CN117463295A

CN117463295A - Porous microsphere, static phase medium containing porous microsphere and adsorption chromatography column

Info

Publication number: CN117463295A
Application number: CN202310591755.2A
Authority: CN
Inventors: 陈晖�; 许铭贤; 杜宗翰; 李志贤; 周正三
Original assignee: Taiwan Advanced Nano Template Technology Inc
Current assignee: Taiwan Advanced Nano Template Technology Inc
Priority date: 2022-07-28
Filing date: 2023-05-24
Publication date: 2024-01-30
Also published as: TW202404677A; TW202404676A; TWI824976B

Abstract

The present invention relates to a static phase medium for adsorption chromatography in the form of porous microspheres suitable for being packed in a chromatography column. The porous microspheres are made of crosslinked polymer materials and are formed with a plurality of interconnected macropores to form a porous network. The porous microspheres of the present invention have a specific ratio of the diameter of the internal porous network to the particle size of the microspheres, and the porous network communicates with the outside through a plurality of openings to allow the convective transport of molecules through the porous network within the microspheres to achieve low backpressure values at high flow rates of the mobile phase, and higher adsorption loading for the molecules and uniformity at elevated flow rates.

Description

Porous microsphere, static phase medium containing porous microsphere and adsorption chromatography column

Technical Field

The present invention relates to a static phase medium for adsorption chromatography, and more particularly, to a static phase medium fabricated into a porous microsphere shape of a polymer, which is suitable to be packed in a chromatography column to separate molecules with high flux, high efficiency and low back pressure.

Background

The recent global pandemic of the new coronavirus Covid-19 has seen a dramatic increase in the need for purification of biomolecules using adsorption chromatography for vaccine development and production. Adsorption chromatography is a type of fluid chromatography that separates a component of 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 are formed with a tortuous network of micropores, several nanometers to tens of nanometers in diameter, allowing low molecular weight solutes present in the mobile phase to diffuse into and out of the micropores. As shown in fig. 1, these micropores are typically 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 in separating small molecules, they perform poorly in separating macromolecules because macromolecules cannot enter small-sized micropores. In other words, macromolecules can only bind to the surface of the resin beads, resulting in a low adsorption capacity. The low separation rate is particularly detrimental for biomolecules that are susceptible to enzymatic degradation or other damaging conditions. Resin-based chromatography has other drawbacks in that the resolution decreases with increasing flow rate, as diffusion within the beads is the rate determining step in the adsorption process, and insufficient convective flow between the resin beads results in a high pressure drop across the chromatography column. All these disadvantages result in a decrease in separation efficiency and unsatisfactory molecular productivity. Generally, chromatography using conventional resin beads as the static phase medium takes several days to complete, and is therefore very time-consuming and costly.

FIG. 2 shows a conventional adsorption chromatography column comprising a hollow elongated tubular body with caps at each of its upper and lower ends for allowing the liquid mobile phase to flow through the column from top to bottom. The interior of the column is filled with porous material, which can be block-shaped porous monoliths, particles 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 of the adsorption amount, and when the adsorption reaches saturation, the adsorption can not be carried out again. These porous materials are called adsorption capacity (expressed in mg/mL) for a particular sample to adsorb the amount required to saturate the packing material per unit volume. In measuring the adsorption capacity, the mobile phase in which the specific sample is dissolved 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 packing material in the column. The mobile phase flowing out from below the column was detected by a UV detector. Since the mobile phase initially flowing out is adsorbed by the filler material, only a very low absorption value at the outlet is observed, corresponding to a low sample concentration. When the adsorption amount reaches the upper limit, the absorption signal of the sample is observed to start to rise, which indicates that the packing material in the pipe column reaches the upper limit of the adsorption amount, so that the concentration of the sample in the mobile phase at the outlet starts to rise. When the adsorption amount reaches the upper limit, the UV absorption value of the sample finally reaches the maximum value (QB 100). The amount of sample injected when the UV absorption at the outlet reaches 10% of the maximum (QB 10) is calculated by means of the ascending curve, called dynamic adsorption capacity (dynamic binding capacity, DBC; expressed in mg/mL). The liquid mobile phase flows through the column at a fixed or time-varying flow rate (v, typically expressed in cm/hr), and the pressure differential created between the upper and lower openings of the column during the flow process is referred to as the back pressure (Δp, typically expressed in MPa).

