TWI870023B - Porous microspheres and stationary phase medium and chromatographic column comprising same - Google Patents

Abstract

The invention relates to a stationary phase medium for adsorption chromatography, which is in form of porous microspheres suitable for being packed into a chromatographic column. The porous microspheres are made of cross-linked polymeric material and formed with interconnected macropores to constitute a porous network. The invented porous microspheres have a specific ratio of porous network diameter to microsphere particle size, and the porous network is in fluid communication with the ambient via multiple openings, so that molecules are convectively transported through the porous network. Accordingly, the invention shows low back pressure and high binding capacity to molecules at high mobile phase velocities.

Description

A porous microsphere, a stationary medium containing the porous microsphere, and an adsorption chromatography column

The present invention relates to a static phase medium for adsorption chromatography, and more particularly to a static phase medium in the form of polymer porous microspheres, which is suitable for being filled in a chromatography column to separate molecules with high throughput, high efficiency and low back pressure.

With the global pandemic of Covid-19 in recent years, the demand for the use of adsorption chromatography to purify biomolecules in vaccine development and production has increased dramatically. Adsorption chromatography is a type of fluid chromatography that separates a component in a mixture by selectively adsorbing the component from a mobile phase to a solid stationary phase. Porous resin beads have been widely used as the stationary phase in adsorption chromatography. Typical resin beads form a tortuous network of micropores with diameters ranging from a few nanometers to tens of nanometers, allowing low molecular weight solutes present in the mobile phase to diffuse in and out of the micropores. As shown in Figure 1, these micropores are usually located near the outer surface of the resin beads and are not connected to each other. Most of the adsorption surface is located inside the resin beads and can only be reached by diffusion. While conventional resin beads have proven to be very useful for separating small molecules, they perform poorly for separating large molecules because they cannot access the small size of the micropores. In other words, large molecules are bound only to the surface of the resin beads, resulting in low adsorption capacity. Slow separation rates are particularly detrimental for biomolecules that are susceptible to enzymatic degradation or other destructive conditions. Resin-based chromatography has other disadvantages. Since diffusion within the beads is the rate-determining step in the adsorption process, resolution decreases with increasing flow rate, and the lack of convective flow between the resin beads results in high pressure drops across the entire chromatography column. All of these disadvantages result in reduced separation efficiency and unsatisfactory molecular productivity. Generally speaking, chromatography processes using traditional resin beads as stationary media take several days to complete, which is very time-consuming and costly.

Figure 2 shows a commonly used adsorption chromatography column, which includes a hollow elongated tube with caps at the upper and lower ends to allow the liquid mobile phase to flow through the column from top to bottom. The interior of the column is filled with porous materials, which can be block-shaped porous monoliths, micro particulates or microspheres. The porous material filled in the column has an adsorption effect on one or more substances, and has an upper limit to its adsorption amount. When the adsorption reaches saturation, it can no longer be adsorbed. The amount of these porous materials required to achieve saturation adsorption of a specific sample in a unit volume of filling material is called the binding capacity (expressed in mg/mL). When measuring the adsorption loading, the mobile phase containing the specific sample is usually passed through the column from the upper opening, so that the sample is adsorbed by the functional groups on the surface of the filling material in the column. The mobile phase flowing out from the bottom of the column is detected by a UV detector. Since the sample is adsorbed by the filling material, only a very low absorption value corresponding to the low sample concentration will be observed at the outlet of the mobile phase that flows out at the beginning. When the adsorption reaches the upper limit, the absorption signal of the sample will be observed to begin to rise, indicating that the filling material inside the column has reached the upper limit of the adsorption, so the sample concentration in the mobile phase at the outlet begins to increase. When the adsorption reaches the upper limit, the UV absorption value of the sample will finally reach the maximum value (QB100). The amount of sample injected when the UV absorbance at the outlet reaches 10% of the maximum value (QB10) is calculated from the rising curve, which is called the dynamic binding capacity (DBC; expressed in mg/mL). The liquid mobile phase flows through the column at a fixed or time-varying flow rate ( ν , usually expressed in cm/hour), and the pressure difference between the upper and lower openings of the column during the flow process is called the back pressure (Δ p , usually expressed in MPa).

There are two important requirements in the application of chromatography purification: First, it is hoped that the back pressure generated during the purification process is low, or the mechanical strength of the internal filling material is high enough to withstand high back pressure. Low back pressure can avoid exceeding the upper limit of the pressure resistance of the chromatography column device or the internal filling material, thereby increasing the operating flow rate and improving the efficiency of the process purification at the application end. Second, it is hoped that in the purification application of the chromatography column, the DBC of the filling material will not decrease as the flow rate of the mobile phase increases, and its adsorption capacity can be maintained while increasing the flow rate.

