Stationary phase medium for adsorption chromatography and method for manufacturing the same

Abstract

The present invention relates to a stationary phase medium for adsorption chromatography in the form of porous particles suitable for packing into chromatography columns. These porous particles are made of a cross-linked polymer material and have numerous interconnected macropores forming a porous network through which a mobile phase fluid can flow by convection. These porous particles are substantially devoid of diffusion pores, so that mass transport through the porous network is governed solely by convection. These porous particles are fabricated into irregular particle configurations with roughened outer surfaces, so that convection flow between the porous particles is not hindered during the chromatography process.

Description

Technical Field

[0001] The present invention relates to a stationary phase medium for adsorption chromatography, and more particularly to a stationary phase medium in the form of polymer porous particles, which is suitable for being packed in a chromatography column to separate macromolecules with high throughput, high efficiency and low back pressure. Background Art

[0002] Adsorption chromatography is a type of liquid chromatography that separates a component in a mixture by selectively adsorbing it from a mobile phase to a solid stationary phase. Porous resin beads have been widely used as the stationary phase in adsorption chromatography. Typical resin beads have a tortuous network of micropores with diameters ranging from a few nanometers to tens of nanometers, allowing low molecular weight solutes present in the mobile phase to diffuse in and out of the micropores. Figure 1 As shown, these micropores are typically located near the outer surface of the resin beads and are not interconnected. Most of the adsorption surface is located within the resin beads, accessible only by diffusion. While conventional resin beads have proven effective for separating small molecules, they perform poorly for separating large molecules because they cannot enter the small micropores. In other words, large molecules bind only to the resin bead surface in a thin layer, resulting in low binding capacity. This low separation rate is particularly detrimental for biomolecules susceptible to enzymatic degradation or other destructive conditions. Resin-based chromatography also has other drawbacks. Because diffusion within the beads is the rate-determining step in the adsorption process, resolution decreases with increasing flow rate. Insufficient convective flow between the beads results in a high pressure drop across the entire chromatography column. All of these drawbacks result in reduced separation efficiency and unsatisfactory macromolecule productivity. Chromatography processes using traditional resin beads as stationary media typically take several days to complete, making them very time-consuming and costly.

[0003] The industry has made significant efforts to address these shortcomings. U.S. Patent No. 5,228,989 proposes so-called perfusion chromatography resins, which contain macropores with diameters of 0.6-0.8 microns for convective transport of substances, and numerous diffusion micropores to provide adsorption capacity. However, the diffusion transport in and out of the perfusion resin beads during the adsorption process still reduces the overall production rate. In addition, perfusion resin beads are typically manufactured in the form of spherical particles with a narrow size distribution. When these beads are densely packed together in a column for use in, for example, high-performance liquid chromatography, the convection channels between the resin beads are very narrow, which in turn leads to high backpressure.

[0004] Unlike resin beads, which are manufactured in the form of fine particles, porous monolithic columns in block form are disclosed in, for example, U.S. Patents Nos. 7,026,364, 11,118,024, and 11,236,184 for use as stationary media in adsorption chromatography. Due to the rapid mass transfer capabilities enabled by the convective macropores formed within these porous monolithic columns, these columns are suitable for use as matrices for separating macromolecules. These monolithic columns can be fabricated using conventional methods, such as high internal phase emulsion templating and colloidal crystal templating. The former involves preparing a water-in-oil emulsion, polymerizing an external phase containing monomers, and removing the internal phase, while the latter involves infiltrating monomers into the interstitial spaces of a colloidal crystal template, polymerizing the monomers into a polymer matrix, and then removing the template. However, fabricating a defect-free polymer monolith is a difficult task, as it is prone to breakage due to uneven thermal expansion during polymerization. Monolithic columns with multiple defects may result in loss of resolution and reduced recovery of target substances.

[0005] Therefore, the relevant industry still needs a static phase medium that is not only made in the form of high-molecular-weight porous particles suitable for filling chromatography columns with low back pressure, but also mainly composed of convective macropores and essentially without diffusion micropores, so that large molecules can more easily access the interior of the particles and allow convective transport of substances through the particles. Summary of the Invention

[0006] To overcome the aforementioned shortcomings, the present invention provides a stationary phase medium for adsorption chromatography in the form of a group of porous particles suitable for packing into chromatography columns. Each porous particle is made of a cross-linked polymer material and has numerous interconnected macropores forming a porous network through which a mobile phase fluid can flow by convection. Because the porous particles herein lack substantially any diffusion pores, solutes or analytes carried by the mobile phase fluid are transported through the porous network solely by convection. The porous network has a sufficiently large diameter to permit convective flow of the mobile phase fluid and possesses an extremely large specific surface area, making it easily accessible and accessible to macromolecules for attachment. More importantly, the porous particles herein are fabricated with an irregular particle shape and a rough outer surface, so that convection between the porous particles during chromatography is not hindered or slowed, unlike the disadvantages that can occur with conventional resin beads exhibiting a uniform spherical structure.