There are two important requirements in chromatographic purification applications: first, it is desirable that the back pressure generated during the purification process is low, or that the mechanical strength of the internal packing material is high enough to withstand the high back pressure, and that the low back pressure can avoid exceeding the upper limit of the withstand pressure of the chromatographic column device or the internal packing material, thereby improving the operating flow rate and the purification efficiency of the application-side process. Second, it is desirable in chromatographic column purification applications that the DBC of the packing material does not decrease as the flow rate of the mobile phase increases while still maintaining its adsorption capacity.

Many efforts have been made in the industry to address the above-mentioned needs. FIG. 3A shows backpressure data for two representative conventional anion exchange columns, CIMmultus ^TM QA column (available from BIA Separations, stluvinia) was packed with a monolithic column based on polymethyl methacrylate and having a pore size of 2 microns, whereas Capto ^TM The Q column (available from Danaher Corporation, usa) was packed with porous agar gel (agaros) microspheres, the surface of which had a number of diffuse micropores of about 20-50 nm in diameter, open at one end and not communicating with each other. FIG. 3A shows CIMmultus filled with bulk monolith material ^TM QA columns (labeled with the symbol diamond-solid) create a large back pressure as the flow phase passes through the monolith material within the column and the back pressure increases linearly with increasing flow rate (slope 1.27X10) ^-3 MPa hr cm ^-1 ) Is unfavorable for the stability of the product in use. Capto filled with porous agar colloidal microspheres, on the other hand ^TM Q column (marked by symbol) gives a backpressure which rises slowly with increasing flow rate (slope 8.3X10) ^-5 MPa hr cm ^-1 ) The product stability is better. However, the porous agar gel microspheres are limited by weak structural strength, and can cause structural deformation and hole collapse due to pressure rise, thereby causing the phenomenon of pipe column blockage. As shown in FIG. 3A, after the flow rate exceeds 500cm/hr, capto ^TM The back pressure of the Q-string can rise rapidly (see, e.g., nweke, M.C.et al., mechanical characterisation of agarose-based chromatography resins for biopharmaceutical manufacture, J.chromatogrA, (2017), 1530: 129-137), greatly limitingThe application range and the efficiency of the pipe column. Since the conventional chromatographic columns described above are irreversibly damaged by the rise in back pressure, the recommended operating flow rate must be less than the recommended value (600 cm/hr) in the product specification.

Fig. 3B shows dynamic adsorption capacity (DBC) data of the two aforementioned representative conventional anion exchange columns for separating molecules, wherein Thyroglobulin (TGY) is used as a representative molecule. Capto filled with porous agar colloidal microspheres ^TM In the case of Q columns (labeled with symbol (S)), when the mobile phase with TGY flows through the microspheres in the column, the TGY in the mobile phase is mainly transported by diffusion (mass transfer), i.e., the TGY is adsorbed after diffusing from the porous structure of the microsphere surface. Since the molecular size of TGY is larger than the diffusion pores at the microsphere surface, its diffusion into the microporous structure is significantly reduced (DBC for TGY is 1-2 mg/mL). Therefore, such columns are not suitable for use in the purification of biomolecules. On the other hand, CIMmultus filled with bulk monolithic column material ^TM The QA column has larger internal pores (about 2 microns in diameter) and because it is a monolithic column structure, the mobile phase flows completely inside the structure, so TGY is transported in convection (concentration) and adsorbed by the internal surface of the monolithic column, and can be more efficiently adsorbed in direct contact with the internal surface of the monolithic column structure than diffusion, thus the adsorption load for molecules is far better than microsphere products (DBC for TGY is 22 mg/mL).

Accordingly, there remains a need in the relevant art for a static phase medium in the form of polymeric porous particles that are suitable for packing into chromatography columns, that has low back pressure and/or is structurally non-disrupted at high operating flow rates of the mobile phase, and that has DBC that does not decrease as the flow rate of the mobile phase increases, yet maintains good adsorption loading for molecules at high operating flow rates.