The industry has made a lot of efforts to meet the above needs. Figure 3A shows the back pressure data of two representative commonly used anion exchange columns, where the CIMmultus ^TM QA column (purchased from BIA Separations, Slovenia) is filled with a monolithic column with a pore size of 2 μm and a polymethyl methacrylate as the main material, while the Capto ^TM Q column (purchased from Danaher Corporation, USA) is filled with porous agarose microspheres, whose surface has many diffusion micropores with a diameter of about 20-50 nm, open at one end and not connected to each other. Figure 3A shows that the CIMmultus ^TM QA column filled with blocky monolithic column material (marked with the symbol ◆) generates a huge back pressure when the mobile phase passes through the monolithic column material in the column, and the back pressure increases linearly with the increase of flow rate (slope is 1.27× ^10-3 MPa hr cm ^-1 ), which is not conducive to the stability of product use. On the other hand, the Capto ^TM Q column filled with porous agarose colloidal microspheres (marked with the symbol ▲) has a back pressure that increases slowly with the increase of flow rate (slope is 8.3× ^10-5 MPa hr cm ^-1 ), reflecting better product stability. However, porous agarose colloidal microspheres are limited by their weak structural strength. When the pressure rises, the structure will be deformed and the pores will collapse, which will cause the column to be blocked. As shown in Figure 3A, when the flow rate exceeds 500 cm/hr, the back pressure of the Capto ^TM Q column will rise rapidly (see, for example, Nweke, MC et al. , Mechanical characterisation of agarose-based chromatography resins for biopharmaceutical manufacture, J. Chromatogr. A , (2017), 1530: 129-137), which greatly limits the scope of use and efficiency of the column. Since the above-mentioned commonly used chromatography columns will cause irreversible damage due to the increase in back pressure, it is recommended in their product descriptions that the operating flow rate must be less than the recommended value (600 cm/hr).

Figure 3B shows the dynamic adsorption capacity (DBC) data of the two representative conventional anion exchange columns mentioned above when used to separate molecules, with thyroglobulin (TGY) as a representative molecule. For the Capto ^TM Q column filled with porous agarose colloidal microspheres (marked with the symbol ▲), when the mobile phase containing TGY flows through the microspheres in the column, the TGY in the mobile phase mainly transfers mass by diffusion, that is, TGY diffuses from the porous structure on the surface of the microspheres and is then adsorbed. Since the molecular size of TGY is larger than the diffusion pores on the surface of the microspheres, its efficiency of diffusion into the microporous structure is significantly reduced (the DBC for TGY is 1-2 mg/mL). Therefore, this type of column is not suitable for the purification of biological molecules. On the other hand, the CIMmultus ^TM QA column filled with block-shaped monolithic column material (marked with the symbol ◆) has larger internal pores (about 2 microns in diameter). Because it is a monolithic column structure, the mobile phase flows completely inside the structure, so TGY is transported by convection and adsorbed by the inner surface of the monolithic column. Compared with diffusion, it can directly contact the surface inside the monolithic column structure and be adsorbed more efficiently. Therefore, the adsorption capacity of molecules is much better than that of microsphere products (DBC for TGY is 22 mg/mL).

Therefore, the relevant industry still needs a static phase medium that is made into a form of polymer porous particles suitable for filling in a chromatography column, has low back pressure and/or its structure will not be destroyed at a high operating flow rate of the mobile phase, and its DBC will not decrease as the flow rate of the mobile phase increases, and can still maintain a good adsorption loading for molecules at a high operating flow rate.

In order to overcome the above-mentioned shortcomings, the present invention provides a static medium for adsorption chromatography, which is in the form of a group of porous microspheres suitable for filling in a chromatography column. Each porous microsphere is made of a cross-linked polymer material and has many interconnected macropores to form a porous network. The porous network provides an extremely large specific surface area as an adsorption surface, allowing molecules to easily approach and attach. More importantly, the porous microspheres in this case have a specific ratio of the internal porous network diameter to the microsphere particle diameter, and the porous network is connected to the outside through multiple openings to allow molecules to be transported through the porous network inside the microspheres by convection, so as to achieve low back pressure values at high flow rates of the mobile phase, and have a higher adsorption loading for the molecules and can remain consistent when the flow rate is increased, thereby overcoming long-standing industry problems in related technical fields.

Therefore, the first aspect of the present invention provides a static medium for adsorption chromatography, which is particularly suitable for separating molecules. The static medium comprises: a plurality of porous microspheres, each of which has a plurality of spherical macropores formed therein, wherein the spherical macropores are interconnected via connecting holes to form an open porous network, wherein the porous network is fluidically connected to the outside through a plurality of openings located on the outer surface of the microsphere; _{wherein each of the porous microspheres satisfies the following formula (1): dpores /dmicrospheres} ≧ ( 0.45/n)………………(1) wherein dpores is the equivalent diameter of the porous network, _{dmicrospheres} is the particle size of the porous _microspheres , and n is the number of openings in the microspheres through which the porous network connects to the outer surface, and n≧2, where n is an integer.

The second aspect of the present invention provides a method for preparing the aforementioned static phase medium, which comprises the following steps: A) In the presence of a polymerization initiator and an emulsification stabilizer, a continuous phase composition comprising at least one monomer and a crosslinking agent is emulsified 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) The first emulsion and a third phase immiscible with the first emulsion are mixed and a shearing device is used to apply shear force to prepare a first macro-droplet emulsion dispersed in the third phase, and then the first macro-droplet emulsion is further micro-dropletized and uniformly dispersed in the third phase through a micro-droplet device to obtain a second emulsion comprising the third phase and monodisperse high internal phase emulsion micro-droplets dispersed in the third phase; and C) The continuous phase is solidified, and the dispersed phase and the third phase are removed to obtain a stationary phase medium in the form of porous microspheres.

