Chromatography resin with composite sponge-like structure

Abstract

Provided are stationary phase media for adsorption chromatography comprising a plurality of composite microspheres made of a cross-linked polymeric material, each comprising an interior comprising multiple spherical macropores interconnected with one another via interconnecting pores to constitute an open porous network in the interior, and an outer surface comprising a plurality of openings through which the porous network of the interior is in fluid communication with the outer surface and a layer of a hydrophilic polymer covalently attached to the outer surface; methods of making the stationary phase media, and related compositions and methods for chromatographic separation of macromolecules.

Description

Docket No. 1580.00257WOCHROMATOGRAPHY RESIN WITH COMPOSITE SPONGE-LIKE STRUCTURECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Serial No. 63 / 682,885 filed Aug 14, 2024, the contents of which are hereby incorporated by reference in their entirety.FIELD OF THE INVENTION

[0002] The invention relates to resins for liquid chromatographic separations, particularly separation of macromolecules.BACKGROUND

[0003] Porous resin beads have been widely used as the stationary phase for adsorption chromatography. Typical resin beads are formed with a network of tortuous micropores having diameters from several to tens of nanometers, thus allowing low molecular weight solutes present in the mobile phase to diffuse in and out of the micropores. However, as shown in FIG.1 , the micropores of conventional resin beads reside near the outer surfaces of the beads and are not interconnected. For resins having this structure, most of the adsorbing surfaces are internal to the solutes and can only be reached via diffusion. While this structure is efficient in separating small molecules, such conventional resins perform poorly for the separation of macromolecules because the small size of the surface micropores excludes large molecules. Thus, macromolecules can only bind to the outermost surfaces of the resin beads, consequently resulting in a low binding capacity. In addition, the slow separation speeds required for such conventional resins are particularly harmful to the biomolecules that are sensitive to enzymatic degradation or other damaging conditions. Conventional resin-based chromatography for macromolecules has additional drawbacks including (1) a decrease in resolution with increases in flow rate because intra-bead diffusion is the rate determining step in the adsorption process; and (2) a high pressure drop across the chromatographic column due to the limited convective flow between the resin beads. All of these drawbacks result in reduced separation efficiency and unsatisfactory productivity in the chromatographic separation of macromolecules. Moreover, chromatographic processes which use the conventional resin beads as stational phase media need days to complete and, thus, are extremely time-consuming and cost-ineffective. The present invention provides an improved resin that addresses these and other problems as discussed in more detail below.Docket No. 1580.00257WOBRIEF SUMMARY

[0004] Provided are stationary phase media comprising a plurality of composite microspheres having improved performance in the liquid chromatographic separation of macromolecules. The stationary phase medium, also referred to herein as a “resin” includes a core sponge-like structure having an interior comprising multiple spherical macropores interconnected with one another via interconnecting pores to constitute an open porous network in the interior, and an outer surface comprising a plurality of openings through which the porous network of the interior is in fluid communication with the outer surface of the microsphere. In accordance with the present invention, the outer surface further comprises a layer of a non-ionic hydrophilic polymer covalently attached to the outer surface such that it does not obstruct the openings. The resins described here may be referred to as having a “composite” sponge-like structure.

[0005] In one aspect, provided is a stationary phase medium for adsorption chromatography comprising: a plurality of composite microspheres made of a cross-linked polymeric material, each comprising an interior comprising multiple spherical macropores interconnected with one another via interconnecting pores to constitute an open porous network in the interior, an outer surface comprising a plurality of openings through which the porous network of the interior is in communication with the outer surface, and a layer of a hydrophilic polymer covalently attached to the outer surface; wherein each of the microspheres satisfies the following Inequality (1): dpOre / dmicrosphere > (0.45 / n) (1), where dpore represents an equivalent diameter of the porous network, dmicrosphere represents a diameter of the microsphere, and n represents the number of openings on the outer surface, with n being an integer and n > 2.

[0006] The stationary phase medium may also include where the cross-linked polymeric material is selected from the group consisting of polyacrylates, polymethacrylates, polyacrylamides, polystyrenes, polypyrroles, polyethylenes, polypropylenes, polyvinyl chloride and silicones, optionally where the cross-linked polymeric material is a polymethacrylate. The stationary phase medium may also include where the composite microspheres are monodisperse and have a porosity ranging from 70% to 90%. The stationary phase medium may also include where the composite microspheres have a dpore of greater than 150 nm, greater than 300 nm, or greater than 500 nm. The stationary phase medium may also include where the composite microspheres have a dmicrosphere of less than 500 microns, less than 300 microns, or less than 200 microns. The stationary phase medium may also include where the composite microspheres have a diameter of 10-100 microns, optionally 30-50 microns, and a pore size of 0.5-Docket No. 1580.00257WO2.5 microns, optionally 1-2 microns. The stationary phase medium may also include where the hydrophilic polymer is a non-ionic hydrophilic polymer, optionally where the non-ionic hydrophilic polymer is a polyethylene, a polymethyl acrylate, a polyethylene glycol, or a polysaccharide. The stationary phase medium may also include where the non-ionic hydrophilic polymer is a polyethylene glycol copolymer, optionally an epichlorohydrin-glycerol copolymer. The stationary phase medium may also include where the outer surface is functionalized with epoxy groups to provide an epoxy content of from 10-50 pmol / g for the composite microspheres of the stationary phase medium. The stationary phase medium may also include where the outer surface is modified with a surface functional group. The stationary phase medium may also include where the surface functional group is an ionic group, a hydrophobic group, a reactive group, a mixed mode group, an affinity ligand, or a combination of any of the foregoing. The stationary phase medium may also include where the surface functional group includes an ionic group selected from the group consisting of a quaternary amine, diethylaminoethyl, sulfonyl and carboxymethyl. The stationary phase medium may also include where the surface functional group includes a hydrophobic group selected from the group consisting of an alkyl and an aryl, optionally where the hydrophobic group is selected from a C4-C18 alkyl. The stationary phase medium may also include where the surface functional group includes a reactive group selected from the group consisting of epoxy, aldehyde and succinimide ester group. The stationary phase medium may also include where the surface functional group includes a mixed mode group which includes a hydrophobic group selected from the group consisting of an alkyl and an aryl and an ionic group selected from the group consisting of a quaternary amine, diethylaminoethyl, sulfonyl and carboxymethyl. The stationary phase medium may also include where the surface functional group includes an affinity ligand selected from Protein A, Protein G, oligo dT, an affinity ligand that binds to a virus such as an adeno-associated virus (AAV) or a lentivirus, an affinity ligand that binds a nucleic acid, such as double stranded RNA or DNA, and an affinity ligand that binds exosomes. Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.

[0007] Also provided are methods for producing the stationary phase medium as described herein. In an aspect, the method comprises adding a hydrophilic polymer to a mixed solution of water, an alcohol and a polyether to form a hydrophilic polymer solution with a polymer concentration of 0.1-10% (w / v); dispersing an amount of porous microspheres in theDocket No. 1580.00257WO hydrophilic polymer solution to form a microsphere solution, wherein the porous microspheres have an interior comprising multiple spherical macropores interconnected with one another via interconnecting pores to constitute an open porous network in the interior, and an outer surface comprising a plurality of openings through which the porous network of the interior is in fluid communication with the outer surface; continuously mixing the microsphere solution at a temperature of from 15-80 °C for 0.5-24 hours; following removal of the liquid phase, adding a 1-5 M aqueous potassium hydroxide solution to form a second microsphere solution; and continuously mixing the second microsphere solution at 15-80 °C for another 0.5-24 hours to obtain the stationary phase medium.

[0008] The method may also include where the alcohol is isopropanol, the polyether is a polyalkylene glycol and the molar ratio of water: alcohol: poly ether is 2:1 :5. The method may also include where the hydrophilic polymer is a polyethylene, polymethyl acrylate, polyethylene glycol, or polysaccharide. The method may also include where the hydrophilic polymer is a polyethylene glycol copolymer, optionally an epichlorohydrin-glycerol copolymer. The method may also include where the concentration of the hydrophilic polymer is less than 3% (w / v) or less than 1% (w / v). The method may also include where the composite microspheres forming the stationary phase medium are functionalized with epoxy groups by contacting a solution of the composite microspheres in deionized water or an aprotic organic solvent with an epoxy-functionalizing agent, optionally where the epoxy-functionalizing agent is a mono-epoxy reagent, such as epichlorohydrin or a di-epoxy reagent, such as butanediol diglycidyl ether.

[0009] In accordance with aspects of the foregoing methods, the porous microspheres may be formed by a process comprising the steps of: A) in the presence of a polymerization initiator and an emulsion stabilizer, emulsifying a continuous phase composition comprising at least one monomer and a crosslinking agent with a dispersed phase composition comprising a solvent to obtain a first emulsion comprising a continuous phase and a dispersed phase dispersed in the continuous phase; B) mixing the first emulsion with a third phase that is immiscible with the first emulsion by applying shear force using a shear device to form a first macro-drop emulsion dispersed in the third phase, and then micronizing the first macro-drop emulsion with a droplet generating device to disperse the first macro-drop emulsion uniformly in the third phase, thereby obtaining a second emulsion containing the third phase and a plurality of monodisperse, high internal phase emulsion droplets dispersed in the third phase; and C)Docket No. 1580.00257WO curing the continuous phase and removing the dispersed phase and the third phase to obtain the stationary phase medium in form of porous microspheres; wherein each of the porous microspheres is formed in its interior with multiple spherical macropores interconnected with one another via interconnecting pores to constitute an open porous network, and formed on its outer surface with multiple openings through which the porous network is in fluid communication with the ambient; and wherein each of the porous microspheres satisfies the following Inequality (1): dpOre / dmicrosphere > (0.45 / n) (1), where dpore represents an equivalent diameter of the porous network, dmicrosphere represents a diameter of the porous microsphere, and n represents the number of openings on the outer surface, with n being an integer and n > 2.

[0010] The method may also include where the step of forming the first macro-drop emulsion dispersed in the third phase includes applying shear force with a mechanical stirring device or a three-dimensional aperture array. The method may also include where the droplet generating device is selected from a sieve plate perforated with narrow channels and a three-dimensional aperture array. The method may also include further includes a step D, subsequent to the step C, of sieving the porous microspheres obtained in step C through one or more Taylor screens to exclude oversized, undersized, or broken microspheres. Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.

