, under certain conditions and concentrations, the biomacromolecules locally form an ordered arrangement to create a liquid crystal state, and the polysaccharide compounds are arranged using the arrangement scheme of the polymers as a model. These polysaccharide compounds further copolymerize with the biomacromolecules with the help of crosslinking agents to form a stable microparticle structure. At this time, the pressure resistance of the polymer microparticles can be further improved without damaging the ordered structure of the formed polymer microparticles. Simultaneously, in the manufacturing process of polymer microparticles, polysaccharide compounds (e.g., agarose) are used to solidify the emulsified emulsion droplets upon cooling, providing structural support for subsequent cross-linked polymer formation, thereby simplifying the manufacturing process.

[0054] In accordance with the spirit of the present invention, a preferred method for obtaining a chromatographic medium having at least a partially ordered pore structure is, as shown in Figure 5(a), a chromatographic medium 200 having an at least locally ordered internal structure, comprising aspherically asymmetric rigid nanoparticles and further composed of polymer microparticles 301 in which molecules are at least partially ordered. In accordance with the spirit of the present invention, a chromatographic medium 200 having an at least locally ordered internal structure with at least partially ordered pores can also be formed by stacking polymer microparticles 301 with ordered pores and microparticles 501 with disordered pores, as shown in Figure 5(b). Furthermore, it can also be formed by stacking polymer microparticles 301 with ordered pores and non-porous microspheres 502, as shown in Figure 5(c). Microspheres with disordered pores generally include polysaccharide microspheres (e.g., agarose microspheres), while common non-porous microspheres include inorganic silicon dioxide microspheres and hydroxyl apatite.

[0055] In accordance with the spirit of the present invention, the production of a chromatography medium containing polymer microparticles includes dispersing rigid nanoparticles alone or with an appropriate amount of amorphous monomer or oligomer to form a dissolved liquid crystal solution, forming emulsion droplets by emulsification, and then forming polymer porous microspheres through procedures such as solidification and crosslinking. 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 they may directly participate in the crosslinking reaction and chemically bond to become part of the polymer.

[0056] When the chromatography medium is formed by crosslinking a material that is at least partially crosslinkable and contains rigid nanoparticles, at least one of the rigid nanoparticles has a non-spherically symmetric shape in solution, and the rigid nanoparticles form at least locally ordered structures within the chromatography medium.

[0057] Specifically, as shown in Figure 5(d), the chromatography medium 200 having an internal structure that is at least locally ordered is an overall structure formed by an orderly arrangement of polymers. However, the present invention is not limited to this, and the above chromatography medium may include other foreseeable ranges.

[0058] In accordance with the spirit of the present invention, when the overall structure formed by crosslinking rigid nanoparticles is used as a chromatography medium, unlike the method for producing polymer porous microspheres, a dissolved liquid crystal solution with local or overall order is formed, and then the crosslinking agent is added directly to carry out on-site polymerization. Specifically, the procedure involves adding a dispersion containing rigid nanoparticles to a pre-loaded stainless steel column, adding the crosslinking agent dropwise while controlling the temperature, adjusting the pH, and drying after the reaction, thereby obtaining at least rigid nanoparticles and a structure in which the rigid nanoparticles are at least locally ordered within the overall structure.

[0059] Methods for packing chromatography media typically include high-pressure homogeneity packing, wet packing, dry packing, and high-density high-viscosity packing. High-pressure homogeneity packing is a method for producing a uniformly packed column by suspending the packing material in a suitable homogeneous solution and rapidly pushing it into the column at a very high flow rate using a high-pressure pump before it settles. This is a common packing method for chromatography in analysis and manufacturing, and is also the method used in this invention. Wet packing is typically used to obtain a uniform and dense column bed for high-efficiency analytical columns packed with small particle size packing material of 3-10 microns. Dry packing is typically used for large particle size powder packing material (often used in chromatography manufacturing), for example, packing material with a particle size of 50 microns or more. High-density high-viscosity packing is applicable when the density of the chromatography packing material is very high and there is a high requirement for uniformity of the column bed. Typically, a high-density or high-viscosity homogeneous solution is prepared, the packing material is suspended in the homogeneous solution, and external pressure is applied to cause the packing material to settle and deposit in layers.