[0007] Therefore, a first aspect of the present invention provides a stationary medium for adsorption chromatography, which is particularly suitable for separating macromolecules. The stationary medium comprises:

[0008] a plurality of porous particles made of a cross-linked polymer material, each having a Feret diameter in the range of 25 to 500 micrometers and a porosity of 70% to 90%, each particle having a plurality of spherical macropores with a diameter in the range of 3 to 10 micrometers, wherein the spherical macropores are interconnected via connecting pores to form a porous network, and the average diameter of the porous network is in the range of 0.2 to 6 micrometers; and

[0009] The porous particles exhibit an irregular particle morphology with a Feret aspect ratio distribution ranging from 1.0 to 3.5 and a standard deviation ranging from 1.2 to 2.2.

[0010] A second aspect of the present invention provides a method for manufacturing the aforementioned stationary medium, comprising the following steps:

[0011] A. preparing a porous monolithic column made of a cross-linked polymer material and having a porosity of 70% to 90%, wherein the porous monolithic column is formed with a plurality of spherical macropores having diameters ranging from 3 μm to 10 μm, and the spherical macropores are interconnected via connecting pores to form a porous network, and the average diameter of the porous network is within the range of 0.2 μm to 6 μm;

[0012] B. subjecting the porous monolith to mechanical grinding, so that the porous monolith is ground into a first population of porous particles having a particle size distribution of less than 1,000 microns; and

[0013] C. sorting the first population of porous particles based on particle size to obtain a second population of porous particles having a Feret diameter in a range of 25 μm to 500 μm, wherein the second population of porous particles exhibits an irregular particle morphology, a Feret aspect ratio distribution in a range of 1.0 to 3.5, and a standard deviation in a range of 1.2 to 2.2.

[0014] In a preferred embodiment, the porous particles have substantially no pores having a diameter less than 100 nm as measured by mercury intrusion porosimetry.

[0015] In a preferred embodiment, the porous network has an average diameter in the range of 0.5 μm to 3.0 μm. In a more preferred embodiment, the porous network has an average diameter in the range of 1.2 μm to 2.4 μm.

[0016] In a preferred embodiment, the static medium is surface-modified to have ion exchange functional groups. In a more preferred embodiment, the ion exchange functional groups are selected from the group consisting of quaternary ammonium, diethylaminoethyl, sulfonyl, and carboxymethyl.

[0017] In a preferred embodiment, the cross-linked polymer material is selected from the group consisting of polyacrylate, polymethacrylate, polyacrylamide, polystyrene, polypyrrole, polyethylene, polypropylene, polyvinyl chloride and polysilicone. In a more preferred embodiment, the cross-linked polymer material is selected from polymethacrylate.

[0018] In a preferred embodiment, at least 70% of the spherical macropores of each porous particle are arranged in a closest-packed manner.

[0019] Furthermore, compared to static media produced by conventional methods, the static media produced by the aforementioned method possesses the following structural features: an internal porous network formed during the porous monolithic column preparation process, and irregular particle configurations and a roughened outer surface imparted by the mechanical grinding step. Therefore, a third aspect of the present invention provides a static media produced by the aforementioned method.

[0020] A fourth aspect of the present invention provides a chromatography column comprising a hollow elongated tube filled with the aforementioned static medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of a commonly used resin bead.

[0022] Figure 2 is a schematic diagram of adsorption chromatography performed according to the present invention.

[0023] Figure 3 A schematic diagram showing that macromolecules with different adsorption characteristics take different times to leave the column.

[0024] Figure 4A Shows the size distribution of the two populations of particles obtained after sieving; and Figures 4B-4F 1 are several scanning electrographic images of porous particles according to one embodiment of the present invention.

[0025] Figure 5 is a scanning electrographic image showing the interior of a porous particle according to one embodiment of the present invention;

[0026] Figure 6 is a schematic diagram illustrating mass transport through a porous particle according to one embodiment of the present invention.

[0027] Figure 7 A flow chart showing a method for manufacturing porous particles according to one embodiment of the present invention is shown.

[0028] Figure 8 This is a pore size distribution curve of porous particles measured by mercury intrusion porosimetry.

[0029] Figure 9 is a bar graph comparing the dynamic binding capacity of a chromatography column according to an embodiment of the present invention and two conventional ion exchange columns.

[0030] Figure 10A is a bar graph comparing the recovery of hemagglutinin (HA) using a chromatography column according to an embodiment of the present invention and two conventional ion exchange columns.

[0031] Figure 10B is a bar graph comparing the DNA removal capabilities of a chromatography column according to an embodiment of the present invention and two conventional ion exchange columns.

[0032] Description of the figure number:

[0033] Still phase......2

[0034] Mobile phase... 3

[0035] Macromolecules……4. DETAILED DESCRIPTION

[0036] Unless otherwise stated, the following terms used in this specification and the scope of the patent application have the definitions given below. Please note that the singular terms "one" and "one" used in this specification and the scope of the patent application are intended to cover one and more than one of the items contained, such as at least one, at least two or at least three, and do not mean that there is only a single item contained. In addition, open conjunctions such as "including" and "having" used in the scope of the patent application are used to indicate the combination of elements or components described in the claim, and do not exclude other components or ingredients not stated in the claim. It should also be noted that the term "or" generally includes "and / or" in its meaning, unless the content clearly indicates otherwise. The terms "about" or "substantially" used in this specification and the scope of the patent application are used to modify any errors that may vary slightly, but such slight changes do not change their essence.