[0011] Also provided is a stationary phase medium for adsorption chromatography produced by the methods described herein.

[0012] Also provided is a chromatographic column, which includes a hollow tubular body packed with the stationary phase medium described herein, and which is equipped with at least one fluid inlet port and at least one fluid outlet port. The chromatographic column may also include where in operation, the column can be operated at ultra-high flow rates of greater than 1000 cm / hr, greater than 1500 cm / hr, or greater than 2000 cm / hr, while maintaining low backpressure, such as less than 0.4 MPa, less than 0.6 MPa, or less than 1.0 MPa.

[0013] Also provided is a method of performing a chromatographic separation of a macromolecule, where the method includes applying a solution comprising the macromolecule to a column packed with a stationary phase medium as described herein and operating the column at a flow rate of greater than 1000 cm / hr, greater than 1500 cm / hr, or greater than 2000 cm / hr. Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.Docket No. 1580.00257WOBRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 is a schematic illustration of a conventional resin bead outside the scope of the present invention showing micropores near the outer surface of the bead and a lack of interconnected pores.

[0015] FIG. 2 illustrates a conventional adsorption chromatographic column which includes a hollow elongated tube with covers at the top and bottom openings, allowing a liquid mobile phase to flow through the column from top to bottom.

[0016] FIG. 3A shows the backpressure profiles of two representative conventional anion exchange column, CIMmultus™ QA column (BIA Separations, Slovenia) filled with poly(methyl methacrylate)-based monolithic material with a pore size of 2 microns, diamonds; and Capto™ Q column (Danaher Corporation, USA) packed with porous agarose beads which are formed on outer surfaces with non-interconnected diffusive micropores, each having a diameter of approximately 20-50 nanometers and a single opening to the ambient, triangles.

[0017] FIG. 3B shows the dynamic binding capacity of the two representative conventional anion exchange columns discussed in FIG. 3 A (CIMmultus™ QA, filled with a PMMA-based monolithic material, diamonds; and Capto™ Q packed with porous agarose beads which are formed on outer surfaces with non-interconnected diffusive micropores, each having a diameter of approximately 20-50 nanometers, triangles) with thyroglobulin (TGY) serving as the reference molecule.

[0018] FIG. 4 illustrates the structure of porous microspheres having a sponge-like structure, also referred to herein as the monomer-derived sponge-like microspheres.

[0019] FIG. 5 illustrates column flow rate and backpressure for columns packed with a stationary phase medium comprising either porous microspheres having a sponge-like structure, also referred to herein as the monomer-derived sponge-like structure (circles), or the composite sponge-like structure of the present invention (open squares).

[0020] FIG. 6 is a micrograph (SEM) showing a smooth outer layer of a composite spongelike structure described herein having a relatively thick coating where the pores are fully filled and encapsulated by the smooth structure. This visually exemplifies the smooth structure, but is not representative of the composite microspheres that are utilized for chromatography resins. The much thinner coating, on the order of nanometer thickness, of such composite microspheres would not be visible with SEM.Docket No. 1580.00257WO

[0021] FIG. 7 is a schematic illustrating the differences in surface texture between the monomer-derived sponge-like structure and the composite sponge-like structure of the present invention.

[0022] FIG. 8 shows line graphs where the x-axis represents the volume of mobile phase and the y-axis represents UV absorbance or electrical conductivity indicative of detected molecular signals for columns packed with microspheres of different pore sizes, 1.6 pm, 1.0 pm, and 50 nm.

[0023] FIG. 9 illustrates methods for composite formation.

[0024] FIG. 10 is a schematic diagram of the composite sponge-like structure A+B.

[0025] FIG. 11 is a bar graph showing total protein adsorption for a column packed with either porous microspheres having a sponge-like structure, (monomer-derived sponge-like structure), or composite microspheres having a composite sponge-like structure A+B (composite sponge-like structure A+B).

[0026] FIG. 12 is a schematic diagram of the composite sponge-like structure A+B+C.

[0027] FIG. 13 is a schematic diagram of the composite sponge-like structure A+B+C+D.

[0028] FIG. 14 is a combination bar graph and line graph showing dynamic binding capacity (DBC) and zeta potential for representative resins.

[0029] FIG. 15 is a schematic diagram of the composite sponge-like structure A+B+C+D+E.

[0030] FIG. 16 is a bar graph showing lysozyme static binding capacity (SBC, mg / ml) for representative resins prepared with DEAE structures (A+B+C+D).

[0031] FIG. 17 is a bar graph showing polyA binding capacity for representative resins prepared with a A+B+C+D+E structure.DETAILED DESCRIPTION

[0032] Adsorption chromatography is a type of fluid chromatography for separation of a component in a mixture by selective adsorption from a mobile phase onto a solid stationary phase. The structure of conventional resin beads is illustrated in FIG. 1. As illustrated in the figure, macromolecules are too large to enter into the surface pores of conventional resins and as a consequence bind only to the surface, resulting in low binding capacity for macromolecules. FIG. 2 illustrates a conventional adsorption chromatographic column, which includes a hollow elongated tube with covers at the top and bottom openings, allowing a liquidDocket No. 1580.00257WO mobile phase to flow through the column from top to bottom. The interior of the column is packed with porous material, which may be in the form of block-shaped porous monoliths, micro particulates, or microspheres. The porous material packed inside a conventional column may exhibit an adsorption effect on one or more substances, with a maximum binding capacity. Once the binding capacity reaches saturation, no further adsorption is possible. The amount of a specific sample (mg) that can be taken up by the porous material per unit volume (mL) of the porous material is referred to as the binding capacity (expressed in mg / mL). To measure the binding capacity, a mobile phase containing the specific sample is typically loaded through the top opening of the column and allowed to flow through the column, allowing the sample to be adsorbed by the surface functionality of the porous material. The flow-through mobile phase exiting from the bottom of the column can be monitored, for example using a UV detector. Initially, the flow-through mobile phase will exhibit low UV absorbance at the bottom outlet, corresponding to a low sample concentration. When the binding capacity reaches its maximum limit, an increase in the UV absorbance of the sample is observed, indicating that the porous material packed inside the column has reached its maximum binding capacity, leading to an increase in the sample concentration in the mobile phase at the outlet. Once the binding capacity reaches its maximum limit, the UV absorbance of the sample will eventually reach its maximum (QB 100). By analyzing the rising curve of the absorbance, the amount of the sample loaded at which the UV absorbance at the outlet reaches 10% of its maximum value (QB 10) is referred to as the dynamic binding capacity (DBC, usually expressed in mg / mL). The liquid mobile phase flows through the column at a fixed or variable flow rate (v, usually expressed in cm / h), and the pressure difference generated between the top and bottom openings of the column during the flowing is called backpressure (A / ?, usually expressed in MPa).

[0033] There are two important requirements in the application of chromatographic purification. Firstly, it is required that the backpressure generated during the purification process is sufficiently low, or alternatively that the mechanical strength of the material packed in the column is sufficiently high to withstand high backpressure. A low backpressure helps avoid exceeding the pressure limits of the chromatographic column and the material packed therewithin, thereby improving the working flow rate and enhancing the purification efficiency. Secondly, it is desired that the dynamic binding capacity (DBC) of the material packed within the chromatographic column does not decrease considerably during the purification as the flowDocket No. 1580.00257WO rate of the mobile phase increases. That is to say, the DBC can be maintained while increasing the flow rate, thus allowing for high throughput without compromising the binding capacity.

[0034] Efforts have been made in the art to address the requirements above. FIG. 3A shows the backpressure profiles of two representative conventional anion exchange columns. CIMmultus™ QA column (purchased from BIA Separations, Slovenia) is filled with poly(methyl methacrylate)-based monolithic material with a pore size of 2 microns, whereas Capto™ Q column (purchased from Danaher Corporation, USA) is packed with porous agarose beads which are formed on outer surfaces with non- interconnected diffusive micropores, each having a diameter of approximately 20-50 nanometers and a single opening to the ambient. FIG. 3A demonstrates that CIMmultus™ QA column filled with block-shaped monolithic material (denoted by diamonds) generates significant backpressure as the mobile phase passes through the column, and the backpressure linearly increases with the flow rate (with a slope of 1.27x l0'3MPa hr cm'1). This is disadvantageous for operation stability. On the other hand, Capto™ Q column packed with porous agarose beads (denoted by triangles) exhibits a slower increase in backpressure with increasing flow rate (with a slope of 8.3x1 O'5MPa hr cm'1), indicative of better operation stability. However, the porous agarose beads are limited by their poor mechanical strength and tend to undergo structural deformation and pore collapse under increased pressure, resulting in a problem of column blockage. As shown in FIG. 3 A, when the flow rate exceeds 500 cm / hr, the backpressure of Capto™ Q column rapidly increases, significantly limiting its usage and efficiency (please refer to, for example, Nweke, M.C. et al., Mechanical characterisation of agarose-based chromatography resins for biopharmaceutical manufacture, J. Chromatogr. A, (2017), 1530: 129-137). Since these conventional chromatographic columns tend to suffer from irreversible damages caused by the increased backpressure, their product instructions recommend operating at flow rates below the highest recommended value (600 cm / hr).

[0035] FIG. 3B shows the dynamic binding capacity (DBC) of the aforementioned two representative conventional anion exchange columns for molecular separation, with thyroglobulin (TGY) serving as the reference molecule. Regarding Capto™ Q column packed with porous agarose beads (denoted by the symbol A), when a mobile phase containing TGY flows through the beads in the column, the mass transfer of TGY primarily occurs through diffusion. In other words, TGY diffuses into the micro-porous structures of the beads from the surfaces of the beads, where it gets adsorbed. However, since TGY has a larger molecular sizeDocket No. 1580.00257WO compared to the diameters of the diffusive pores formed on the bead surfaces, its diffusion into the micro-porous structures is considerably hindered (resulting in a DBC for TGY of 1 -2 mg / mL). Therefore, columns of this type are not suitable for purification of biomolecules. On the other hand, regarding CIMmultus™ QA column packed with block-shaped monolithic material (denoted by the symbol ♦), it has relatively large internal pores (approximately 2 microns in diameter). Owing to the monolithic interior structure of the column which allows the mobile phase to flow entirely therewithin, TGY is transported by convection and directly adsorbed onto the internal surface of the monolith. This direct contact with the internal surface of the monolithic structure makes the adsorption of molecules more efficient compared to diffusion, resulting in a significantly higher binding capacity for molecules than the bead-type columns (with a DBC for TGY of 22 mg / mL).