[0060] The present invention also discloses a chromatography apparatus, and Figure 6 is a schematic diagram of the structure of a columnar chromatography apparatus according to the present invention. As shown in Figure 6(a), the chromatography apparatus comprises a support 600 and a chromatography medium 200 which is filled inside the support or coated on the surface of the support and has an internal structure that is at least locally ordered, and the chromatography apparatus also further comprises a sample inlet 601 and a sample outlet 602 located at both ends of the support.

[0061] As shown in Figure 6(b), the separation principle of the chromatography apparatus of the present invention is as follows: when porous polymer microparticles 301 separate molecules of different sizes (molecular weights) (e.g., proteins), small protein molecules 603 can enter the stationary phase through small and large pores, large protein molecules 604 can enter the stationary phase through large pores only, and very large protein molecules 605 cannot enter the stationary phase and remain in the gaps between the stationary phase particles, later being eluted with the fluid phase. In principle, very large protein molecules are eluted first, large protein molecules next, and small protein molecules last, thereby achieving the objective of effectively separating the mixture.

[0062] Furthermore, since the polymer microparticles in the present invention have at least locally ordered characteristic directional arrangements and pore arrangements, the pathways through which molecules of the same size diffuse into and out of the polymer microparticles are regular and clear. Compared to stationary phases with irregular internal arrangements, the peak appearance time of the sample molecules to be separated in the chromatography apparatus of the present invention is shortened, i.e., the retention time is further reduced.

[0063] As a preferred technical option, the support is selected from one of the following: a cylindrical wall material 701 (shown in Figure 7(a)), a flat substrate 702 (shown in Figure 7(b)), or a curved hollow tube, as shown in Figure 7. Specifically, as shown in Figure 6, the cross-sectional shape of the support 600 in the A-A' direction may be one of the following polygons: circular (shown in Figure 8(a)), elliptical (shown in Figure 8(b)), rectangular (shown in Figure 8(c)), or hexagonal (shown in Figure 8(d)). If the support is a flat substrate, the chromatography medium is coated to a certain thickness on the surface of the flat substrate, and if the support is a cylindrical wall material or a curved hollow tube, the chromatography medium is filled inside the support. As a preferred technical option, the material of the support is selected from one of the following: plastic, glass, ceramic, metal, or a composite material thereof.

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

[0065] Step 1: The above polymer microparticles are manufactured. The embodiments of the present invention include a method for manufacturing polymer microparticles. First, a dispersion is formed by dispersing a biopolymer and a polysaccharide compound. Specifically, a dispersion is formed by dispersing a biopolymer and a polysaccharide compound in water to form a dispersed phase solution. Next, the dispersion is emulsified to form emulsion droplets. There are many emulsification methods, but membrane emulsification is included. Membrane emulsification refers to a method in which the dispersed phase enters the continuous phase directly through the pores of a microporous membrane during the emulsification process, and emulsion droplets are formed at the ends of the pores and successively extruded. Finally, a crosslinking agent is added to the emulsion droplets to crosslink the biopolymers within the emulsion droplets and form polymer microparticles.

[0066] Second step: The manufactured polymer microparticles are weighed to a fixed amount and, preferentially, washed 2-3 times by extraction filtration. The washed polymer microparticles are then sufficiently swollen in a homogeneous solution to form a suspension. The homogeneous solution is ultrapure water or phosphate buffer of different concentrations, and is not particularly limited in this invention, but in the following examples, the homogeneous solution used is always ultrapure water.

[0067] Third step: The suspension obtained in the second step is packed into the column and mounted using the homogenization method. Ultrapure water is selected as the fluid phase. Specifically, the suspension is poured into a homogenization tank, and the stationary phase is allowed to settle freely. After settling is complete, the fluid phase flow rate is increased by 0.5 ml / min every 10 minutes, while observing the relationship between flow rate and pressure in real time. When the pressure reaches 0.2-0.25 megapascals, the increase in flow rate is stopped, and it is observed whether there is any change in pressure. If there is no significant increase, it is considered that the loading is solid and there is no damage to the packing material, and finally a chromatography apparatus containing polymer microparticles can be formed.