[0037] Figure 2 and Figure 3The adsorption chromatography method according to the present invention is shown, wherein a solid static phase 2 and a liquid mobile phase 3 are used, and the macromolecules 4 are separated by adsorptive interactions of various macromolecules 4 with the static phase 2. The adsorption chromatography may be of a type known in the relevant technical field, including but not limited to ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography and reverse phase chromatography. The term "static phase" used in this case may refer to an immobilized solid phase carrier that allows the mobile phase 3 to flow through it during the chromatography process so that the macromolecules 4 can be retained by the static phase 2. Here, the static phase 2 comprises a group of porous particles filled in a chromatography column. The term "static medium" used in this case is intended to cover porous particles in a filled state or a non-filled state. As Figure 2 As shown, the mobile phase 3 is fed from the top of the column and flows downward to the bottom of the column. Figure 3 As shown, macromolecules 4 that are more easily attracted to the stationary phase 2 will remain in the column longer, while macromolecules 4 that are less easily adsorbed by the stationary phase 2 will leave the column faster. As a result, macromolecules 4 with different adsorption characteristics can be collected separately. Adsorption chromatography in this case can be performed in a bind-and-elute mode, in which the target macromolecule is retained on the stationary phase and then eluted with an appropriate eluent, or in a flow-through mode, in which unwanted molecules and impurities are adsorbed by the stationary phase, while the target macromolecule is allowed to elute.

[0038] Figures 4B-4F and Figure 5 The porous particles according to the present invention are shown. It is obvious that each of these porous particles has many spherical giant pores stacked on each other, such as Figure 5 These macropores are interconnected and in fluid communication via connecting pores, such as Figure 5Indicated by the dashed circle. As measured under an electron microscope, these porous particles have a Feret diameter ranging from 25 to 500 microns, preferably from 25 to 300 microns, for example from 25 to 100 microns, while the spherical macropores have a diameter ranging from 3 to 10 microns. As used herein, the term "Feret diameter" refers to the longest distance between the two most distant points on the outer contour of a particle. In individual porous particles, these interconnected macropores and their interconnected pores form a continuous porous network. As measured by capillary flow porometry, the average diameter of this porous network is within the range of 0.2 to 6.0 microns, preferably from 0.5 to 3.0 microns, for example from 1.2 to 2.4 microns. As described below, the particle size of the porous particles, as well as the diameters of the macropores and porous network, can be adjusted by controlling the parameters and conditions of the porous particle manufacturing method.

[0039] According to the aforementioned electron microscopy and capillary flow porometer measurements, the porous network formed in each porous particle is large enough to allow convective flow of the mobile phase fluid through the porous network. These porous particles are also substantially free of diffusion pores, which means that, as measured by mercury intrusion porosimetry, more than 90%, preferably more than 95%, more preferably more than 98%, for example more than 99% of the complete porous particles, and the porous network formed therein, are substantially free of micropores with a diameter of less than 100 nanometers. The advantageous result is that the transport of substances through the porous network is entirely by convection, as described later in Example 5 and shown in Figure 9 As further shown in Figures 4B-4F SEM images and Figure 6 As shown in the schematic diagram, the porous network is open to the surrounding environment and provides a large specific surface area, making it easy for large molecules to access and be retained. With these structural features, the stationary phase medium disclosed in this application allows the mobile phase to flow through it at high speed while providing a high adsorption capacity for large molecules.

[0040] The particles disclosed in this case have high porosity, with macropores evenly distributed throughout the particles, ensuring high mass transfer and low backpressure during the separation process. The porosity of porous particles is defined as the percentage of pore volume relative to the total volume of the particle, which can be calculated using the following formula:

[0041] 1-[(weight of porous particles / density of continuous phase) / apparent volume of porous particles]

[0042] Porosity can also be calculated by scanning electron microscopy (SEM) cross-sectional images of the porous particles using ImageJ software (National Academy of Sciences, Bethesda, MD, USA). In one embodiment, the particles have a porosity in the range of about 70% to about 90%, preferably 74% to 90%.