In a preferred embodiment, the dpore _size of the porous microspheres is greater than 150 nm, more preferably greater than 300 nm, and even more preferably greater than 500 nm.

In a preferred embodiment, the d _microspheres of the porous microspheres are less than 500 microns, more preferably less than 300 microns, and even more preferably less than 200 microns.

In a preferred embodiment, the static medium has ion exchange functional groups after surface modification. In a more preferred embodiment, the ion exchange functional groups are selected from the group consisting of quaternary ammonium, diethylaminoethyl, sulfonyl and carboxymethyl.

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

In a more preferred embodiment, the porous microspheres are monodisperse and have a porosity of 70% to 90%.

The third aspect of the present invention provides a static medium made by the above method.

The fourth aspect of the present invention provides a chromatography column, which includes a hollow elongated tube filled with the aforementioned static medium.

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

FIG4 shows an adsorption chromatography column 10 as an embodiment of the present invention, which comprises a hollow elongated tube having at least one fluid input end 12 and at least one fluid output end 14. In a specific example, the tube is made of a material selected from the group consisting of stainless steel, titanium, quartz, glass and hard plastics such as polypropylene, and is made into a cylindrical, rectangular or polygonal tube, wherein a solid static phase 20 is filled in the column 10. When the fluid mobile phase 30 flows through the column, the molecules are separated by the adsorptive interaction between various fluid molecules and the static phase 20. The adsorption chromatography may be of a type known in the relevant technical field, including but not limited to ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography and reverse phase chromatography, and of course the fluid also includes liquid or gas. The term "static phase" used in this case may refer to an immobilized solid phase carrier, which allows the mobile phase 30 to flow through it during the chromatography process so that the molecules can be retained by the static phase 20. Here, the static phase 20 includes a group of porous microspheres 40 filled in the chromatography column 10. The term "static medium" used in this case is intended to cover the porous microspheres 40 in a filled state or a non-filled state. The mobile phase 30 is fed from the top of the column 10 so that it flows downward to the bottom of the column 10. As shown in FIG4 , molecules that are more easily attracted to the stationary phase 20 will be retained in the column 10 for a longer time, while molecules that are less easily adsorbed by the stationary phase 20 will flow to the bottom of the column 10 faster and leave. As a result, molecules with different adsorption characteristics can be collected separately. The adsorption chromatography in this case can be performed in a bind-and-elute mode, in which the target molecules are retained on the stationary phase medium and then eluted with an appropriate eluent, or in a flow-through mode, in which unwanted molecules and impurities are adsorbed by the stationary phase medium, while the target molecules are allowed to flow out.

Figures 5A-5C are electron microscope images of porous microspheres according to the present invention. It is obvious that these porous microspheres are spherical in nature, and can be manufactured with roughly uniform particle size monodispersity (narrowly distributed size) by controlling the manufacturing parameters, and d _microsphere is defined as the median diameter of the distribution in the following text. The size distribution of porous microspheres can be measured using traditional laser scattering or diffraction techniques, such as using a laser diffraction particle size analyzer to make laser light incident on microspheres suspended in a liquid phase for measurement. Many spherical giant pores stacked on each other are formed inside these porous microspheres, as indicated by the solid circle in Figure 5C. These giant pores are interconnected and fluidically connected through connecting holes, as indicated by the dotted circles in FIG5C. In individual porous microspheres, these interconnected giant pores, the connecting holes connected thereto, and the many openings on the surface of the microspheres (as indicated by the arrows in FIG5C) together constitute a continuous three-dimensional porous network, and the porous network is open to the outside world through these openings and is fluidically connected. In other words, the porous network is connected to the outside world through n openings, and n≧2. In this way, the porous microspheres of the present invention allow molecules to enter the interior of the microsphere from one opening and leave the microsphere from another opening, as shown in the schematic diagram of FIG6. The equivalent diameter of the porous network, hereinafter referred to _as dpore , can be measured by commonly used porosimetry, including but not limited to, for example, mercury intrusion porosimetry, capillary flow porometry, and electron microscopy. As described below, the size of the porous microspheres, as well as the diameter of the porous network, can be adjusted by controlling the parameters and conditions of the porous microsphere manufacturing method.

Since the porous microspheres in the present invention are monodisperse, when they are filled in a chromatography column, they tend to be stacked in the form of the densest packing, that is, the adjacent microspheres are tangent to each other, and the centers of any three tangent microspheres form an equilateral triangle, the coordination number of each microsphere is 12, and a plurality of approximately triangular voids are left between the microspheres. Preferably, at least 50% of the porous microspheres in the chromatography column, more preferably at least 60%, for example at least 75% of the porous microspheres are arranged in the form of the densest packing. The densest packing includes but is not limited to three-dimensional hexagonal closest packing (hcp), three-dimensional face-centered cubic packing (fcc), or a combination thereof. The pore system of a packed bed composed of densely packed microspheres mainly includes voids generated by the microspheres and the three-dimensional porous network inside the microspheres. In this paper, the diameter of the void is referred to as dvoid , _and there is the following relationship between it and the _{microsphere particle size (dmicrosphere} ) : dvoid = 0.225 _dmicrosphere ………… (2) Therefore _, dvoid can be represented _{by dmicrosphere} . According to Washburn's equation: Pd = -4γ _cosθ …………(3) Where P = pressure; d = pore diameter; γ = surface tension; θ = contact angle. When the mobile phase and the type of porous microspheres remain unchanged, γ and θ are constants, and d is inversely proportional to P , and d is contributed by d _holes and d _microspheres . This shows that the larger the particle size of the porous microspheres filled in the column ( d _microspheres ), or the larger the equivalent diameter of the porous network ( d _holes ), the lower the back pressure caused by the porous microspheres in the column. In this case, a specific d _hole / d _microsphere size ratio is given to the porous microspheres so that low back pressure values can be achieved at high flow rates of the mobile phase, and a higher adsorption capacity for large biomolecules can be maintained at a consistent level when the flow rate is increased.