[0068] When the chromatography medium is formed by overall crosslinking with at least partially crosslinkable materials including rigid nanoparticles, unlike the method for producing polymer nanoparticles, the biopolymer and polysaccharide compounds are dispersed to form a dispersion, and then the crosslinking agent is added directly to carry out on-site polymerization. The specific procedure is as follows: A dispersion of cellulose nanocrystals and agarose is added to a pre-loaded stainless steel column, and the temperature is controlled to 80°C with a heating jacket to prevent the dispersion from solidifying. Next, the crosslinking agent (1,4-butanediol diglyceryl ether) is added dropwise, followed by the addition of a 0.5 M sodium hydroxide solution, and the mixture is reacted at 38°C for 12 hours after the addition is complete. Finally, this overall column is lyophilized or critically dried to obtain a porous overall column bed. Unreacted material is washed away with a large amount of ethanol and water, and then lyophilized or critically dried again to obtain a porous overall gel chromatography apparatus.

[0069] The structure, production, and separation effect of the chromatography medium and chromatography apparatus of the present invention, as well as the method of its production, will be described in detail below based on specific examples. In the embodiments of the present invention, all ratios are expressed by mass unless otherwise specified.

[0070] (Alternative example 1) Polymer 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, and cooled to form a dispersion containing solidified emulsion droplets. After washing to remove the emulsifier and liquid paraffin, the weight of the resulting gel was measured and added to 10 ml of aqueous solution containing 500 microliters of the crosslinking agent 1,4-butanediol diglyceryl ether, and the reaction was stirred for 12 hours. Next, 500 microliters of aqueous solution containing 40 wt% sodium hydroxide and 5 wt% sodium borohydride was added to the above reaction mixture and the reaction was stirred for 8 hours. Then, 500 microliters of epoxychloropropane and 500 microliters of an aqueous solution containing 40 wt% sodium hydroxide and 5 wt% sodium borohydride were mixed and added to the reaction mixture, and the reaction was stirred for a further 12 hours. After the reaction was complete, the mixture was thoroughly washed. As shown in Figure 9, under orthogonal polarizing microscope, the polymer particles exhibit radial optical anisotropy (black cross of chiral malta), indicating that their internal structure and pore distribution are radial.

[0071] (Alternative example 2) Unlike the method of Example 1, polymer particles containing 4% cellulose nanocrystals and 2% agarose were prepared. The other procedures and experimental conditions were the same as in Example 1. As shown in Figure 10, under orthogonal polarizing microscope, the polymer particles exhibit radial optical anisotropy (Malta's black cross), revealing their radial internal structure and pore distribution.

[0072] (Alternative example 3) Unlike the method in Example 1, polymer microparticles containing 2% cellulose nanocrystals and 4% agarose were prepared. The other procedures and experimental conditions were the same as in Example 1. As shown in Figure 11, no clear radial optical anisotropy (Malta's black cross) was observed in the polymer microparticles under orthogonal polarizing microscope, indicating a weakened tendency toward radial internal pore structure.

[0073] (Alternative example 4) Unlike the method in Example 1, polymer microparticles containing 1% cellulose nanocrystals and 5% agarose were prepared. The other procedures and experimental conditions were the same as in Example 1. As shown in Figure 12, under orthogonal polarizing microscope, no clear radial optical anisotropy (Malta's black cross) was observed in the polymer microparticles, indicating a weakened tendency toward radial internal pore structure.

[0074] (Examples 1-4) The polymer microparticles produced in Preliminary Examples 1-4 were sieved to retain polymer microparticles with a particle size of 60 to 90 microns, and washed three times using an extraction filtration method. The washed polymer microparticles were dispersed in ultrapure water and allowed to swell sufficiently. The resulting suspension was injected into a 5*100 mm hollow column using the homogenization method described above, and the corresponding chromatography apparatus was fabricated.

[0075] (Example 5) The polymer microparticles produced in Preliminary Example 2 were sieved to retain polymer microparticles with a particle size of 60 to 90 microns, and washed three times using an extraction filtration method. The washed polymer microparticles were dispersed in ultrapure water and allowed to swell sufficiently. The resulting suspension was injected into a 16*400 mm hollow column using the homogenization method described above to produce a chromatography apparatus.

[0076] (Proportional Relations 1) Unlike the method in Preliminary Example 1, agarose balls with a solid content of 6% were prepared. The other procedures and experimental conditions were the same as 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. The other procedures were the same as in Examples 1-4.