[0043] If further Figures 4B-4F As shown, the porous particles of the present invention are fabricated into irregular particle configurations with rough outer surfaces. The term "irregular" as used herein means that the porous particles deviate from a spherical shape to a certain degree. According to the present invention, the degree of irregularity is expressed as the Feret aspect ratio, which is calculated as the quotient obtained by dividing the length of the major axis of a particular porous particle by the length of its minor axis. Since the Feret aspect ratio of a perfect sphere is 1, the calculated value is usually greater than 1. The irregularity of the particles is calculated using particle image analysis, which shows that the porous particles of the present invention have a Feret aspect ratio distribution ranging from 1.0 to 3.5 and a standard deviation ranging from 1.2 to 2.2. As described below, the irregular configuration of the porous particles can be achieved by mechanically grinding the porous monolith into particles, which provides several advantages. First, compared to block-shaped monoliths and spherical particles, porous particles of the same type but with an irregular configuration have a larger surface area and thus a higher adsorption capacity. Second, it can be intuitively inferred that the irregular particles in this case have a rough outer surface, making them easily interlocked when packed into a chromatography column, resulting in a packed bed with high mechanical strength that can withstand the backpressure generated during the separation process. Third, compared to spherical particles in a packed bed, the irregular particles in the packed bed have wider convection flow channels. Therefore, during the separation process, the convection flow between the irregular particles in this case will not be hindered or slowed down. In some specific examples, the average diameter of the channels between the particles ranges from 2 microns to 110 microns.

[0044] The porous particles in this embodiment are made of a cross-linked polymer material. Polymer materials suitable for use in this invention are well known in the relevant art and include, but are not limited to, polyacrylates, polymethacrylates, polyacrylamides, polystyrene, polypyrrole, polyethylene, polypropylene, polyvinyl chloride, and polysilicone. In a preferred embodiment, the porous particles are made of polymethacrylate.

[0045] With its fast kinetics, high porosity, superior mechanical properties, and low backpressure, the static media of this invention can be used to separate macromolecules with extremely large molecular sizes, including those with a hydrodynamic radius exceeding 10 nm and preferably exceeding 50 nm. These include, but are not limited to, proteins, nucleic acids, viroids, viruses, viral vectors, virus-like particles (VLPs), extracellular vesicles (EVs), and liposomes.

[0046] The present invention further contemplates a chromatography column filled with the static medium of the present invention. The chromatography column comprises a hollow, elongated tubular body for housing a packed bed. Suitable materials and configurations for the hollow, elongated tubular body are well known in the art of chromatography. In one embodiment, the tubular body is made of a material selected from the group consisting of stainless steel, titanium, quartz, glass, and a rigid plastic such as polypropylene, and is fabricated in a cylindrical, rectangular, or polygonal tubular form.

[0047] In some embodiments, the stationary medium is chemically modified to carry functional groups or ligands for adsorbing macromolecules. For example, in embodiments where the stationary medium is used as an ion exchanger, the porous particles, including the porous network formed therein, are surface-modified to carry ion-exchange functional groups, such as quaternary ammonium as a strong anion exchanger, diethylaminoethyl (DEAE) as a weak anion exchanger, sulfonyl groups as a strong cation exchanger, and carboxymethyl groups as a weak cation exchanger.

[0048] Figure 7 A flow chart of a method for manufacturing a stationary medium according to the present invention is shown, which includes step A: preparing a porous polymer monolith; step B: grinding the monolith into a first group of porous particles; and step C: sorting the first group of porous particles according to particle size to obtain a second group of porous particles with a Feret diameter of 25 microns to 500 microns.

[0049] Step A involves preparing a porous polymer monolith having the same pore characteristics as the porous particles described above. Specifically, the porous monolith is made of a cross-linked polymer material, has a porosity ranging from 70% to 90%, and is formed with numerous spherical macropores with diameters ranging from 3 to 10 microns. These spherical macropores are interconnected via connecting pores to form a porous network, and the average diameter of the porous network is within the range of 0.2 to 6.0 microns, preferably within the range of 0.5 to 3.0 microns, for example, within the range of 1.2 to 2.4 microns.

[0050] The term "monolithic column" refers to a solid porous three-dimensional structure that is not granular in nature. Methods suitable for preparing porous polymer monolithic columns are known in the relevant art.

[0051] In a preferred embodiment, step A is performed using a high internal phase emulsion template method. The general process principles of the high internal phase emulsion template method involve preparing a high internal phase emulsion (HIPE) in which an internal phase (or dispersed phase) composed of emulsified droplets is dispersed in an external phase (or continuous phase) with a volume fraction of the internal phase exceeding 74.05%. The external phase containing monomers is then polymerized, and the internal phase template is removed.

[0052] A practical approach can be found, for example, in U.S. Patent No. 11,236,184, assigned to the applicant of the present application. This approach involves vigorously stirring a continuous phase component and an immiscible dispersed phase component using a high-speed homogenizer to uniformly disperse the dispersed phase in the continuous phase, thereby forming a water-in-oil emulsion. The water-in-oil emulsion can optionally be subjected to external sedimentation to increase the volume fraction of the dispersed phase relative to the continuous phase in the emulsion, thereby forming a high-internal-phase emulsion. In this case, the continuous phase is typically the phase where the polymerization reaction occurs and may include at least one monomer and a crosslinker, and optionally an initiator and an emulsion stabilizer, while the dispersed phase may include a solvent and an electrolyte.