The mass transfer mechanism in the filling material can be roughly divided into two types: diffusion and convection. As shown in Figure 1, when the pores of the porous microspheres are small or the internal pores are not connected, the mobile phase will only flow to the outside of the microspheres, and the substances in the mobile phase can only diffuse into the inside of the microspheres for adsorption. As the flow rate of the mobile phase increases, it will be difficult for the substances to diffuse into the internal structure of the porous microspheres, resulting in a decrease in the microsphere surface that the substances can contact, thereby resulting in a decrease in the adsorption amount. In contrast, when the internal pores of the porous microspheres are larger and interconnected, the mobile phase will tend to flow through the microspheres by convection, and the substances will directly enter the microspheres through the mobile phase, with better adsorption efficiency. As shown in FIG6 , porous microspheres 40 of the present invention are filled in a chromatography column, and a plurality of spherical giant pores 41 are formed in each microsphere 40. The spherical giant pores 41 are interconnected via connecting holes to form an open porous network 42. The porous network 42 is fluidically connected to the outside through a plurality of openings 43 located on the outer surface of the microsphere 40. The mobile phase can flow through the gaps between the microspheres 40 and the three-dimensional porous network 42 located inside the microsphere 40.

According to Darcy's law, which describes the flow of liquids through porous media: ……………(4) Wherein η : viscosity of mobile phase; Δ p : back pressure; Q: flow rate; A: flow channel cross-sectional area; ΔL: flow channel length. When the column, filling material and mobile phase remain unchanged, the flow channel cross-sectional area A is proportional to the flow rate Q. Assuming that the holes of the porous medium are circular, A = πr ^2. According to the definition of d _holes mentioned above, r ∝ 0.5 d _holes . Therefore, the flow rate will be proportional to d _holes . In the present invention, the mobile phase flows through a column filled with porous microspheres, and its total flow rate is the sum of the gaps between the microspheres and the porous network inside the microspheres, that is, _Qtotal = Q _gaps + Q _holes , and the flow rate is proportional to the hole size. According to formula (2), d _void = 0.225 d _microsphere , when the size ratio d _hole / d _microsphere decreases, Q _void will increase, so that the material transfer tends to be adsorbed by diffusion. In this case, as the flow rate of the mobile phase increases, the adsorption amount of the porous microsphere will decrease. Conversely, when the size ratio d _hole / d _microsphere increases, Q _hole will increase, so that the material transfer tends to be adsorbed by convection. In this case, as the flow rate of the mobile phase increases, the adsorption amount of the porous microsphere will not substantially decrease. As mentioned above, the porous network of individual microspheres in this case can be connected to the outside world through n openings, and n ≧ 2, where n is an integer. In other words, n is the number of openings in the porous network of the microsphere that connect to the outer surface, which can be calculated by taking images of the porous microspheres with a scanning electron microscope. Therefore, assuming that there are n/2 openings located in the inflow direction of the mobile phase and another n/2 openings located in the outflow direction of the mobile phase, then (n/2) d _holes ≧ d _gaps , that is, d _holes   ≧(0.45/n) d _microspheres . It is expressed as the size ratio d _pore / d _microsphere : d _pore / d _microsphere ≧(0.45/n)………………(1). The porous microparticles in this case satisfy the above formula (1), so that the mobile phase can flow through the porous network inside the microspheres in a convection manner, and reduce the gaps between the microspheres to achieve high flow rate and low back pressure, and have a high and stable adsorption amount.

In a preferred embodiment, _the dpore of the porous microspheres of the present invention can be greater than 150 nm, more preferably greater than 300 nm, such as greater than 500 nm. In a preferred embodiment, _{the dmicrosphere} of the porous microspheres of the present invention can be less than 500 μm, more preferably less than 300 μm, such as less than 200 μm, so as _to reduce the value of dvoid .

The microspheres disclosed in this case have high porosity, and the macropores are evenly distributed in the microspheres. The porosity of porous microspheres is defined as the percentage of the pore volume relative to the total volume of the microspheres, which can be calculated by the following formula: 1-[(weight of porous microspheres/density of continuous phase)/apparent volume of porous microspheres] The porosity can also be calculated by taking cross-sectional images of porous microspheres with a scanning electron microscope and then using ImageJ software (National Academy of Sciences, Bethesda, Maryland, USA). In one embodiment, the porosity of these microspheres is at least greater than 50%, or greater than 70%, but in order to maintain the strength of the microspheres, the porosity does not exceed 90%.