[0036] The present invention provides resins with improved performance in the liquid chromatographic separation of macromolecules. The resins described here include a core sponge-like structure having an interior comprising multiple spherical macropores interconnected with one another via interconnecting pores to constitute an open porous network in the interior, and an outer surface comprising a plurality of openings through which the porous network of the interior is in fluid communication with the outer surface. In accordance with the present invention, the outer surface further comprises a layer of a non-ionic hydrophilic polymer covalently attached to the outer surface such that it does not obstruct the openings. Accordingly, the resins described here are referred to as having a “composite” sponge-like structure, which includes the core structure described in US 20240033713 with modifications of the outer surface as described herein. Methods for making the core spongelike structure are provided in US 20240033713, which is hereby incorporated herein by reference in its entirety. The core sponge-like structure may be spherical in form, as described in US 20240033713, or formed as a microparticulate as described in US 20230338924, which is hereby incorporated herein by reference in its entirety. Resins having a sponge-like structure with a spherical shape are described herein for illustration.

[0037] The core monomer-derived sponge-like structure is formed of a cross-linked polymeric material. Suitable polymeric materials include, but are not limited to, polyacrylates, polymethacrylates, polyacrylamides, polystyrenes, polypyrroles, polyethylenes, polypropylenes, polyvinyl chloride and silicones. In one aspect, the cross-linked polymericDocket No. 1580.00257WO material is a polymethacrylate. In aspects where the core monomer-derived sponge-like structure is in the form of a microsphere, the microsphere satisfies the following Inequality (1): dpore / dmicrosphere (0.45 / n) (1) where dpore represents an equivalent diameter of the porous network, dmicrosphere represents a diameter of the porous microsphere, and n represents the number of openings on the outer surface, with n being an integer and n > 2.

[0038] Methods for making porous microspheres having the monomer-derived sponge-like structure are described in US 20240033713. Briefly, the fabrication of the porous microspheres involves emulsifying two immiscible phases to obtain a first emulsion, dispersing the first emulsion in a third phase by, for example, passing the first emulsion through a perforated sieve plate to obtain uniformly sized, spherical-shaped, high internal phase emulsion (HIPE) droplets suspended in the third phase, and then curing the emulsion droplets to produce the porous microspheres. The method comprises Step A: preparing the first emulsion; Step B: dispersing the first emulsion in the third phase to obtain a second emulsion containing monodisperse HIPE droplets; and Step C: curing the HIPE droplets to obtain porous microspheres.

[0039] Step A involves preparing the first emulsion. The term “emulsion” is used herein to refer to a mixture of a continuous phase (i.e., an external phase) and a dispersed phase (i.e., an internal phase) immiscible with the continuous phase. As used herein, the term “continuous phase” may refer to a phase constituted by a single composition which is contiguous throughout the emulsion. The term “dispersed phase” may refer to a phase constituted by mutually separated units of a composition dispersed in the continuous phase, with each and every unit in the dispersed phase being surrounded by the continuous phase. The dispersed phase mainly includes a solvent. The solvent can be any liquid that is immiscible with the continuous phase. In the embodiment where the continuous phase is highly hydrophobic, the solvent may include, but be not limited to water, fluorocarbon liquids and other organic solvents that are immiscible with the continuous phase. Preferably, the solvent is water. According to the invention, the continuous phase is usually the one in which polymerization occurs and may comprise at least one monomer, a crosslinking agent, and optionally an initiator and an emulsion stabilizer, whereas the dispersed phase may comprise a solvent and an electrolyte. In preferred embodiments, the first emulsion is a water-in-oil emulsion.Docket No. 1580.00257WO

[0040] The at least one monomer is meant to encompass any monomers and oligomers that are capable of forming a polymer through polymerization. In one preferred embodiment, the at least one monomer comprises at least one ethylenically unsaturated monomer or acetylenically unsaturated monomer suitable for free radical polymerization, namely, organic monomers with carbon-to-carbon double bonds or triple bonds, which include but are not limited to acrylic acids and the esters thereof, such as hydroxyethyl acrylate; methacrylic acids and the esters thereof, such as glycerol methacrylate (GMA), hydroxyethyl methacrylate (HEMA), methyl methacrylate (MMA); acrylamides; methacrylamides; styrene and its derivatives, such as chloromethylstyrene, divinylbenzene (DVB), styrene sulfonate; silanes, such as dichlorodimethylsilane; pyrroles; vinyl pyridine; and combinations thereof.

[0041] The term “crosslinking agent” as used therein may refer to a reagent that chemically bridges the polymer chains formed by polymerization of the at least one monomer. In preferred embodiments, the “crosslinking agent” is a crosslinking monomer which can be dissolved along with the at least one monomer in the continuous phase and usually has multiple functional groups to enable the formation of covalent bonds between the polymer chains of the at least one monomer. Suitable crosslinking agents are well known in the art and can be selected depending upon the type of the at least one monomer, which include but are not limited to oil-soluble crosslinking agents, such as ethylene glycol dimethacrylate (EGDMA), polyethylene glycol dimethacrylate (PEGDMA), ethylene glycol diacrylate (EGDA), triethylene glycol diacrylate (TriEGDA), divinylbenzene (DVB); and water-soluble crosslinking agents, such as N,N- diallylacrylamide, N,N'-methylenebisacrylamide (MBAA). As known to those having ordinary skill in the art, the amount of the crosslinking agent used is positively correlated to the mechanical strength of the porous microspheres produced, that is, the higher the degree of crosslinking, the higher the mechanical strength of the porous microspheres. Preferably, the crosslinking agent is present in an amount about 5 to 50% by weight, such as in an amount about 5 to 25% by weight, of the continuous phase.

[0042] The term “emulsion stabilizer” as used herein may refer to a surface-active agent suitable for stabilizing a HIPE and preventing the mutually separated units of the dispersed phase of the emulsion from coalescence. The emulsion stabilizer can be added to the continuous phase composition or the dispersed phase composition prior to preparing the emulsion. The emulsion stabilizer suitable for use herein may be a nonionic surfactant, or an anionic or a cationic surfactant. In the embodiment where the emulsion is a water-in-oilDocket No. 1580.00257WO emulsion, the emulsion stabilizer preferably has a hydrophilic-lipophilic balance (HLB) of 3 to 14, and more preferably has a HLB of 4 to 6. In preferred embodiments, a non-ionic surfactant is used herein as the emulsion stabilizer, and the useful types thereof include, but are not limited to polyoxyethylated alkylphenols, polyoxy ethylated alkanols, polyoxyethylated polypropylene glycols, polyoxyethylated mercaptans, long-chain carboxylic acid esters, alkanolamine condensates, quaternary acetylenic glycols, polyoxyethylene polysiloxanes, N- alkylpyrrolidones, fluorocarbon liquids and alkyl polyglycosides. Specific examples of the emulsion stabilizer include, but are not limited to sorbitan monolaurate (trade name Span®20), sorbitan tristearate (trade name Span®65), sorbitan monooleate (trade name Span®80), glycerol monooleate, polyethylene glycol 200 dioleate, polyoxyethylene-polyoxypropylene block copolymers (such as Pluronic® F-68, Pluronic® F-127, Pluronic® L-121, Pluronic® P- 123), castor oil, mono-ricinoleic acid glyceride, distearyl dimethyl ammonium chloride, and dioleyl dimethyl ammonium chloride.

[0043] The term “initiator” may refer to a reagent capable of initiating polymerization and / or crosslinking reaction of the at least one monomer and / or the crosslinking agent. Preferably, the initiator used herein is a thermal initiator which is an initiator capable of initiating the polymerization and / or crosslinking reaction upon receiving heat. The initiator can be added to the continuous phase composition or the dispersed phase composition before preparing the HIPE. According to the invention, the initiators which may be added to the continuous phase composition include, but are not limited to azobisisobutyronitrile (AIBN), azobisisoheptonitrile (ABVN), azobisisovaleronitrile, 2,2-bis[4,4-bis(tert-butylperoxy)cyclohexyl]propane, benzyl peroxide (BPO) and lauroyl peroxide (LPO), whereas the initiators which may be added to the dispersed phase composition include, but are not limited to persulfates, such as ammonium persulfate and potassium persulfate. The emulsion herein may further include a photoinitiator which can be activated by ultraviolet light or visible light to initiate the polymerization and / or crosslinking reaction and, alternatively, a suitable photoinitiator may be used to replace the thermal initiator.

[0044] The process of obtaining the first emulsion through emulsification involves uniformly mixing the at least one monomer with the crosslinking agent to form a continuous phase composition, and uniformly mixing the solvent with the electrolyte to form a dispersed phase composition. Subsequently, the continuous phase composition and the dispersed phase composition are mixed with agitation in a predetermined ratio, such as in a volume ratio of 5:95Docket No. 1580.00257WO to 40:60, so as to make the dispersed phase evenly dispersed in the continuous phase. In one embodiment, the dispersed phase composition may be slowly added dropwise to the continuous phase composition, while being vigorously agitated to form the emulsion. In an alternative and preferred embodiment, an entire batch of the dispersed phase composition is directly added to the continuous phase composition at one time, while being vigorously agitated to form the emulsion. In the preferred embodiment where the dispersed phase composition is added in a single batch, a high-speed homogenizer may be used to vigorously stir and, therefore, apply a high shear force to the emulsion, so that the separated units of the dispersed phase could have a uniform size. As well known in the art, the size and uniformity of the separated units of the dispersed phase may be tuned by adjusting parameters such as the volume fraction of the dispersed phase relative to the continuous phase, the feeding rate of the dispersed phase composition, the type and concentration of the emulsion stabilizer, and the agitation rate and agitation temperature.

[0045] In one embodiment, the first emulsion obtained through the emulsification step above is a high internal phase emulsion (HIPE). The term “high internal phase emulsion”, or abbreviated as “HIPE”, is used herein to refer to an emulsion, in which the internal phase has a volume fraction of more than 74.05% (v / v). According to the invention, in Step B, the first emulsion is mixed with the third phase and then passed through a droplet generating device to uniformly disperse the first emulsion in the third phase, resulting in a second emulsion containing monodisperse HIPE droplets dispersed in the third phase.