[0053] The at least one monomer is intended to encompass any monomers and oligomers that can form a polymer through polymerization. In a preferred embodiment, the at least one monomer comprises at least one ethylenically unsaturated monomer or acetylenically unsaturated monomer suitable for free radical polymerization, i.e., an organic monomer having a carbon-carbon double bond or triple bond, including but not limited to acrylic acid and its esters, such as hydroxyethyl acrylate; methacrylic acid and its esters, such as glycerol methacrylate (GMA), hydroxyethyl methacrylate (HEMA), and methyl methacrylate (MMA); acrylamides; methacrylamides; styrene and its derivatives, such as chloromethylstyrene, divinylbenzene (DVB), and styrene sulfonate; silanes, such as dichlorodimethylsilane; pyrroles; vinylpyridine, and combinations thereof.

[0054] The term "crosslinker" as used herein refers to an agent that forms a chemical bridge between the polymer backbones formed by the polymerization reaction of the at least one monomer. In a preferred embodiment, the "crosslinker" is a crosslinking monomer that can be co-soluble with the at least one monomer in the continuous phase and typically has multiple functional groups to form covalent bonds between the polymer backbones formed by the polymerization of the at least one monomer. Suitable crosslinkers are well known in the art and can be selected based on the type of the at least one monomer. They include, but are not limited to, oil-soluble crosslinkers such as ethylene glycol dimethacrylate (EGDMA), polyethylene glycol dimethacrylate (PEGDMA), ethylene glycol diacrylate (EGDA), triethylene glycol diacrylate (TriEGDA), and divinylbenzene (DVB); and water-soluble crosslinkers such as N,N-diallylacrylamide and N,N'-methylenebisacrylamide (MBAA). As known to those skilled in the art, the amount of crosslinking agent used is positively correlated with the mechanical strength of the resulting porous monolithic column. Specifically, the higher the degree of crosslinking, the higher the mechanical strength of the porous monolithic column. Preferably, the crosslinking agent comprises approximately 5 to 50% by weight of the continuous phase, for example, approximately 5 to 25% by weight.

[0055] The term "emulsion stabilizer" as used herein refers to a surfactant suitable for stabilizing high internal phase emulsions to prevent the merging of droplet units in the dispersed phase. The emulsion stabilizer can be added to the continuous phase or the dispersed phase before preparing the emulsion. Emulsion stabilizers suitable for use in the present invention can be nonionic surfactants, or anionic or cationic surfactants. In the specific embodiment where the high internal phase emulsion is a water-in-oil emulsion, the emulsion stabilizer preferably has a hydrophilic-lipophilic balance (HLB) of 3 to 14, more preferably an HLB of 4 to 6. In a preferred embodiment, the present invention uses a nonionic surfactant as an emulsion stabilizer. Applicable types include, but are not limited to, polyoxyethylene alkylphenols, polyoxyethylene linear alkanols, polyoxyethylene polypropylene glycols, polyoxyethylene thiols, long-chain carboxylates, alkanolamine condensates, quaternary acetylene glycols, polyoxyethylene polysiloxanes, N-alkyl pyrrolidones, fluorocarbon liquids, and alkyl polyglycosides. Specific examples of emulsion stabilizers include, but are not limited to, sorbitan monolaurate (trade name 20), Sorbitan tristearate (trade name 65), Sorbitan monooleate (trade name 80), glyceryl monooleate, polyethylene glycol 200 dioleate, polyoxyethylene-polyoxypropylene block copolymer (e.g. F-68, F-127, L-121, P-123), castor oil, glyceryl monoricinoleate, distearyldimonium chloride, and dioleyldimonium chloride.

[0056] "Initiator" refers to an agent capable of initiating polymerization and / or crosslinking reactions of at least one of the aforementioned monomers and / or crosslinking agents. Preferably, the initiator used in the present invention is a thermal initiator, i.e., an initiator capable of initiating the aforementioned polymerization and / or crosslinking reactions upon heating. The initiator can be added to the continuous phase composition or the dispersed phase composition prior to preparing the high internal phase emulsion. According to the present invention, suitable initiators for addition to the continuous phase composition include, but are not limited to, azobisisobutyronitrile (AIBN), azobisisoheptanenitrile (ABVN), azobisisovaleronitrile, 2,2-bis[4,4-di(tert-butylperoxy)cyclohexyl]propane (BPO), and lauroyl peroxide (LPO), while suitable initiators for addition to the dispersed phase composition include, but are not limited to, persulfates, such as ammonium persulfate and potassium persulfate. The high internal phase emulsion of the present invention may also contain a photoinitiator activated by ultraviolet or visible light to initiate the aforementioned polymerization and / or crosslinking reactions. A suitable photoinitiator can even replace the aforementioned thermal initiator.

[0057] The dispersed phase mainly comprises a solvent. The solvent can be any liquid that is immiscible with the continuous phase. In the specific example where the continuous phase has high hydrophobicity, the solvent includes but is not limited to water, fluorocarbon liquids (fluorocarbonliquids) and other organic solvents that are immiscible with the continuous phase. Preferably, the solvent is water. In this example, the dispersed phase can further comprise an electrolyte that can substantially dissociate into free ions in the solvent, including salts, acids and bases that are soluble in the solvent. Preferably, the electrolyte comprises alkali metal sulfates, such as potassium sulfate, and alkali metal and alkaline earth metal chlorides, such as sodium chloride, calcium chloride and magnesium chloride.