The porous microspheres in this case are made of cross-linked polymer materials. The polymer materials suitable for the present invention include but are not limited to polyacrylate, polymethacrylate, polyacrylamide, polystyrene, polypyrrole, polyethylene, polypropylene, polyvinyl chloride and polysilicone. In a preferred embodiment, the porous microspheres are made of polymethacrylate.

With fast kinetics, high porosity, high mechanical properties and low back pressure, the static media of the present invention can be used to separate molecules with larger sizes, including molecules with sizes greater than 15 nm, including but not limited to proteins (e.g., thyroglobulin (size is about 17 nm), nucleic acids (mRNA, 100 nm; DNA plasmids, 80-200 nm), viroids, viruses (e.g., adeno-associated virus (AAV), 20 nm; lentivirus, 80-100 nm), viral vectors, virus-like particles (VLPs), extracellular vesicles (EVs) (e.g., exosomes, 30-100 nm) and liposomes.

In some embodiments, the static medium is chemically modified to have functional groups or ligands useful for adsorbing molecules. For example, in embodiments where the static medium is used as an ion exchanger, the porous microspheres, including the porous network formed therein, are surface modified to have ion exchange functional groups, such as quaternary ammonium as a strong anion exchanger, diethylaminoethyl (DEAE) as a weak anion exchanger, sulfonyl as a strong cation exchanger, and carboxymethyl as a weak cation exchanger.

The preparation of the static phase medium of the present invention involves, after emulsifying two immiscible phases to obtain a first emulsion, dispersing the first emulsion through a sieve plate provided with holes in a third phase to obtain microspherical high internal phase emulsion droplets of uniform size suspended in the third phase, and then solidifying these emulsion droplets to produce a static phase medium in the form of porous microspheres. FIG7 shows a flow chart of the preparation method of the static phase medium according to the present invention, the method comprising step A: preparing a first emulsion; step B: dispersing the first emulsion in a third phase to obtain a second emulsion containing monodisperse high internal phase emulsion droplets; and step C: solidifying the high internal phase emulsion droplets to obtain porous microspheres.

Step A involves preparing a first emulsion. The term "emulsion" used in the present invention means a mixture consisting of a continuous phase (or external phase) and a dispersed phase (or internal phase) that is immiscible with the continuous phase. The "continuous phase" referred to in the present invention refers to an interconnected phase composed of the same composition, while the "dispersed phase" is a phase composed of many mutually isolated component units distributed in the aforementioned continuous phase, and each isolated unit in the dispersed phase is surrounded by the continuous phase. According to the present invention, the continuous phase is usually a phase where a polymerization reaction occurs, which may contain at least one monomer and a crosslinking agent, and optionally contains an initiator and an emulsifier stabilizer, while the dispersed phase may include a solvent and an electrolyte. In a preferred embodiment, the first emulsion is an oil-in-water emulsion.

The at least one monomer is intended to include any monomers and oligomers that can form a macromolecule through a polymerization reaction. In a preferred embodiment, the at least one monomer comprises at least one ethylenically unsaturated monomer or acetylenically unsaturated monomer suitable for free radical polymerization, i.e., an organic monomer having a carbon-carbon double bond or a triple bond, including but not limited to acrylic acid and its esters, such as hydroxyethyl acrylate; methacrylic acid and its esters, such as glyceryl 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; vinylpyridine, and combinations thereof.

The term "crosslinking agent" used in this case means a reagent that forms a chemical bridge between the polymer backbones formed by the polymerization reaction of the at least one monomer. In a preferred embodiment, the "crosslinking agent" is a crosslinking monomer that can be co-soluble with the at least one monomer in the continuous phase and usually has multiple functional groups to form a covalent bond between the polymer backbones formed by the polymerization of the at least one monomer. Suitable crosslinking agents are well known in the art to which the present invention belongs, and can be selected depending on the type of the at least one monomer, including but not limited to oil-soluble crosslinking agents, such as ethylene glycol dimethyl acrylate (EGDMA), polyethylene glycol dimethyl acrylate (PEGDMA), ethylene glycol diacrylate (EGDA), triethylene glycol diacrylate (TriEGDA), divinylbenzene (DVB); and water-soluble crosslinking agents, such as N,N-diallyl acrylamide, N,N'-methylenebisacrylamide (MBAA). As known to those skilled in the art, the amount of the crosslinking agent is positively correlated with the mechanical strength of the porous monolithic column, i.e., the higher the degree of crosslinking, the higher the mechanical strength of the porous monolithic column. Preferably, the crosslinking agent accounts for about 5 to 50% by weight, for example, about 5 to 25% by weight, in the continuous phase.

In addition to monomers and crosslinkers, the continuous phase may optionally contain other substances to modify the physical and/or chemical properties of the resulting porous microstructure. Examples of these substances include, but are not limited to, magnetic metal particles, such as Fe ₃ O ₄ particles; polysaccharides, such as cellulose, dextran, agarose, agar, alginates; inorganic materials, such as silicon oxide; and graphene. For example, the addition of Fe ₃ O ₄ particles can enhance the mechanical strength of the porous microstructure and impart ferromagnetism to it.