[0046] As used herein, the term “third phase” may refer to a phase in which the HIPE can be stably dispersed and is immiscible with the continuous phase of the HIPE. The third phase primarily comprises a solvent, which may include but be not limited to water, fluorocarbon liquids, and other organic solvents that are immiscible with the continuous phase. Preferably, the solvent is water. In preferred embodiments, the second emulsion is a water-in-oil-in-water emulsion. The third phase may further comprise an electrolyte which can substantially dissociate free ions in the solvent and may include salts, acids, and bases that are soluble in the solvent. Preferably, the electrolyte may be an alkali metal sulfate, such as potassium sulfate, or an alkali metal or alkaline-earth metal chloride salt, such as sodium chloride, calcium chloride, and magnesium chloride. The third phase may further comprise an emulsion stabilizer as defined above.Docket No. 1580.00257WO

[0047] In preferred embodiments, the first emulsion may be added to the third phase, and the mixture thus obtained may be subjected to shear force generated by a shear device to form a first macro-drop emulsion dispersed in the third phase. The shear device may be selected from a mechanical stirring device or a three-dimensional aperture array. Afterwards, the first macrodrop emulsion is further micronized and uniformly dispersed in the third phase using a droplet generating device to obtain a second emulsion containing the third phase and a plurality of monodisperse, high internal phase emulsion droplets dispersed in the third phase. The droplet generating device is adapted to generate a large number of monodisperse HIPE droplets, which may be a sieve plate perforated with narrow channels (whose configuration is not limited to straight, approximate straight, smooth curve, or approximate smooth curve) or, alternatively, a three-dimensional aperture array. The sieve plate perforated with channels may be made of any inert material that does not undergo physical and chemical reactions with the first emulsion and the second emulsion, and examples of the inert material may include carbon fiber, ceramics, glass, quartz, silicon wafers, plastics, e.g., polyvinyl chloride (PVC), polyoxymethylene (POM), polycarbonate (PC), polyphenylene oxide (PPO), PA6 / 66 nylon, polycarbonate (PC) / acrylonitrile butadiene styrene (ABS) composites, polyethylene terephthalate (PET), polyetherimide (PEI), polymethyl methacrylate (PMMA), polyphenylene sulfide (PPS), polyethylene (PE), polypropylene (PP), polystyrene (PS) and ethylene vinyl acetate (EVA), and metal material, e.g., stainless steel, Ti, Al and Al-Mg alloys.

[0048] The HIPE droplets dispersed in the third phase will spontaneously form into spherical shape due to their inherent cohesive force. The size of the HIPE droplets may be adjusted by selecting the channel size of the droplet generating device.

[0049] In Step C, the HIPE droplets may be further subjected to heat, and / or exposed to light with an appropriate wavelength, or added with a polymerization promoter, so as to allow the at least one monomer and / or the crosslinking agent to complete polymerization and / or crosslinking reaction, whereby the HIPE droplets are cured into a shaped mass. The term “cure” or “curing” as used herein may refer to a process of converting the HIPE droplets into a structure with a stable free-standing configuration. The dispersed phase and the third phase are removed afterwards from the cured HIPE droplets, thus forming a stationary phase medium in form of porous microspheres. In the embodiment where the first emulsion is a water-in-oil emulsion, the cured HIPE droplets may be dried directly, preferably dried under vacuum, to thereby facilitate rupturing the mutually separated units of the dispersed phase to generate theDocket No. 1580.00257WO interconnecting pores. The size and uniformity of the macropores in the porous microspheres can be adjusted by changing the agitation speed and / or the agitation temperature during the preparation of the first emulsion, whereas the size of the interconnecting pores and, therefore, the equivalent diameter of the porous networks formed in the porous microspheres, can be modified by altering the volume ratio of the dispersed phase to the continuous phase in the emulsion.

[0050] In a preferred embodiment, the porous microspheres obtained in step C are sieved through one or more Taylor screens to exclude oversized, undersized, or broken microspheres, and the microspheres within desired size ranges are collected.

[0051] Monomer-derived sponge-like structures in the form of microspheres may be referred to herein simply as “porous microspheres” or “porous microspheres having a sponge-like structure” or “microspheres having a sponge-like structure”, as differentiated from the composite microspheres of the present invention.

[0052] In accordance with the present invention, composite microspheres comprise a covalently attached layer of a hydrophilic polymer which provides advantageous features, including the ability to withstand ultra-high flow rates such as >1000 cm / hr, >1500 cm / hr, or even >2000 cm / hr, while maintaining low backpressure values, such as <0.4 MPa, <0.6 MPa, or <1.0 MPa, thereby providing improved efficiency and reduced costs.

[0053] FIG. 5 shows a column flow rate and backpressure chart obtained utilizing porous microspheres having a sponge-like structure, as disclosed in US 20240033713, and composite microspheres having a composite sponge-like structure provided herein. The data show that the composite microspheres achieve lower backpressure under high flow conditions compared to the microspheres having a sponge-like structure. Thus, the data show that the composite sponge-like structure described herein can withstand a considerably increased flow rate under the same backpressure levels, thereby meeting industrial production demands more efficiently.

[0054] FIG. 7 is a schematic illustration showing one of the advantages of the composite sponge-like structure provided herein. The left diagram shows that the monomer-derived sponge-like structure has a relatively uneven surface at the microscopic level. As such, when a fluid passes by or through this sponge-like structure, it will experience greater resistance, which will in turn limit the flow rate and backpressure performance. To address this, the composite sponge-like structure herein is formed with a smooth layer achieved by covalent attachment of a hydrophilic polymer to the outer surface of the monomer-derived sponge-likeDocket No. 1580.00257WO structure. The resulting composite sponge-like structure described herein has a reduced surface roughness and an effectively decreased fluid resistance, thereby achieving a superior flow rate and backpressure performance compared to the unmodified monomer-derived sponge-like structure. The advantages of the composite structure in achieving lower backpressure under high flow conditions are illustrated in FIG. 5, discussed above.

[0055] The composite sponge-like structure herein can take any shape and may be packed into columns for various applications. For example, it may be configured in form of a spherical sponge-like structure that conforms to the dimensions of the columns. In a preferred embodiment, it is in the form of a sphere or a near-spherical particle (which may be referred to herein as a composite microsphere), with a preferred diameter of 30-50 pm and a pore size of 0.5-2.5 pm, more preferably a pore size of 1-2 pm. The specific surface area thereof is 2- 20 cm3 / g. The definitions and measurement methods for particle diameter and pore size are discussed in US 20240033713. In brief, the particle size distribution of the composite spongelike structure may be measured using conventional laser scattering or diffraction techniques, such as using a laser diffraction particle size analyzer to measure the scattered laser light from microspheres suspended in a liquid phase. The pore size of the composite sponge-like structure may be determined by commonly used porosimetry techniques, including but not limited to mercury intrusion porosimetry, capillary flow porometry, and electron microscopy.

[0056] The thickness of the smooth structure can be altered by adjusting the composite formation process to range from several nanometers to up to about 500 nanometers. This is illustrated by FIG. 6 which shows a scanning electron microscopy (SEM) image of a composite microsphere having a relatively thick coating of hydrophilic polymer where the hydrophilic polymer forming the smooth structure has penetrated the pores. This is substantially thicker than that which would be utilized as a chromatography resin, which would generally be in the range of from about 2-200 nm thickness, and not visible with SEM. Unmodified porous microspheres, also referred to as monomer-derived microspheres, are visible at the lower right and left edges of the photograph in FIG. 6.

[0057] The composite microspheres described herein for use as a chromatography resin will generally include a thin hydrophilic layer of only from about 2-200 nanometer (nm) thickness so as not to obstruct the surface pores of the composite microstructure. This structure would not be visible under SEM. For example, in aspects the composite microspheres include a hydrophilic layer of from about 2 to 200 nm, or 2 to 100 nm, or 2-50 nm, or preferably fromDocket No. 1580.00257WO about 2-25 nm thickness; or in other aspects the composite microspheres include a hydrophilic layer having a thickness of about 5 nm, 10 nm, 20 nm, 50 nm, 75 nm, or 100 nm. In accordance with aspects of the composite microspheres described herein for use in chromatography, the pores may contain the hydrophilic polymer forming the smooth structure but the pores are not obstructed. The thickness of the hydrophilic polymer layer will be less than or equal to 5%, 3%, or 2% of the unmodified porous microsphere pore size. For example, if the unmodified porous microsphere has a pore size of 1 micron, then the composite microspheres formed therefrom according to the present invention would have a pore size of between 0.9 micron and 1 micron. Thus, the hydrophilic polymer forming the smooth structure may penetrate the pores of the outer surface of the composite microsphere without obstructing the pores.

[0058] When the composite sponge-like microspheres described herein are used in adsorption chromatography, they are packed in a chromatographic column, with each being formed with interconnected macropores to constitute an open porous network. The porous network provides an extremely large specific surface area as an adsorbing surface, where molecules can easily approach and adhere. It is more important to note that the composite sponge-like microspheres herein possess a characteristic ratio of the diameter of the internal porous network to the particle size of the microspheres, and the porous network is in communication with the ambient through multiple openings, such that molecules are transported through the internal porous network via convection. Such architecture may achieve low backpressure at high flow rates of the mobile phase and provide a high binding capacity for molecules that remains constant even with increasing flow rates. The invention overcomes long-standing problems in the related art accordingly. The pore size of the composite sponge-like microspheres may influence the paths through which molecules in the fluid travel.