[0077] (Proportional Relations 2) Unlike the method in Preliminary Example 1, agarose balls with a solid content of 6% were prepared. The other procedures and experimental conditions were the same as 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. The other procedures were the same as in Example 5.

[0078] To verify that the chromatography apparatus provided in this invention has excellent separation capabilities, sample separation was performed using multiple standards in chromatography apparatuses proportional to those used in each example. The protein purification apparatus used for the chromatography column was an AKTA PURE. Ultrapure water was used as the liquid phase for the separation of acetone, and a mixed buffer of PBS solution and sodium chloride solution was used as the liquid phase for the separation of bovine serum albumin and cytochrome C.

[0079] (Example of experiment) The chromatography apparatuses obtained in Examples 1-4 and Proportional 1 were connected to protein purifiers, and samples were injected at a flow rate of 0.1 ml / min. The sample was 25 microliters of 0.5% acetone solution. The ultrapure water in the chromatography apparatus was replaced with a mixed buffer of 0.2 mmol / L PBS solution and 0.15 mol / L sodium chloride, and then the sample was injected at a flow rate of 0.1 ml / min. The separation effect of the samples was tested using 25 microliters of 0.8 mg / ml bovine serum albumin (BSA) solution and 25 microliters of 0.5 mg / ml cytochrome C solution, respectively. The results are shown in Table 1 and Figure 13.

[0080] The chromatography apparatuses of Example 5 and Proportional 2 were connected to protein purification apparatuses, and samples were injected with a flow rate controlled to 1 ml / min. The separation effect was tested with 300 microliters of 1% acetone solution and 300 microliters of 1 mg / ml Blue Dextran 2000 solution. The ultrapure water in the chromatography apparatus was replaced with a 0.2 mmol / L PBS solution and a 0.15 mol / L sodium chloride mixed buffer, and then the samples were injected with a flow rate controlled to 1 ml / min. The separation effect was tested with 300 microliters of 0.8 mg / ml bovine serum albumin solution, 300 microliters of 0.5 mg / ml cytochrome C solution, and 300 microliters of 1 mg / ml IgG solution. The results are shown in Table 2 and Figure 14.

[0081] Table 1: Separation effect data using a chromatography apparatus with proportionality 1 for Examples 1-4 [Table 1]

[0082] Table 2 Separation effect data using chromatography apparatus with proportionality 2 compared to Example 5 [Table 2]

[0083] Since acetone molecules can pass through all spaces in the chromatographic medium of a chromatography apparatus (including gaps between particles and pores within particles), the theoretical plate number and symmetry can generally be calculated using the retained volume value of acetone to evaluate whether the chromatographic apparatus packing meets the application criteria. Typical evaluation criteria are a theoretical plate number of ≥3000 and an asymmetry range of 0.8 to 1.5. Based on this, the data in Table 1 and Figure 13 indicate that, with the exception of the data in Example 4, all samples in the other examples meet the packing criteria. Analysis of the sample peak symmetry revealed that the asymmetry of the sample peak in Example 3 is close to or exceeds 1.5, indicating a significant trailing effect. This suggests that the pore size of these packings is minimal and unsuitable for separating samples with relatively large molecular weights. Overall, the polymer particles obtained in Preliminary Examples 1 and 2 are interchangeable with conventional chromatographic packings of the same solid content.

[0084] A large-volume column chromatography method was employed to measure pore volume. In this method, blue dextran 2000 (2000 kDa) is the standard with the highest molecular weight. When this standard passes through the column, it does not enter the pores of the polymer microparticles, but passes through the gaps between the microparticles. The retained volume of this standard is the volume of the interparticle gaps. Acetone (58 kDa) is the standard with the lowest molecular weight. This standard can enter all spaces in the stationary phase of the chromatography apparatus, including the gaps between microparticles and the pores inside the microparticles. The retained volume of this standard is the sum of the volumes of the interparticle gaps and microparticle pores. Therefore, the difference between the retained volumes of acetone and blue dextran 2000 is the volume of the microparticle pores.

[0085] Based on the data in Table 2, the pore volume of the polymer microparticles used in Example 5 was 17.301 mL. The pore volume of the disordered pore agarose balls used in proportionality 2 was 18.237 mL. Therefore, the disordered pore volume of the agarose balls was 0.936 mL greater than the ordered pore volume of the polymer microparticles.