The term "emulsification stabilizer" used in this case refers to a surfactant that is suitable for stabilizing a high internal phase emulsion to prevent the dispersed phase units separated by the continuous phase in the emulsion from merging with each other. The emulsification stabilizer can be added to the continuous phase composition or the dispersed phase composition before preparing the emulsion. The emulsification stabilizer suitable for the present invention can be a non-ionic surfactant, or an anionic or cationic surfactant. In the specific example where the high internal phase emulsion is an oil-in-water emulsion, the emulsification stabilizer preferably has a hydrophilic-lipophilic balance (HLB) of 3 to 14, and more preferably has an HLB value of 4 to 6. In a preferred embodiment, the present invention uses a non-ionic surfactant as an emulsifying stabilizer, and applicable types include but are not limited to polyoxyethylene alkoxyphenols, polyoxyethylene linear alkanols, polyoxyethylene polypropylene glycols, polyoxyethylene thiols, long-chain carboxylates, chain alkanolamine condensates, quaternary acetylene glycols, polyoxyethylene polysiloxanes, N-alkyl pyrrolidones, fluorocarbon liquids, and alkyl polyglycosides. Specific examples of emulsion stabilizers include, but are not limited to, sorbitan monolaurate (trade name ^Span® 20), sorbitan tristearate (trade name ^Span® 65), sorbitan monooleate (trade name ^Span® 80), glyceryl monooleate, polyethylene glycol 200 dioleate, polyoxyethylene-polyoxypropylene block copolymers (e.g., ^Pluronic® F-68, ^Pluronic® F-127, ^Pluronic® L-121, ^Pluronic® P-123), castor oil, glyceryl monoricinoleate, distearyl dimethyl ammonium chloride, and dioleyl dimethyl ammonium chloride.

"Initiator" means a reagent that can trigger the polymerization reaction and/or crosslinking reaction of the at least one monomer and/or crosslinking agent. Preferably, the initiator used in the present invention is a thermal initiator, that is, an initiator that can trigger the polymerization reaction and/or crosslinking reaction after being heated. The initiator can be added to the continuous phase composition or the dispersed phase composition before preparing the high internal phase emulsion. According to the present invention, initiators suitable for adding to the continuous phase composition include but are not limited to azobisisobutyronitrile (AIBN), azobisisoheptanenitrile (ABVN), azobisisovaleronitrile, 2,2-bis[4,4-di(tert-butylperoxy)cyclohexyl]propane (BPO), and lauryl peroxide (LPO), and initiators suitable for adding to the dispersed phase composition include but are not limited to persulfates, such as ammonium persulfate and potassium persulfate. The high internal phase emulsion of the present invention may also further include a photoinitiator activated by ultraviolet light or visible light to initiate the aforementioned polymerization reaction and/or crosslinking reaction, and even a suitable photoinitiator may replace the aforementioned thermal initiator.

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

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

The process of obtaining the first emulsion by emulsification is to firstly uniformly mix the monomer and the crosslinking agent to form a continuous phase composition, and uniformly mix the solvent and the electrolyte to form a dispersed phase composition. Then, the continuous phase composition and the dispersed phase composition are mixed in a predetermined ratio, for example, the continuous phase composition and the dispersed phase composition are mixed in a volume ratio of 5:95 to 40:60, and agitated so that the dispersed phase is uniformly dispersed in the continuous phase. In a specific embodiment, the dispersed phase composition can be slowly added dropwise to the continuous phase composition, and violent agitation is applied to prepare the emulsion. In another and preferred embodiment, the entire batch of dispersed phase components can be added directly to the continuous phase components at once, and violent agitation is applied to form an emulsion. In the preferred embodiment of adding the dispersed phase components in batches, a high-speed homogenizer can be used for violent agitation to apply high shear force to the emulsion, thereby making the dispersed phase size of each isolated unit uniform. As is well known to those of ordinary skill in the relevant art, the size and uniformity of each isolated unit in the dispersed phase can be adjusted by changing the volume ratio of the dispersed phase to the continuous phase in the emulsion, the addition rate of the dispersed phase components, the type and concentration of the emulsifier stabilizer, and the agitation rate and temperature.

In one embodiment, the first emulsion obtained by the above emulsification is a high internal phase emulsion. The term "high internal phase emulsion" or "HIPE" used in the present invention means that the volume fraction of the internal phase in an emulsion exceeds 74.05%. According to the present invention, in step B, after mixing the first emulsion with the third phase, the first emulsion can be uniformly dispersed in the third phase through a droplet device to obtain a second emulsion containing the third phase and monodisperse high internal phase emulsion droplets dispersed in the third phase.

The "third phase" referred to in the present invention refers to a phase that can stably disperse the high internal phase emulsion and is immiscible with the continuous phase of the high internal phase emulsion. The third phase mainly contains a solvent, and the solvent includes but is not limited to water, fluorocarbon liquids and other organic solvents that are immiscible with the continuous phase. Preferably, the solvent is water. In a preferred embodiment, the second emulsion is a water-in-oil-in-water emulsion. The third phase may further include an electrolyte that can substantially dissociate free ions in the solvent, including salts, acids and bases that are soluble in the solvent. Preferably, the electrolyte contains sulfates of alkaline metals, such as potassium sulfate, and chlorides of alkaline metals and alkaline earth metals, such as sodium chloride, calcium chloride and magnesium chloride. The third phase may also further include the emulsifying stabilizer described above.