[0059] In the experiment shown in FIG. 8, composite microspheres having different pore sizes of either 1.6 microns (top graph), 1.0 microns (middle graph) or 50 nanometers (bottom graph) were packed into columns, and three types of molecules of different sizes — NaCl (0.2- 0.35 nm), BSA (7 nm), and H7N9 virus (100 nm) — were passed through the packed columns. In this example, the outer surface of the composite microspheres was coated with an epichlorohydrin-glycerol copolymer, using a process as described in Example 1 and the composite microspheres were functionalized as described in Example 11. In the figure, the x- axis represents the volume of mobile phase, and the y-axis represents UV absorbance for BSA and H7N9 virus or electrical conductivity for NaCl, indicative of the target molecules. For allDocket No. 1580.00257WO pore sizes, the elution volumes of NaCl, BSA, and the virus followed the trend of NaCl > BSA > Virus. In general, for molecules of the same size, smaller pore sizes cause the molecules to diffuse into the structure via the openings, which takes more time and results in lower purification efficiency. In contrast, larger pore sizes allow molecules to be transported directly into the structure by convection, more effectively increasing the available surface area for molecular interaction and thereby improving purification efficiency. The data for microspheres with 1.6 pm pore size show the smallest difference in retention time among the molecules. As the pore size decreases to 1.0 pm, the elution volumes of BSA and the virus significantly decrease, indicating that the smaller pore size causes BSA and the virus to preferentially travel around the microspheres and exhibit diffusion-dominated behavior. At a pore size as small as 50 nm (bottom plot in figure), which is far smaller than the particle size of the virus, BSA and the virus show even lower and closer elution volumes, indicating that both particles can only pass around the outer surfaces of the microspheres.

[0060] FIG. 9 illustrates two methods for making composite microspheres in accordance with the present invention, coating / crosslinking and direct composite formation. In the method of coating followed by crosslinking, molecules, oligomers, or polymers are first used to coat the surface of the structure via non-covalent interactions. Then, a crosslinking reaction is performed by chemically bonding the electrophiles (E) and nucleophiles (N) present on the molecules to form covalent linkages and generate a composite structure. This method is not limited by the functionalities originally present on the surfaces of the monomer-derived microspheres. The electrophiles (E) include, but are not limited to, R-Ots (tosylates), R-NO2, R-F, R-Cl, R-Br, R-I, and epoxide groups (where R represents the rest of the molecular structure). The nucleophiles (N) include, but are not limited to, R-OH, R-0 , R-SH, R-S , R- NH3, R-NH2, R-CN, R-COOH, and R-COO (where R represents the rest of the molecular structure). In the method of direct composite formation functional molecules or polymers are chemically synthesized and directly bonded to the surface of the monomer-derived structure through covalent bonds (A-B), thereby introducing functional groups (F) onto the surface. This method requires the presence of corresponding functional groups on the monomer-derived structure, depending on the chemical synthesis method used. In a preferred embodiment, the coating followed by crosslinking method is used to form the smooth structure.

[0061] While hydrophilic coatings have been described for the prevention of non-specific hydrophobic binding to resins during chromatographic separations, see for example USDocket No. 1580.00257WO5,030,352, it was completely unexpected that the hydrophilic polymer forming the smooth structure of the composite microspheres described herein would provide increased strength and rigidity to the particles allowing them to withstand high column flow rates of greater than 1000 cm / hr, greater than 1500 cm / hr, or greater than 2000 cm / hr without increasing backpressure, for example while maintaining a low backpressure, such as less than 0.4 MPa, less than 0.6 MPa, less than 1.0 MPa, or less than 1.2 MPa. This is illustrated in FIG. 5. The figure shows column flow rate and backpressure for columns packed with a stationary phase medium comprising either uncoated porous microspheres having a sponge-like structure, also referred to herein as the monomer-derived sponge-like structure (solid circles), or the composite microspheres of the present invention (open squares). As illustrated in the figure, a column packed with composite microspheres according to the present invention and operated at flow rates of from about 1000- 2000 cm / hr maintained a low backpressure of about 1 MPa or less. In contrast, uncoated microspheres exhibited a steep rise in backpressure at flow rates above about 1300 cm / hr.

[0062] Example 1 : Method for forming a layer of A+B composite sponge-like structure layer: A schematic diagram of the composite sponge-like structure A+B is shown below (FIG. 10). In a first step, a non-covalent coating is applied to monomer-derived sponge-like microspheres by adding a specific polymer (15-250 g) to a mixed solution of water / isopropanol / poly ethylene glycol (PEG) (2 / 1 / 5) at a concentration of 0.1-10% (w / v) followed by uniformly dispersing an amount of monomer-derived sponge-like microspheres in the aqueous solution and mixed continuously at 15-80 °C for 0.5-24 hours. Next chemical crosslinking is performed using a strong base such as potassium hydroxide (KOH) to promote nucleophilic substitution or deprotonation, leading to covalent bond formation. After removing the solution, a 1-5 M aqueous potassium hydroxide solution was added and the mixture was stirred continuously at 15-80 °C for another 0.5-24 hours. After washing, a composite sponge-like structure was obtained. In this example, nucleophiles may be provided by PEG or another polyether or polyalkylene glycol rich in hydroxyl groups (R-OH), or by alkoxide anions (R-O ) formed by KOH deprotonation of hydroxyl groups, or by amine groups (R-NH2) of the polymer; and electrophiles may be provided by the monomer-derived sponge-like microspheres, for example epoxide groups of the polymer. The hydrophilic or hydrophobic nature of the specific polymer used would affect the hydrophilicity and mechanical strength of the resulting composite sponge-like structure. Examples of the specific polymer include but are not limited to polyethylenes, polymethyl acrylates, polyethylene glycols, or polysaccharides. In a preferredDocket No. 1580.00257WO embodiment, it is a polyethylene glycol copolymer, preferably an epichlorohydrin-glycerol copolymer. The concentration of the specific polymer used would affect the thickness of the smooth layer, and an overly high concentration could result in the filling and clogging of the microsphere pores (FIG. 6). In aspects, the polymer concentration is less than 10% (w / v), more preferably less than 3% (w / v), and most preferably less than 1% (w / v), based on the total volume of the mixed solution. Furthermore, it is noted that the composite sponge-like structure herein not only exhibits excellent flow rate and backpressure characteristics, but also allows surface hydrophilic / hydrophobic modification through selection of the specific polymer. This is an important factor for biomedical chromatographic purification. Therefore, the composite sponge-like structure herein realizes the above-mentioned flow rate and backpressure advantages while providing a hydrophilic / hydrophobic surface. As shown in FIG. 11, the composite sponge-like structure with a layer of smooth structure provides a highly hydrophilic surface. In chromatographic purification technology, high-salt buffer solutions are commonly used as mobile phases to elute biomolecules adsorbed on the packed adsorbents. The hydrophobic regions of biomolecules tend to be exposed due to dehydration in high-salt buffer solutions, making them prone to bind with hydrophobic surface of the structure, resulting in precipitation and aggregation, which hinders desorption and lowers recovery and, thus, reduces purification efficiency. Measurements showed a significant decrease in lysozyme adsorption in high-salt buffer solutions (FIG. 11), indicating that the higher the surface hydrophilicity of the composite structure, the more effective it is in reducing nonspecific adsorption during biomolecular purification, and therefore more favorable for achieving improved purification efficiency.

[0063] Accordingly, a column product packed with the composite sponge-like structure herein exhibits low backpressure performance under high flow rate and provides excellent surface hydrophilicity. This makes it suitable for various chemical and biomedical chromatographic purification applications. Based on the composite sponge-like structure, various surface modifications can be applied to create a functionalized structure bearing reactive functional groups on its surface. Such a structure exhibits high chemical reactivity and can serve as an anchoring platform for subsequent specific materials. The modification can also enhance surface structural irregularity, introduce amination, impart positive charges, and incorporate highly reactive functional groups onto the surface, thereby expanding the application potential of the composite sponge-like structure.Docket No. 1580.00257WO

[0064] Monolayer surface modification (C) on the composite sponge-like structure (A+B) (the composite structure refers to A+B; the subsequent modification treatments may be monolayer or multilayer): a schematic diagram of the structure A+B+C is shown below (FIG. 12).

[0065] The composite sponge-like structure (A+B) described above is further subjected to specific surface treatments to introduce reactive functional groups on its surface. These functional groups exhibit high chemical reactivity, enabling the structure to serve as an anchoring platform for subsequent specific materials. These treatments can also enhance surface structural irregularity, introduce amination, impart positive charges, and incorporate highly reactive functional groups onto the surface, thereby expanding the application potential of the composite sponge-like structure. Common functional groups used in the active layer include epoxy groups, carbodiimides (e.g., N,N'-dicyclohexylcarbodiimide, N,N'-di(propan-2- yl)methanediimide, N,N'-diisopropylcarbodiimide, 3-[(ethylimino)methylidene]amino-N,N- dimethylpropan-1 -amine), carbonyldiimidazoles (e.g., carbonyldiimidazole, di(lH-imidazol-l- yl)methanethione), thiols (-SH), amines, N-hydroxysuccinimide, maleimide, hydroxybenzotriazole, l-hydroxy-7-azabenzotriazole, pentafluorophenol, alkenes, alkynes, azides, aziridines, aldehydes, isocyanides, isocyanates, isothiocyanates, and their derivatives. Established analytical methods can be used for measurement of the reactive functional groups on the active layer.