[0086] Furthermore, data from Table 2 showed that the retention volume of IgG in the ordered pore stationary phase in Example 5 was 1.5 mL faster than that of the disordered pore agarose ball with a ratio of 2. Accordingly, the retention times of bovine serum albumin and cytochrome C in Example 5 were 2.24 mL and 2.58 mL faster, respectively, than with the ratio of 2. Simultaneously, Figure 14 also demonstrated that the samples effluxed faster in the more ordered pore packing, resulting in a smaller half-width of the protein standards, sharper peak shapes, and higher column efficiency.

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

[0088] The series of detailed descriptions provided above are merely a concrete description of the feasibility of the present invention and do not limit the scope of protection of the present invention. Equivalent practices and modifications made without departing from the technical spirit of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A chromatography medium wherein at least a portion of the chromatography medium is composed of a polymer material formed by crosslinking at least partially crosslinkable substances including rigid nanoparticles, at least one of the rigid nanoparticles has a non-spherically symmetric shape in solution, and the rigid nanoparticles form at least locally ordered arrangement structures in the chromatography medium. The chromatography medium further comprises a polysaccharide compound that does not have a non-spherically symmetric shape in solution, wherein the polysaccharide compound copolymerizes with the rigid nanoparticles to form the chromatography medium.

2. The chromatography medium according to claim 1, wherein the chromatography medium is a deposition of the polymer material, at least a portion of the polymer material is polymer microparticles, the polymer microparticles are formed by crosslinking of at least partially crosslinkable material including rigid nanoparticles, at least one of the rigid nanoparticles has a non-spherically symmetric shape in solution, and the rigid nanoparticles form at least locally ordered arrangement structures within the polymer microparticles.

3. The chromatography medium according to claim 2, wherein the polymer microparticles contain one or more regions in which rigid nanoparticles are arranged in an ordered manner, and the molecular arrangements between the multiple regions are unrelated, related, or partially related.

4. The chromatography medium according to claim 1, wherein the chromatography medium is formed by crosslinking of at least a partially crosslinkable material comprising rigid nanoparticles, at least one of the rigid nanoparticles has a non-spherically symmetric shape in solution, and the rigid nanoparticles form at least locally ordered arrangement structures in the chromatography medium.

5. The chromatography medium according to claim 1, wherein the shape of the non-spherically symmetric rigid nanoparticles is rod-shaped, strip-shaped, sheet-shaped, needle-shaped, or linear, with the characteristic direction being the long axis of the molecule.

6. The chromatography medium according to claim 2, wherein the entire interior of the chromatography medium is arranged in an ordered manner, and the characteristic directions of the non-spherically symmetric rigid nanoparticles are distributed along the radial direction of the polymer nanoparticles, along the bipolar axis direction of the polymer nanoparticles, or distributed so as to form a plurality of concentric circles inside the polymer nanoparticles.

7. The chromatography medium according to claim 5, wherein in an orderedly arranged local region, the characteristic directions are arranged in parallel, fan-shaped, or spiral.

8. 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.

9. The chromatography medium according to claim 8, wherein the rigid nanoparticles having a non-spherically symmetric shape are cellulose nanocrystals or cellulose nanofibers.

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

1.

11. The chromatography medium according to claim 1, wherein the polysaccharide compound is at least one selected from agar, agarose, dextran, starch, chitosan, or trehalose.

12. The chromatography medium according to claim 1, wherein the separation range of the chromatography medium is 50-300,000 kDa.

13. A chromatography apparatus, Support and The chromatographic medium is either filled inside the support or coated on the surface of the support. A chromatography apparatus characterized in that the chromatography medium includes the chromatography medium described in claim 1.

14. The chromatography apparatus according to claim 13, wherein the support is one of a cylindrical wall material, a flat substrate, or a curved hollow tube.

15. The chromatography apparatus according to claim 14, wherein the axial cross-sectional shape of the cylindrical wall material is one of a circle, an ellipse, or a polygon.

16. The chromatography apparatus according to any one of claims 14 to 15, wherein the material of the support is selected from one or more of plastic, glass, ceramic, and metal.

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