[0058] A polymerization accelerator may be added to the high internal phase emulsion. "Accelerator" refers to an agent capable of accelerating the polymerization and / or crosslinking reaction of the at least one monomer and / or crosslinker. Typical examples of accelerators include, but are not limited to, N,N,N',N'-tetramethylethylenediamine (TEMED), N,N,N',N",N"-pentamethyldiethylenetriamine (PMDTA), tris(2-dimethylamino)ethylamine, 1,1,4,7,10,10-hexamethyltriethylenetetramine, and 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane. These accelerators promote the decomposition of initiators such as ammonium persulfate into free radicals, thereby accelerating the polymerization and / or crosslinking reaction. The accelerator is preferably added in an amount of 10-100 mol% of the initiator.

[0059] The prepared HIPE is heated and exposed to light of an appropriate wavelength to allow the at least one monomer and the cross-linking agent to complete polymerization and cross-linking reactions, thereby solidifying the HIPE into a monolithic column. Subsequently, the dispersed phase and unreacted reagents are removed from the monolithic column by Soxhlet extraction, for example, using isopropanol, methanol, ethanol, tetrahydrofuran, acetone, or methyl ethyl ketone. The monolithic column can be directly dried, preferably under vacuum, to cause the droplets in the dispersed phase to break and form connecting pores. When the porous monolithic column is prepared from HIPE, the size and uniformity of the macropores in the monolithic column can be adjusted by changing the stirring rate and / or stirring temperature during the HIPE preparation process, while the size of the connecting pores and the minimum diameter of the porous network formed in the monolithic column can be adjusted by changing the volume ratio of the dispersed phase to the continuous phase.

[0060] In another preferred embodiment, step A is performed using a colloidal crystal template method, which includes allowing uniformly sized polymer nanospheres to self-assemble to produce a template having a three-dimensional ordered microstructure, then allowing a monomer-containing composition to infiltrate the interstitial spaces of the template, polymerizing the monomers to form a monolithic column, and finally removing the template from the monolithic column by Soxhlet extraction or supercritical fluid extraction. The monomer-containing composition referred to herein can have the same composition as the continuous phase composition. Possible methods of the colloidal crystal template method can be found in, for example, U.S. Patent Nos. 6,414,043 and 11,118,024.

[0061] The porous monoliths prepared using the colloidal crystal template method advantageously possess a highly regular porous structure, wherein uniformly sized macropores are arranged in a closest-packed pattern, with each macropore interconnected with adjacent macropores via 12 connecting pores. Preferably, at least 70% of the macropores in the porous monolith are arranged in a closest-packed pattern, more preferably at least 80%, and most preferably at least 90%, for example at least 95%, of the macropores. Examples of closest-packed patterns include three-dimensional hexagonal closest packing (hcp), three-dimensional face-centered cubic packing (fcc), or combinations thereof. As disclosed in U.S. Patent No. 11,118,024, the size of the macropores in the monolith can be adjusted by varying the size of the nanospheres used to generate the template. The size of the connecting pores, as well as the minimum diameter of the porous network formed in the monolith, can be amplified by increasing the contact area between the ordered nanospheres through controlled deformation.

[0062] The porous monolith obtained in step A is shaped like the reaction vessel in which the polymerization reaction took place. Typically, the porous monolith takes the form of a polymer block with a volume of less than or equal to 200 cubic centimeters. In step B, the porous monolith is subjected to mechanical milling to fragment and grind it into a first population of porous particles. The first population of porous particles has a size distribution below 1,000 microns, meaning that at least 50%, preferably at least 70%, more preferably at least 80%, and for example, at least 90% of the particles have a Feret diameter below 1,000 microns. The term "mechanical milling" as used herein may refer to the reduction of the size of a solid by friction, collision, impact, shear, or other mechanical action. Mechanical milling is typically performed using high-energy milling in a conventional milling apparatus, such as a disc mill, ball mill, rotary knife mill, double-roll mill, granulator, turbine mill, or combinations thereof. During the mechanical milling process, the first population of porous particles is imparted with an irregular shape and a rough outer surface.

[0063] In step C, the first population of porous particles obtained in step B is separated by size to produce a second population of porous particles having a Feret's diameter within the range of 25 μm to 500 μm. This means that at least 70%, preferably at least 80%, more preferably at least 90%, and for example, at least 95%, of the separated particles have a Feret's diameter within the range of 25-500 μm. In a preferred embodiment, the first population of porous particles is screened through a series of Taylor sieves to collect particles within a desired size range, such as <25 μm, 25-75 μm, 75-150 μm, and 150-500 μm. In a preferred embodiment, the screening is performed in a vibrating screen having at least three vertically stacked screens, with the upper screens having larger mesh sizes than the lower screens. The first population of porous particles can be fed into the vibrating screen from above, and after screening, a second population of porous particles having the desired size distribution can be obtained from one of the screens. In some cases, grinding balls made of a hard material, such as zirconium oxide or glass, may be added to individual screens to break the porous particles into smaller sizes, thereby improving yield. Measurements made using particle image analysis under an electron microscope revealed that the second population of porous particles had a statistically significant Feret aspect ratio distribution ranging from 1.0 to 3.5, with a standard deviation ranging from 1.2 to 2.2.