[0066] Example 2: In this example, further compositing was performed on an A+B composite sponge-like structure to form a monolayer of material C (referred to as a composite structure with an additional modification layer), resulting in an A+B+C application structure. The C layer illustrated in Example 2 comprises epoxy groups. To a well-mixed solution of the composite sponge-like structure dispersed at a concentration below 80 vol% and deionized water or an aprotic organic solvent, an epoxy-functionalizing agent was added in an amount of 0.5-150 g. Afterwards, a sodium hydroxide aqueous solution (0.1-3 M) was added, and the mixture is stirred continuously at 15-80 °C for 24 hours. After washing, an epoxyfunctionalized application structure was obtained. The epoxy-functionalizing agent may be selected from mono-epoxy reagents, such as epichlorohydrin; di-epoxy reagents, such as butanediol diglycidyl ether, ethylene glycol diglycidyl ether, poly(ethylene glycol) diglycidyl ether, diglycidyl aniline, diglycidyl 4,5-epoxycyclohexane-l,2-dicarboxylate, 3,4- epoxycyclohexylmethyl-3,4-epoxy cyclohexanecarboxylate, bis[(3,4-epoxycyclohexyl)methyl] adipate, 3,5,3’,5’-tetramethyl-4,4’-di(2,3-epoxypropoxy)bibenzene, four methyl phthalate 1Docket No. 1580.00257WO glycidyl ester, hexahydrophthalic anhydride diglycidyl ether, adipic acid diglycidyl ether, sorbitol diglycidyl ether, dimer acid diglycidyl ester, 1 ,4-cyclohexanedimethanol diglycidyl ether, 1,2-cyclohexanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, neopentyl glycol diglycidyl ether, dibromo neopentyl glycol diglycidyl ether, resorcinol diglycidyl ether; and poly-epoxy reagents, such as glycerol triglycidyl ether, 4,5-epoxycyclohexane-l,2-dicarboxylic acid diglycidyl ester, triglycidyl ether of meta-aminophenol, trimethylolpropane triglycidyl ether, propoxylated triglycidyl ether, N-diglycidyl-4-glycidyloxyaniline, 4,4'- methylenebis(N,N-diglycidylaniline), pentaerythritol tetraglycidyl ether, and castor oil polyglycidyl ether. The epoxy group content on the modified layer C was influenced by reaction temperature, reaction time, and sodium hydroxide concentration. Depending on the conditions, the epoxy content of the resulting epoxy-functionalized application structure can range from 10-50 pmol / g. In a preferred embodiment, the concentration of the composite sponge-like structure was less than 70 vol%, the amount of epoxy reagent used was 0.5-100 g, sodium hydroxide was at a concentration of less than 2 M, and the reaction was performed at 20-50°C for 24 hours. In the most preferred embodiment, the concentration of the composite sponge-like structure was less than 50 vol%, the epoxy reagent used was 0.5-75 g, sodium hydroxide concentration was at a concentration of less than 1.5 M, and the reaction was carried out at 25-35°C for 24 hours. The epoxy content in the most preferred embodiment was 25 pmol / g.

[0067] Example 3 : In this example, further compositing was performed on an A+B composite sponge-like structure to form a monolayer of material C, resulting in an A+B+C application structure. The C layer illustrated in Example 3 comprises carbonyldiimidazolyl (CDI) groups. To a well-mixed solution of the composite sponge-like structure dispersed at a concentration below 80 vol% in an aprotic organic solvent, a CDI-functionalizing agent (0.5- 1000 mg) was added. The mixture was stirred continuously at 15-80 °C for 24 hours. After washing, a CDI-functionalized application structure was obtained. The CDI-functionalizing agent may be selected from N,N'-dicyclohexylcarbodiimide, N,N'-di(propan-2- yl)methanediimide, N,N'-diisopropylcarbodiimide, 3-[(ethylimino)methylidene]amino-N,N- dimethylpropan-1 -amine, and the derivatives thereof. The CDI content on the surface modification layer C was affected by conditions such as reaction temperature, reaction time, and the amount of CDI reagent used. Depending on the conditions, the resulting CDI- functionalized application structure may exhibit a CDI content of 10-100 pmol / mL. In aDocket No. 1580.00257WO preferred embodiment, the concentration of the composite sponge-like structure was less than 70 vol%, the CDI-functionalizing reagent was used in an amount of 0.5-300 mg, and the reaction was carried out at 20-75 °C for 12 hours. In the most preferred embodiment, the concentration of the composite sponge-like structure was less than 50 vol%, the CDI- functionalizing reagent was used in an amount of 0.5-50 mg, and the reaction was performed at 25-35°C for 6 hours. The CDI content in the most preferred embodiment was 50 pmol / mL.

[0068] Example 4: In this example, further compositing was performed on an A+B hydroxyltype composite structure to form a monolayer of material C, resulting in an A+B+C application structure. The C layer illustrated in Example 4 comprises imide groups. To a well-mixed solution of the composite sponge-like structure dispersed at a concentration below 80 vol% in a 0.1 M fatty acid methyl ester sulfonic acid buffer solution, an imide-functionalizing agent was added in an amount of 0.1-100 mg. Optionally, an additional imide reagent (0.1-100 mg) may be added. The mixture was stirred continuously at 15-80°C for 0.5-24 hours. After washing, an imide-functionalized sponge-like structure with reactive imide groups was obtained. The imide-functionalizing agent may be selected from N,N'-dicyclohexylcarbodiimide, N,N'- diisopropylcarbodiimide, N,N'-di(propan-2-yl)methanediimine; 3- [(ethylimino)methylidene]amino-N,N-dimethylpropan-l-amine and the derivatives thereof. The optional imide reagent may be selected from N-hydroxysuccinimide, maleimide, hydroxybenzotriazole, l-hydroxy-7-azabenzotriazole, pentafluorophenol and the derivatives thereof. The imide group content in the resulting imide-functionalized sponge-like structure may range from 10-100 pmol / mL. In a preferred embodiment, the composite sponge-like structure was at a concentration of less than 70 vol%, the imide-functionalizing agent was used in an amount of 0.1-80 mg, and optionally, another imide reagent was added in an amount of 0.1-80 mg. The mixture was stirred at 20-50 °C for 0.5-20 hours. In the most preferred embodiment, the composite sponge-like structure concentration was at a concentration of less than 50 vol%, the imide-functionalizing agent was used in an amount of 0.1-75 mg, and optionally, another imide reagent was added in an amount of 0.1-75 mg. The reaction was conducted at 25-35 °C for 0.5-18 hours under constant stirring.

[0069] This example involves further compositing on an A+B+C application structure by adding a D modification layer, resulting in an A+B+C+D application structure. A schematic diagram is shown in FIG. 13. In the above-described A+B composite sponge-like structure having an activated C layer, various chemical synthesis processes can be employed to bond aDocket No. 1580.00257WO functional molecule to the structure, thereby forming an A+B+C+D structure with dual modification layers for use in a variety of resin chromatography applications. The functional molecule may include amines, such as 3-(diethylamino)propylamine (DEAP), diethylamine (DE), trimethylamine (TMA), and taurine; polyamines, such as tetraethylenepentamine (TEPA), polyethylenimine (PEI), and poly(vinylpyrrolidone) (PVP); polysaccharides, such as dextran, DEAE-dextran, Q-dextran, and agarose; and poly(meth)acrylamides, such as polyacrylamide, polyacrylamide-co-quaternary ammonium (meth)arcrylamide, polyacrylamide- co-DEAE (meth)arcrylamide, and polyacrylamide-co-sulfonate (meth)arcrylamide.

[0070] Example 5: In this example, further compositing was performed on an A+B+C application structure by adding a D modification layer, resulting in an A+B+C+D application structure. The D layer illustrated in Example 5 comprises diethylaminoethyl (DEAE) groups. DEAE is a commonly used functional group in cation exchange chromatography, and polyethylenimine (PEI) is a polyamine polymer widely used in the related art, which has repeating units consisting of one amine group (-NHz) and two aliphatic groups (CH2-CH2). PEI can be classified in terms of its structure as linear, branched, or dendritic, with a molecular weight ranging from 600 Da to 100 kDa. Due to its high cationic charge density, PEI has been widely used in various industrial applications such as water treatment, detergents, cosmetics, and adhesives. Here, PEI was composited onto a sponge-like structure. In one embodiment, an epoxy-functionalized application structure (A+B+C, with the epoxy layer C prepared according to Example 2) at a concentration of less than 80 vol% was mixed with an aqueous solution of PEI (molecular weight >1 kDa) at a concentration of more than 2%. The mixture was stirred at 20-30°C for 1-24 hours, followed by thorough washing with a high-salt solution to obtain the DEAE-functionalized application structure. In a preferred embodiment, the epoxy-functionalized structure (A+B+C) was used at a concentration below 70 vol%, the PEI aqueous solution had a PEI molecular weight >10 kDa and a concentration >2%, and the mixture was mixed at 20-30°C for 1-16 hours. In the most preferred embodiment, the epoxy- functionalized structure (A+B+C) was used at a concentration below 50 vol%, and a PEI aqueous solution with a molecular weight >60 kDa and a concentration >2% was used. Zeta potential analysis of the surface of the composite structure showed that the surface charge increased from -7 mV (for A+B+C) to +10 mV or more (for A+B+C+D). After packing the resulting application structure into a fixed column, the dynamic binding capacity (DBC, QB 10) for bovine serum albumin (BSA) was measured. The DEAE-functionalized structureDocket No. 1580.00257WO significantly enhanced the BSA binding capacity, achieving 12.7-23.0 mg / mL. Since BSA has negative surface charges in the buffer solution, its adsorption behavior is driven by electrostatic attraction to the positively charged surface of the structure. Thus, the binding capacity of BSA is directly related to the net positive charges on the structure’s surface, and increasing the zeta potential of the structure notably improves the DBC, showing a positive correlation (see FIG. 14).

[0071] Example 6: In this example, further compositing was performed on an A+B+C application structure by adding a D modification layer, resulting in an A+B+C+D application structure. The D layer illustrated in Example 6 comprises a weak anion-exchange functional group. 10 mL of CDI-functionalized application structure (see Example 3) was dispersed in deionized water or an aprotic polar solvent (e.g., acetonitrile, or ACN), with the CDI- functionalized structure being at a concentration of 50%, and 3-(diethylamino)propylamine (DEAP) was added to a concentration of 1-10%. After continuous mixing at room temperature for 8 hours, the reaction solution was removed and the resultant structure was washed with deionized water.

[0072] Example 7: In this example, further compositing was performed on an A+B+C application structure by adding a D modification layer, resulting in an A+B+C+D application structure. The D layer illustrated in Example 7 comprises a weak anion-exchange functional group. 10 mL of epoxy-functionalized application structure (see Example 2) was dispersed in a 50 mM phosphate buffer solution, with the structure concentration set to 50%, and DEAP was added to a concentration of 1% (v / v). The solution was adjusted to pH 8.0 using 1 M HC1 and stirred continuously at 50°C for 16 hours. Afterwards, the reaction solution was removed and the resultant structure was washed with deionized water.

[0073] Example 8: In this example, further compositing was performed on an A+B+C application structure by adding a D modification layer, resulting in an A+B+C+D application structure. The D layer illustrated in Example 8 comprises a strong cation-exchange functional group. lOmL of CDI-functionalized application structure (see Example 3) was dispersed in a 50 mM phosphate buffer solution, with the structure concentration set to 50%, and taurine was added to a concentration of 3%. The solution was adjusted to pH 8.0 using 1 M NaOH and stirred continuously at room temperature for 16 hours. The reaction solution was then removed and the resultant structure was washed with deionized water.Docket No. 1580.00257WO

[0074] Example 9: In this example, further compositing was performed on an A+B+C application structure by adding a D modification layer, resulting in an A+B+C+D application structure. The D layer illustrated in Example 9 comprises a weak anion-exchange functional group. lOmL of epoxy-functionalized application structure (see Example 2) was uniformly dispersed in a 32% sodium sulfate aqueous solution, with the structure concentration set to 50%, and 1 mL of 20% DEAE-dextran solution was added. After stirring the mixture for 5 minutes to ensure uniform blending, the pH was adjusted to 9.5 using 1 M NaOH, and the reaction was allowed to proceed at 50°C for 16 hours under constant stirring. The reaction solution was then removed, and the resultant structure was washed with deionized water.