In a preferred embodiment, the first emulsion can be first added to the third phase, and then a shear force is applied by a shearing device to prepare a first macro-emulsion dispersed in the third phase. The shearing device can be a mechanical stirring device or a 3D structure with pore arrangement. The first macro-emulsion is then further micro-dropletized and uniformly dispersed in the third phase by a droplet device to obtain a second emulsion containing the third phase and monodisperse high internal phase emulsion droplets dispersed in the third phase. The droplet device can be a sieve plate structure with a plurality of fine channels (the channels are not limited to straight lines, approximate straight lines, smooth curves, and approximate smooth curves), or the droplet device can be a 3D structure with pore arrangement, which is used to produce a large number of monodisperse high internal phase emulsion droplets. The sieve plate with the holes can be made of any inert material that does not physically and chemically react with the first and second emulsions, such as carbon fiber, ceramic, glass, quartz, silicon wafer, or plastic materials such as polyvinyl chloride (PVC), polyoxymethylene (POM), polycarbonate (PC), polyphenylene oxide (PPO), PA6/66 nylon plastic, polycarbonate/acrylonitrile-butadiene-styrene copolymer (PC/ABS) composite plastic, polyethylene terephthalate (PET), polyethyleneimine (PEI), polymethyl methacrylate (PMMA), polyphenylene sulfide (PPS), polyethylene (PE), polypropylene (PP), polystyrene (PS), ethylene/vinyl acetate copolymer (EVA), or metal materials such as stainless steel, titanium, aluminum, and aluminum-magnesium alloy.

The high internal phase emulsion droplets dispersed in the third phase will spontaneously form into a substantial spherical shape due to their own cohesive force. The size of the high internal phase emulsion droplets can be adjusted by selecting the channel size of the droplet device.

In step C, the high internal phase emulsion droplets can be further heated and/or exposed to light of appropriate wavelength, or a promoter can be further added to allow the aforementioned at least one monomer and/or crosslinking agent to complete polymerization reaction and/or crosslinking reaction, thereby solidifying and forming. "Solidification" herein means the process of converting the high internal phase emulsion into a structure with a stable free-standing configuration. The dispersed phase and the third phase are then removed from the solidified high internal phase emulsion droplets to produce a stationary phase medium in the form of porous microspheres. In the specific example where the first emulsion is an oil-in-water emulsion, the solidified high internal phase emulsion droplets can be directly dried, preferably under vacuum, which helps to break the isolation units in the dispersed phase to form connecting holes. The size and uniformity of the macropores in the porous microspheres can be adjusted by changing the stirring rate and/or stirring temperature during the preparation of the first emulsion, while the size of the connecting pores and the equivalent diameter of the porous network formed in the porous microspheres can be adjusted by changing the volume ratio of the dispersed phase to the continuous phase.

In a preferred embodiment, the porous microspheres obtained in step C are screened using a Taylor screen in step D to exclude oversized, undersized or broken microspheres, so as to collect microspheres within a desired size range.

Table 1 shows the porous microsphere structure produced by the above-mentioned manufacturing method and satisfying formula (1), which has four sizes: A, B, C, and D. Table 1. No. d _hole d _Microspheres d _Holes / d _Microspheres A 1.0 micron 50 microns 0.020 B 1.4 microns 50 microns 0.028 C 1.8 microns 50 microns 0.036 D 2.2 microns 50 microns 0.044

The porous microspheres A, B, C and D prepared in Table 1 were added to a 1% aqueous solution of tetraethylenepentamine, and heated at 70 ^° C for at least 5 hours. The porous microspheres were filtered out, and then added to a 1% aqueous solution of trimethylammonium chloride, and heated at 70 ^° C for at least 5 hours. The porous microspheres were washed with water to obtain four strong anion exchange agents based on the porous microspheres A, B, C and D.

1 mL of the strong anion exchanger was filled into a polypropylene chromatography column with an inner diameter of 7.4 mm and a height of 3 mm.

Test Result 1: Dynamic Binding Capacity

The dynamic adsorption capacity of thyroglobulin (TGY) on the chromatography columns prepared with the aforementioned A, B, C, and D microspheres was tested. The mobile phase used in this example was 50 mM Tris-HCl, pH 8.5, and 1 mg/mL TGY was added to the mobile phase as the analyte. The dynamic adsorption capacity was tested using an ÄKTA ^TM Pure chromatography system (Cytiva Sweden AB, Uppsala, Sweden). The results are shown in Figure 8.

As shown in Figure 8, under the premise that porous microspheres A, B, C and D have substantially the same particle size ( d _microsphere ), porous microsphere A with the smallest porous network diameter ( d _hole = 1.0 micron) has an adsorption capacity of 5-7 mg/mL, and its adsorption capacity has a significant downward trend as the flow rate of the mobile phase increases. This shows that when the size ratio of the microspheres d _hole / d _microsphere is smaller, TGY tends to be adsorbed in a diffusion manner. As for the porous microspheres _{B (dpore = 1.4 μm), C (dpore} = 1.8 μm) and _D ( dpore = 2.2 μm) with relatively large porous _network diameters, their adsorption capacities are 6-8 mg/mL, 9-11 mg/mL and 14-15 mg/mL, respectively. The decreasing trend of their adsorption capacities slows down with the increase of the flow rate of the mobile phase, and the trend of their adsorption capacities can still be maintained at a higher flow rate. Thus, the material transport mode in the chromatographic column can be adjusted by adjusting the size ratio of _{the porous microspheres dpores/dmicrospheres. That is, increasing the size ratio of the microspheres dpores} / dmicrospheres can _make the _{material tend} to be transported by convection between the mobile phases, making its adsorption behavior less affected by the flow rate.