[0064] The following examples are only provided to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0065] Example 1: Preparation of porous monolithic columns

[0066] Glyceryl methacrylate (GMA; Sigma-Aldrich Corporation, USA), divinylbenzene (DVB; Sigma-Aldrich Corporation, USA) and lauroyl peroxide (LPO; Sigma-Aldrich Corporation, USA) were mixed in a ratio of 50:25:2 (w / w / w) to prepare a continuous phase composition. 6 wt% of PEG-100 was added to the total weight of the continuous phase composition. L-121. An aqueous discontinuous phase composition containing CaCl2 and tetramethylethylenediamine (TMEDA; Sigma-Aldrich Corporation, USA) dissolved in double-distilled water at a ratio of approximately 11:2:1100 (w / w / w) was prepared. The aqueous discontinuous phase composition was then added to the continuous phase composition at a ratio of 3:1 (v / v) and vigorously stirred for 5 minutes using a high-speed homogenizer (Model T25; IKA, Germany) to form a water-in-oil emulsion. The emulsion was placed in an oven (Model DENG YNG DO60). The temperature was gradually increased from room temperature to 80°C over 4 hours and maintained at 80°C for 24 hours to initiate a polymerization reaction, thereby solidifying the emulsion into a monolithic column. The monolithic column was washed by Soxhlet extraction with isopropanol to remove water and unreacted reagents. The porous monolithic column was then dried under vacuum to obtain a dry porous monolithic column. The minimum diameter of the porous network in the monolithic column was measured by capillary flow porometer (PMI Porous Materials Inc., CFP-1100AE).

[0067] Example 2: Preparation of porous particles from monolithic columns

[0068] The porous monolith prepared in Example 1 was ground in a stainless steel grinder (Huilv Technology Co., Ltd., Model NBM-200, Taiwan Province, China) to obtain a first group of porous particles with a particle size distribution below 1,000 μm. The porous particles were then screened in a vibrating sieving machine equipped with a stacked porous sieve plate (Fritz GmbH, Model Analysette 3Pro, Bahnhofstrasse, Germany), and particles with a diameter range of 25-100 μm were separated as a second group of porous particles. Figure 4A In the embodiment shown, the second group of porous particles has a particle size distribution showing a normal distribution, with a median diameter (D50) of 56 μm. Images of the porous particles were taken using a scanning electron microscope (Thermo Fisher Scientific Inc., Phenom Pro), some of which are shown in FIG. Figures 4B-4FSEM images of 45 randomly selected particles were used to calculate the irregularity of the particles, and the second population of porous particles showed a Feret aspect ratio of 1.75±0.59.

[0069] Example 3: Pore Identification of Porous Particles

[0070] The pore size distribution curve of the porous particles obtained in Example 2 was measured by mercury intrusion porosimetry according to ASTM D-4284 (2003). Figure 8 As shown, the porous network formed in the porous particles has an average diameter of 1.26 microns. Figure 8 Furthermore, it was found that over 90% of the porous network lies within the 500-2,000 nm range, with only a very small fraction having diameters below 500 nm. These results indicate that the porous particles in this invention have virtually no diffusive pores with diameters below 100 nm, and that the porous network formed within the particles is sufficiently large to allow convective transport of substances through the particles.

[0071] Example 4: Surface modification of porous particles

[0072] The porous particles prepared in Example 2 were added to a 1% aqueous solution of tetraethylenepentamine and heated at 70°C for at least 5 hours. The porous particles were filtered out and added to a 1% aqueous solution of glycidyltrimethylammonium chloride and heated at 70°C for at least 5 hours. The porous particles were washed with water to obtain a strong anion exchanger, hereinafter referred to as DuloCore. TM QA.

[0073] 1 mL of strong anion exchanger DuloCore TM QA was packed in a polypropylene chromatography column with an inner diameter of 7.4 mm.

[0074] Example 5: Dynamic Binding Capacity

[0075] The dynamic binding capacity of the chromatography column prepared in Example 4 for bovine serum albumin (BSA) was tested and the results were compared with those obtained using two commercial anion exchange columns, Capto TM Q (available from GE Healthcare Life Science, USA) includes a dextran matrix and has a particle size of 90 μm and a diffusion pore size of 50 nm, while CIMmultus TMQA (purchased from BIA Separations) is a monolithic column based on polymethyl methacrylate with a pore size of 2 μm. The mobile phase used in this example was 50 mM Tris-HCl, pH 8.5, and 1 mg / mL BSA was added to the mobile phase as the analyte. The dynamic binding capacity was determined using a KTA™ Pure chromatography system (Cytiva Sweden AB, Uppsala, Sweden). Figure 9 .