[0075] Example 10: In this example, further compositing was performed on an A+B+C application structure by adding a D modification layer, resulting in an A+B+C+D application structure. The D layer illustrated in Example 10 comprises a strong cation-exchange functional group. 10 mL of epoxy-functionalized application structure (see Example 2) was uniformly dispersed in a 15% sodium sulfate aqueous solution, with the structure concentration set to 50%, and 1 mL of 20% quaternary ammonium-containing polyamide copolymer was added. The solution was adjusted to pH 8.0 using 1 M NaOH and stirred continuously at 50°C for 16 hours. The reaction solution was then removed and the resultant structure was washed with deionized water.

[0076] Example 11 : In this example, further compositing was performed on an A+B+C application structure by adding a D modification layer, resulting in an A+B+C+D application structure. The D layer illustrated in Example 11 comprises a strong cation-exchange functional group. lOmL of PEI-functionalized application structure (see Example 5) was uniformly dispersed in a 50 mM phosphate buffer solution, with the structure concentration set to 50%, and glycidyltrimethylammonium chloride (GTMAC) was added to a concentration of 10%. The solution was adjusted to pH 8.0 using 1 M NaOH and stirred continuously at 50°C for 4 hours. The reaction solution was then removed and the resultant structure was washed with deionized water.

[0077] In this example, further compositing was performed on an A+B+C+D application structure by adding an E modification layer, resulting in an A+B+C+D+E application structure. The method for preparing the A+B+C+D+E application structure is as follows. A schematic diagram of the A+B+C+D+E application structure is shown below (FIG. 15).Docket No. 1580.00257WO

[0078] Example 12: In this example, further compositing was performed on an A+B+C+D application structure by adding an E modification layer, resulting in an A+B+C+D+E application structure. The E layer illustrated in Example 12 comprises a functional group for use in hydrophobic interaction chromatography (HIC). In FIG. 15, (A+B) represents composite sponge-like structure (see Example 1), C represents imide-modified layer (see Example 4), and D represents DEAE-modified layer (see Example 5). Hydrophobic interaction chromatography (HIC) is a separation method based on the hydrophobicity of molecules. HIC is a commonly used technique for purifying proteins and other biological molecules while preserving their biological activity. Depending on the hydrophilic / hydrophobic properties of the analyte, C4, Ce, Cs, or phenyl resins can be selected accordingly. In one embodiment, more than 0.1 pmol of butyric acid may be used, and the hydrophobic molecule may be any molecule bearing a terminal carboxylic acid or its carboxylate sodium salt, including but not limited to linear or branched alkyl acids, phenylalkyl acids, and fatty acids with varying chain lengths. The hydrophobic molecule was dissolved in a fatty acid methyl ester sulfonic acid buffer solution (0.05-0.1 M). An equimolar or excess amount of l-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) or N-hydroxysuccinimide (NHS) were also dissolved in the same buffer. The hydrophobic molecule solution and EDC / NHS solution were thoroughly mixed at room temperature (20-30 °C) for 0.5-3 hours. The A+B+C+D DEAE application structure (prepared according to Example 5) was then dispersed in the resultant mixture at a concentration of less than 80 vol%, and stirred at 20-30 °C for 3-16 hours. Afterward, the material was washed thoroughly with high-salt solution to yield the hydrophobicity-functionalized composite microspheres. In this example, it was found that the PEI concentration used to prepare the D layer of the DEAE application structure affects the hydrophobicity of the resulting E-layer (HIC). In a preferred embodiment, the DEAE application structure was prepared using PEI at concentrations >1%, and the resulting HIC application structure showed a lysozyme static binding capacity (SBC) of 3.73 mg / mL in high-salt buffer. In a more preferred embodiment, the DEAE application structure (A+B+C+D) prepared using a PEI concentration greater than 2% resulted in an HIC application structure with a lysozyme static binding capacity (SBC) of up to 4.50 mg / mL. In the most preferred embodiment, the DEAE application structure (A+B+C+D) prepared using a PEI concentration greater than 4% resulted in an HIC application structure with a lysozyme static binding capacity (SBC) of up to 4.03 mg / mL (FIG. 16). For comparison, DEAE application structures prepared under different PEI compositing conditions showed near-zero SBC forDocket No. 1580.00257WO lysozyme in high-salt buffer, indicating no adsorption capacity. However, once these DEAE structures underwent HIC hydrophobic compositing, their SBC for lysozyme increased with rising PEI content — from 3.73 to 4.50 mg / mL — demonstrating a positive correlation between the degree of HIC modification and the PEI concentration used in DEAE structures. This confirms that the surface characteristics of the DEAE structures transitioned from hydrophilic to hydrophobic, resulting in enhanced protein adsorption capacity.

[0079] Example 13: In this example, further compositing was performed on an A+B+C+D application structure by adding an E modification layer, resulting in an A+B+C+D+E application structure. The E layer illustrated in Example 13 comprises a functional group for use in affinity chromatography. Affinity chromatography is based on the specific biological adsorption between biomolecules and ligands on affinity-functionalized microspheres, forming reversible interactions that enable a powerful method for purification. Among these, the development of Oligo dT affinity resins is intended to meet the demands of large-scale mRNA purification in vaccine and gene therapy applications. Oligo dT affinity resins can selectively capture mRNA via their polyadenylate (poly A) tails, effectively separating mRNA from other transcription reaction components (e.g., enzymes and plasmid DNA), thereby achieving purification. An epoxy-functionalized application structure (A+B+C) (see Example 2) can be reacted with hydroxyl-functionalized polymers such as dextran, poly(glycidol), polyethylene glycol (PEG), or polyvinyl alcohol (PVA) to form an A+B+C+D application structure, which can then be reacted with an Oligo dT ligand to obtain the Oligo dT application structure (A+B+C+D+E). In this Example, the epoxy-functionalized structure (A+B+C) was dispersed in an aqueous solution containing 0.1-10 wt% hydroxyl-functionalized polymer and 0.1-0.5 M KOH, and stirred at 20-45°C for 0.5-24 hours to form the A+B+C+D application structure. It was then dispersed in an aprotic organic solvent, and 0.5-1.0 mg of CDI (carbonyldiimidazole) was added and stirred at 20-30 °C for 4-6 hours. After washing, the material was evenly dispersed in a buffer containing 50 mM PBS and 0.6-1.2 M Na2SO4, and the Oligo dT ligand was added in an amount of 0.1-1 pmol per 1 mL of the application structure. The mixture was stirred at 25-45°C for 0.5-24 hours, yielding the Oligo dT application structure (A+B+C+D+E). In a preferred embodiment, dextran or poly(glycidol) was used as the hydroxyl-functionalized polymer. To evaluate its adsorption capacity for poly A, the Oligo dT application structure was packed into a 0.3 mL column (4.6 x 18 mm) and tested for its dynamic binding capacity (DBC) to polyA. In a CDI-modified application structure, A+BDocket No. 1580.00257WO constitute a composite structure prepared according to Example 1, C is a CDI-modified layer prepared according to Example 3, D is a dextran- or poly(glycidol)-modified layer according to Example 13, and E is an Oligo dT-modified layer according to Example 13. Oligo dT was composited on the application structures with different hydroxyl-functionalized polymers, i.e., either dextran or poly(glycidol), the resulting Oligo dT application structures (A+B+C+D+E) showed the following dynamic binding capacities for polyA: 0.35 mg / mL, 0.85 mg / mL, and 1.47 mg / mL. The results indicate that hydroxyl-functionalized polymers can significantly improve polyA dynamic binding capacity. As shown, the presence of hydroxyl-functionalized polymers can significantly enhance the dynamic binding capacity for polyA, and the variation in the types of the hydroxyl-functionalized polymers also affects the dynamic binding capacity.

[0080] While the invention herein disclosed has been described by means of specific embodiments and applications thereof, modifications and variations could be made thereto by those skilled in the art without departing from the scope of the invention set forth in the claims.

[0081] It will be appreciated that the present invention is set forth in various levels of detail in this application. In certain instances, details not necessary for one of ordinary skill in the art to understand the invention, or that render other details difficult to perceive may have been omitted. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting beyond the scope of the appended claims. Unless defined otherwise, technical terms used herein are to be understood as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

[0082] Various features of a process system may be used independently of, or in combination, with each other. It will be appreciated that a system as disclosed herein may be embodied in different forms and should not be construed as limited to the illustrated embodiments of the figures.

[0083] The term “chromatography” refers to separation technologies which employ a mobile phase and a stationary phase to separate one type of molecules from other molecules (e.g., host cell nucleic acids or proteins) in a sample. A liquid mobile phase contains a mixture of molecules and transports these across or through a stationary phase (such as a solid resin, support, or matrix, which terms are used interchangeably). Due to the differential interaction of the different molecules in the mobile phase with the stationary phase, molecules in the mobile phase can be separated. The term “affinity chromatography” refers to a specific type of chromatography in which a ligand having a specific affinity for a target molecule is coupled toDocket No. 1580.00257WO the stationary phase. The ligand interacts with the target molecule in the mobile phase thereby separating it from the mobile phase. Generally, the target molecule is eluted from the stationary phase in a separate step. The terms “solid support” or “solid matrix” or “resin” are used interchangeably to refer to the stationary phase.