Test result 2: back pressure test

FIG9 shows the back pressure curves of four chromatographic columns made of microspheres A, B, C and D at different linear flow rates. The results show that the four chromatographic columns all have low back pressure at high operating flow rates of the mobile phase, and the back pressure values they exhibit at high flow rates (>600 cm/hr) are also much smaller than the effect of flow rate on back pressure values in the conventional art in FIG3A. In this embodiment, even for sample A, the slope of back pressure/flow rate of 7.5x10 ^-5 MPa cm hr ^-1 is much smaller than 130 or 62 x10 ^-5 MPa cm hr ^-1 in the conventional art. , which fully highlights the advantages of the chromatography column made using the microspheres of the embodiment of the present invention. Therefore, the present invention further includes a chromatography column, which is a hollow column and is filled with the aforementioned plurality of porous microspheres. The column has at least one fluid input end and at least one fluid output end, and has a slope value of fluid back pressure relative to fluid flow rate of less than or equal to 50 ^x10-5 MPa cm hr ^-1 or less than or equal to 30 ^x10-5 MPa cm hr ^-1 or less than or equal to 10 ^x10-5 MPa cm hr ^-1 .

Although the present invention has been described with reference to the preferred embodiments above, it should be recognized that the preferred embodiments are only provided for illustrative purposes and are not intended to limit the scope of the present invention. Various changes and modifications that are obvious to those skilled in the relevant art can be made without departing from the spirit and scope of the present invention.

10: column 12: fluid input 14: fluid output 20: static phase 30: mobile phase 40: porous microspheres 41: spherical macropores 42: open porous network 43: opening

The above and other objects, features and effects of the present invention will become apparent by referring to the following preferred embodiment description together with the accompanying drawings, in which:

FIG1 is a schematic diagram of a conventional resin bead;

FIG2 is a schematic diagram of a commonly used adsorption chromatography column;

FIG. 3A is a back pressure curve of two representative commonly used anion exchange columns at different linear flow rates; and FIG. 3B is a dynamic adsorption loading curve of two representative commonly used anion exchange columns at different linear flow rates;

FIG4 is a schematic diagram of an adsorption chromatography column according to an embodiment of the present invention;

5A-5C are scanning electrochemical images of porous particles according to an embodiment of the present invention;

FIG6 is a schematic diagram showing the transport of substances through porous particles according to one embodiment of the present invention;

FIG7 is a flow chart showing a method for manufacturing porous particles according to the present invention;

FIG8 shows the dynamic adsorption loading curves of the chromatography columns at different linear flow rates according to several specific examples of the present invention; and

FIG. 9 shows back pressure curves of chromatography columns according to several specific examples of the present invention at different linear flow rates.

without

Claims (5)

A chromatography column is a hollow column filled with a plurality of porous microspheres, the column having at least one fluid input end and at least one fluid output end, wherein the porous microspheres are prepared by the following steps: A) in the presence of a polymerization initiator and an emulsification stabilizer, a continuous phase composition comprising at least one monomer and a crosslinking agent and a dispersed phase composition comprising a solvent are emulsified to obtain a first emulsion comprising a continuous phase and a dispersed phase dispersed in the continuous phase; B) the first emulsion and a third phase immiscible with the first emulsion are mixed and sheared to obtain a first emulsion; A shearing device applies shear force to prepare a first macro-emulsion dispersed in the third phase, and then a micro-droplet device is used to further micro-drop the first macro-emulsion uniformly in the third phase to obtain a second emulsion containing the third phase and monodisperse high internal phase emulsion micro-droplets dispersed in the third phase; and C) solidifying the continuous phase of the second emulsion and removing the dispersed phase and the third phase to obtain porous microspheres; wherein each of the microspheres has a plurality of spherical macropores, and the spherical macropores are interconnected through connecting holes to form an open porous network (open A porous network is provided, wherein the porous network is fluidically connected to the outside through a _plurality of openings located on the outer surface of the microsphere; _{each of the porous microspheres satisfies the following formula (1): dhole} / dmicrosphere ≧(0.45/n) (1) wherein dhole is _the equivalent diameter of the porous network, dmicrosphere _is the particle size of the porous microsphere, and n is the number of openings in the microsphere through which the porous network connects to the outer surface, and n≧2, where n is an integer. The chromatography column as claimed in claim 1, wherein the slope value of the fluid back pressure relative to the fluid flow rate is less than or equal to 50 x 10 ^-5 MPa/cm hr ^-1 . The chromatography column as claimed in claim 1, wherein the slope value of the fluid back pressure relative to the fluid flow rate is less than or equal to 30 x 10 ^-5 MPa/cm hr ^-1 . The chromatography column as claimed in claim 1, wherein the slope value of the fluid back pressure relative to the fluid flow rate is less than or equal to 10 x 10 ^-5 MPa/cm hr ^-1 . A chromatography column as described in claim 1, wherein at least 50% of the porous microspheres in the chromatography column are arranged in the form of closest packing.