[0076] like Figure 9 As shown, Capto TM For the Q column, its dynamic binding capacity decreases significantly with increasing flow rate of the mobile phase, indicating that increasing flow rate is not conducive to the adsorption of macromolecules from the mobile phase by the dextran particles packed in the column. This seems to be attributed to the fact that the transport of substances within the particles in the conventional column is achieved only by diffusion, and the efficiency of diffusion decreases with increasing flow rate. TM As for QA, its dynamic binding capacity increases slightly with increasing flow rate, but the column manufacturer does not recommend operation at flow rates exceeding 600 cm / h. Regarding the chromatography column of Example 4, the porous particles packed therein exhibited a stable ability to adsorb BSA molecules from the mobile phase, even at ultra-high flow rates such as 2400 cm / h. In other words, the adsorption capacity of the porous particles in this case for BSA molecules is independent of the flow rate of the mobile phase, indicating that BSA molecules are transported within the porous particles by convection. This result also shows that in the chromatography column packed in this case, the transport of BSA within the particles is dominated solely by convection, indicating that the porous particles of the present invention, and the porous network formed therein, do not substantially have diffusive pores, that is, do not substantially have micropores with a diameter of less than 100 nanometers.

[0077] Example 6: Virus purification

[0078] In this example, the ion exchange chromatography column prepared in Example 4 was used in conjunction with Capto TM Q and CIMmultus TM The ability of the conventional column products such as QA to harvest human infectious avian influenza virus (H7N9) was tested according to the method proposed in the paper Tseng YF et al., Vaccine 36 (2018), p.3146-3152. The virus sample was Figures 10A-10B The flow rate indicated was used to load each column. The fraction that passed through the column was collected and used to measure virus recovery, while the host cell DNA was adsorbed by the column.

[0079] like Figure 10A As shown, CIMmultus TM The virus recovery efficiency of the QA column decreases sharply with increasing flow rate. TM The recovery of the QA column remained above 75% throughout the entire flow rate range tested. Figure 10B Display, DuloCore TM The QA column showed a nearly constant DNA removal rate, and even when the flow rate was increased to 2400 cm / h, the DNA removal rate remained high. TM Q and CIMmultus TM QA columns are not recommended for use at flow rates exceeding 600 cm / h.

Claims

1. A stationary medium for adsorption chromatography, characterized in that It includes: a plurality of porous particles made of a cross-linked polymer material, each having a Feret diameter in the range of 25 to 500 micrometers and a porosity of 70% to 90%, each of the particles forming a plurality of spherical macropores with a diameter in the range of 3 to 10 micrometers, wherein the spherical macropores are interconnected via connecting pores to form a porous network, and the average diameter of the porous network is in the range of 0.2 to 6 micrometers; and The porous particles exhibit an irregular particle morphology with a Feret aspect ratio distribution ranging from 1.0 to 3.5 and a standard deviation ranging from 1.2 to 2.

2.

2. The stationary medium according to claim 1, wherein According to mercury intrusion porosimetry, the porous particles substantially do not have micropores with a diameter less than 100 nanometers.

3. The stationary medium according to claim 2, wherein: Wherein the porous network has an average diameter in a range of 0.5 microns to 3.0 microns.

4. The stationary medium according to claim 3, wherein wherein the porous network has an average diameter in a range of 1.2 microns to 2.4 microns.

5. The stationary medium according to claim 2, wherein: The static phase medium is surface-modified to have ion exchange functional groups.

6. The stationary medium according to claim 5, wherein: The ion exchange functional group is selected from the group consisting of quaternary ammonium, diethylaminoethyl, sulfonyl and carboxymethyl.

7. The stationary medium according to claim 6, wherein: The cross-linked polymer material is selected from the group consisting of polyacrylate, polymethacrylate, polyacrylamide, polystyrene, polypyrrole, polyethylene, polypropylene, polyvinyl chloride and polysilicone.

8. The static medium according to claim 7, wherein: The cross-linked polymer material is selected from polymethacrylate.

9. The stationary medium according to claim 2, wherein: At least 70% of the spherical macropores of the porous particles are arranged in a densely packed manner.

10. A chromatography column, characterized in that The invention comprises a hollow elongated tube filled with the static medium as claimed in claim 1 .

11. A method for manufacturing the stationary medium according to claim 1, characterized in that: It includes the following steps: A. preparing a porous monolithic column made of a cross-linked polymer material and having a porosity of 70% to 90%, wherein the porous monolithic column is formed with a plurality of spherical macropores having diameters ranging from 3 μm to 10 μm, and the spherical macropores are interconnected via connecting pores to form a porous network, and the average diameter of the porous network is within the range of 0.2 μm to 6 μm; B. subjecting the porous monolith to mechanical grinding, so that the porous monolith is ground into a first population of porous particles having a particle size distribution of less than 1,000 microns; and C. sorting the first population of porous particles by particle size to obtain a second population of porous particles having a Feret diameter in the range of 25 μm to 500 μm, wherein the second population of porous particles exhibits an irregular particle configuration, has a Feret aspect ratio distribution between 1.0 and 3.5, and a standard deviation in the range of 1.2 to 2.

2.

12. The method according to claim 11, wherein The particle size sorting in step C includes screening the first group of porous particles through a series of Taylor screens.

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