[0084] It should be understood that, as described herein, an “embodiment” (such as illustrated in the accompanying Figures) may refer to an illustrative representation of an environment or article or component in which a disclosed concept or feature may be provided or embodied, or to the representation of a manner in which just the concept or feature may be provided or embodied. However such illustrated embodiments are to be understood as examples (unless otherwise stated), and other manners of embodying the described concepts or features, such as may be understood by one of ordinary skill in the art upon learning the concepts or features from the present disclosure, are within the scope of the disclosure. In addition, it will be appreciated that while the Figures may show one or more embodiments of concepts or features together in a single embodiment of an environment, article, or component incorporating such concepts or features, such concepts or features are to be understood (unless otherwise specified) as independent of and separate from one another and are shown together for the sake of convenience and without intent to limit to being present or used together. For instance, features illustrated or described as part of one embodiment can be used separately, or with one or more other features to yield a still further embodiment. Thus, it is intended that the present subject matter covers such modifications and variations as come within the scope of the appended claims and their equivalents.

[0085] In the foregoing description and the following claims, the following will be appreciated. The phrases “at least one”, “one or more”, and “and / or”, as used herein, are open- ended expressions that are both conjunctive and disjunctive in operation. The terms “a”, “an”, “the”, “first”, “second”, etc., do not preclude a plurality. For example, the term “a” or “an” entity, as used herein, refers to one or more of that entity. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.

[0086] The term “about” when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by ( + ) or ( - ) 10%, 5%, 1%, or any subrange or subvalue there between. Preferably, the term “about” means that the value may vary by + / - 10%.Docket No. 1580.00257WO

[0087] In the claims, the term “comprises / comprising” does not exclude the presence of other elements, components, features, regions, integers, steps, operations, etc. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and / or advantageous. In addition, singular references do not exclude a plurality. Reference signs in the claims are provided merely as a clarifying example and shall not be construed as limiting the scope of the claims in any way. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.

Claims

Docket No. 1580.00257WOCLAIMSWhat is claimed is:

1. A stationary phase medium for adsorption chromatography comprising: a plurality of composite microspheres made of a cross-linked polymeric material, each comprising an interior comprising multiple spherical macropores interconnected with one another via interconnecting pores to constitute an open porous network in the interior, an outer surface comprising a plurality of openings through which the porous network of the interior is in communication with the outer surface, and a layer of a hydrophilic polymer covalently attached to the outer surface; wherein each of the microspheres satisfies the following Inequality (1): dpore / dmicrosphere (0.45 / n) (1) where dpore represents an equivalent diameter of the porous network, dmicrosphere represents a diameter of the microsphere, and n represents the number of openings on the outer surface, with n being an integer and n > 2.

2. The stationary phase medium of claim 1, wherein the cross-linked polymeric material is selected from the group consisting of polyacrylates, polymethacrylates, polyacrylamides, polystyrenes, polypyrroles, polyethylenes, polypropylenes, polyvinyl chloride and silicones, optionally wherein the cross-linked polymeric material is a polymethacrylate.

3. The stationary phase medium of claim 1 or 2, wherein the composite microspheres are monodisperse and have a porosity ranging from 70% to 90%.

4. The stationary phase medium of any one of claims 1 to 3, wherein the composite microspheres have a dpore of greater than 150 nm, greater than 300 nm, or greater than 500 nm.

5. The stationary phase medium of any one of claims 1 to 4, wherein the composite microspheres have a dmicrosphere of less than 500 microns, less than 300 microns, or less than 200 microns.

6. The stationary phase medium of any one of claims 1 to 5, wherein the composite microspheres have a diameter of 10-100 microns, optionally 30-50 microns, and a pore size of 0.5-2.5 microns, optionally 1-2 microns.Docket No. 1580.00257WO7. The stationary phase medium of any one of claims 1 to 5, wherein the hydrophilic polymer is a non-ionic hydrophilic polymer, optionally wherein the non-ionic hydrophilic polymer is a polyethylene, a polymethyl acrylate, a polyethylene glycol, or a polysaccharide.

8. The stationary phase medium of claim 7, wherein the non-ionic hydrophilic polymer is a polyethylene glycol copolymer, optionally an epichlorohydrin-glycerol copolymer.

9. The stationary phase medium of any one of claims 1 to 8, wherein the outer surface is functionalized with epoxy groups to provide an epoxy content of from 10-50 pmol / g for the composite microspheres of the stationary phase medium.

10. The stationary phase medium of any one of claims 1 to 9, wherein the outer surface is modified with a surface functional group.

11. The stationary phase medium of claim 10, wherein the surface functional group is an ionic group, a hydrophobic group, a reactive group, a mixed mode group, an affinity ligand, or a combination of any of the foregoing.

12. The stationary phase medium of claim 11, wherein the surface functional group comprises an ionic group selected from the group consisting of a quaternary amine, diethylaminoethyl, sulfonyl and carboxymethyl.

13. The stationary phase medium of claim 11, wherein the surface functional group comprises a hydrophobic group selected from the group consisting of an alkyl and an aryl, optionally wherein the hydrophobic group is selected from a C4-C18 alkyl.

14. The stationary phase medium of claim 11, wherein the surface functional group comprises a reactive group selected from the group consisting of epoxy, aldehyde and succinimide ester group.

15. The stationary phase medium of claim 11, wherein the surface functional group comprises a mixed mode group which comprises a hydrophobic group selected from the group consisting of an alkyl and an aryl and an ionic group selected from the group consisting of a quaternary amine, diethylaminoethyl, sulfonyl and carboxymethyl.Docket No. 1580.00257WO16. The stationary phase medium of claim 11, wherein the surface functional group comprises an affinity ligand selected from Protein A, Protein G, oligo dT, an affinity ligand that binds to a virus such as an adeno-associated virus (AAV) or a lentivirus, an affinity ligand that binds a nucleic acid, such as double stranded RNA or DNA, and an affinity ligand that binds exosomes.

17. A method for producing the stationary phase medium of claim 1, the method comprising adding a hydrophilic polymer to a mixed solution of water, an alcohol and a polyether to form a hydrophilic polymer solution with a polymer concentration of 0.1-10% (w / v); dispersing an amount of porous microspheres in the hydrophilic polymer solution to form a microsphere solution, wherein the porous microspheres have an interior comprising multiple spherical macropores interconnected with one another via interconnecting pores to constitute an open porous network in the interior, and an outer surface comprising a plurality of openings through which the porous network of the interior is in fluid communication with the outer surface; continuously mixing the microsphere solution at a temperature of from 15-80 °C for 0.5-24 hours; following removal of the liquid phase, adding a 1-5 M aqueous potassium hydroxide solution to form a second microsphere solution; continuously mixing the second microsphere solution at 15-80 °C for another 0.5-24 hours to obtain the stationary phase medium of claim 1.

18. The method of claim 17, wherein the alcohol is isopropanol, the poly ether is a polyalkylene glycol and the molar ratio of water:alcohol:polyether is 2: 1:5.

19. The method of claim 17 or 18, wherein the hydrophilic polymer is a polyethylene, polymethyl acrylate, polyethylene glycol, or polysaccharide.

20. The method of claim 19, wherein the hydrophilic polymer is a polyethylene glycol copolymer, optionally an epichlorohydrin-glycerol copolymer.

21. The method of any one of claims 17 to 20, wherein the concentration of the hydrophilic polymer is less than 3% (w / v) or less than 1% (w / v).

22. The method of any one of claims 17 to 21, wherein the composite microspheres forming the stationary phase medium are functionalized with epoxy groups by contacting a solution ofDocket No. 1580.00257WO the composite microspheres in deionized water or an aprotic organic solvent with an epoxyfunctionalizing agent, optionally wherein the epoxy-functionalizing agent is a mono-epoxy reagent, such as epichlorohydrin or a di-epoxy reagent, such as butanediol diglycidyl ether.

23. The method of any one of claims 17 to 22, wherein the porous microspheres are formed by a process comprising the steps of:A) in the presence of a polymerization initiator and an emulsion stabilizer, emulsifying a continuous phase composition comprising at least one monomer and a crosslinking agent with a dispersed phase composition comprising a solvent to obtain a first emulsion comprising a continuous phase and a dispersed phase dispersed in the continuous phase;B) mixing the first emulsion with a third phase that is immiscible with the first emulsion by applying shear force using a shear device to form a first macro-drop emulsion dispersed in the third phase, and then micronizing the first macro-drop emulsion with a droplet generating device to disperse the first macro-drop emulsion uniformly in the third phase, thereby obtaining a second emulsion containing the third phase and a plurality of monodisperse, high internal phase emulsion droplets dispersed in the third phase; andC) curing the continuous phase and removing the dispersed phase and the third phase to obtain the stationary phase medium in form of porous microspheres; wherein each of the porous microspheres is formed in its interior with multiple spherical macropores interconnected with one another via interconnecting pores to constitute an open porous network, and formed on its outer surface with multiple openings through which the porous network is in fluid communication with the ambient; and wherein each of the porous microspheres satisfies the following Inequality (1): dpore / dmicrosphere (0.45 / n) (1) where dpore represents an equivalent diameter of the porous network, dmicrosphere represents a diameter of the porous microsphere, and n represents the number of openings on the outer surface, with n being an integer and n > 2.

24. The method of claim 23, wherein the step of forming the first macro-drop emulsion dispersed in the third phase comprises applying shear force with a mechanical stirring device or a three-dimensional aperture array.

25. The method of claim 23 or 24, wherein the droplet generating device is selected from a sieve plate perforated with narrow channels and a three-dimensional aperture array.Docket No. 1580.00257WO26. The method of any one of claims 23 to 25, further comprising a step D, subsequent to the step C, of sieving the porous microspheres obtained in step C through one or more Taylor screens to exclude oversized, undersized, or broken microspheres.

27. A stationary phase medium for adsorption chromatography produced by the method of any one of claims 17 to 26.

28. A chromatographic column, comprising a hollow tubular body packed with the stationary phase medium of any one of claims 1 to 16 and equipped with at least one fluid inlet port and at least one fluid outlet port.

29. The chromatographic column of claim 28, wherein in operation, the column can be operated at ultra-high flow rates of greater than 1000 cm / hr, greater than 1500 cm / hr, or greater than 2000 cm / hr, while maintaining low backpressure, such as less than 0.4 MPa, less than 0.6 MPa, or less than 1.0 MPa.

30. A method of performing a chromatographic separation of a macromolecule, the method comprising applying a solution comprising the macromolecule to a column packed with the stationary phase medium of any one of claims 1 to 16, and operating the column at a flow rate of greater than 1000 cm / hr, greater than 1500 cm / hr, or greater than 2000 cm / hr.

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