Disclosure of Invention

To overcome the aforementioned drawbacks, the present invention provides a static phase medium for adsorption chromatography in the form of a population of porous microspheres suitable for packing into a chromatography column. Each porous microsphere is made of a cross-linked polymer material and is formed with a plurality of interconnected macropores to form a porous network. The porous network provides a very large specific surface area as an adsorption surface, allowing molecules to easily access and attach. More importantly, the porous microspheres of the present invention have a specific ratio of the diameter of the internal porous network to the particle size of the microspheres, and the porous network communicates with the outside through a plurality of openings to allow the convective transport of molecules through the porous network within the microspheres to achieve low backpressure values at high flow rates of the mobile phase, and higher adsorption loading for the molecules and maintenance of uniformity at elevated flow rates, thereby overcoming long standing industrial problems in the related art.

Accordingly, in a first aspect the invention provides a static phase medium for adsorption chromatography which is particularly suitable for separating molecules. The static phase medium comprises:

a plurality of porous microspheres, each microsphere having a plurality of spherical macropores formed therein, the spherical macropores being interconnected via connecting pores to form an open porous network (open porous network) in fluid communication with the outside through a plurality of openings at the outer surface of the microsphere;

wherein each porous microsphere satisfies the following formula (1):

d _{holes and holes} /d _{Microsphere(s)} ≧(0.45/n)………………(1)

Wherein d is _{Holes and holes} D is the equivalent diameter of the porous network _{Microsphere(s)} The particle size of the porous microsphere is equal to or larger than 2, n is the number of openings in the microsphere, wherein the openings are communicated with the outer surface of the porous network, and n is an integer.

In a second aspect, the present invention provides a method for manufacturing the aforementioned static phase medium, comprising the steps of:

a) Emulsifying a continuous phase composition comprising at least one monomer and a cross-linking agent with a disperse phase composition comprising a solvent in the presence of a polymerization initiator and an emulsion stabilizer to obtain a first emulsion comprising a continuous phase and a disperse phase dispersed in the continuous phase;

B) Mixing the first emulsion and a third phase which is not miscible with the first emulsion, applying shearing force by a shearing device to prepare a first macroemulsion dispersed in the third phase, and then uniformly dispersing the first macroemulsion in the third phase in a further micro-droplet manner by a micro-droplet device to obtain a second emulsion containing micro-droplets of the third phase and the monodisperse high internal phase emulsion dispersed in the third phase; and

c) Solidifying the continuous phase and removing the dispersed phase and the third phase to obtain a static phase medium in the form of porous microspheres.

In a preferred embodiment, d of the porous microspheres _{Holes and holes} More preferably greater than 150 nm, still more preferably greater than 300 nm, and still more preferably greater than 500 nm.

In a preferred embodiment, d of the porous microspheres _{Microsphere(s)} Less than 500 microns, more preferably less than 300 microns, still more preferably less than 200 microns.

In a preferred embodiment, the static phase medium is surface modified to have ion exchange functionality. In a more preferred embodiment, the ion exchange functionality is selected from the group consisting of quaternary ammonium, diethylaminoethyl, sulfonyl, and carboxymethyl.

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

In a more preferred embodiment, the porous microspheres have a monodispersity and a porosity (porosity) of 70% to 90%.

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

In a fourth aspect, the present invention provides a chromatography column comprising a hollow elongate tubular body filled with the aforementioned static phase medium.

Drawings

The above and other objects, features and functions of the present invention will become apparent from the following description of the preferred embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 is a schematic view of a conventional resin bead;

FIG. 2 is a schematic diagram of a conventional adsorption chromatography column;

FIG. 3A is a graph of backpressure curves for two representative conventional anion exchange columns at different linear flow rates; and FIG. 3B is a graph of dynamic adsorption loading for two representative conventional anion exchange columns at different linear flow rates;

FIG. 4 is a schematic illustration of an adsorption chromatography column according to an embodiment of the invention;

FIGS. 5A-5C are scanning electrograms of porous particles according to one embodiment of the present invention;

FIG. 6 is a schematic diagram showing mass transport through porous particles according to one embodiment of the invention;

FIG. 7 is a flow chart showing a method of manufacturing porous particles according to the present invention;

FIG. 8 shows dynamic adsorption capacity curves for chromatography columns at different linear flow rates according to several embodiments of the invention; and

FIG. 9 shows backpressure curves for chromatography columns at different linear flow rates according to several embodiments of the invention.

Description of the figure:

tubular column … … … 10

Fluid input … … … 12

Fluid output end … … … 14

Static phase … … … 20

Mobile phase … … 30

Porous microspheres … ….

Detailed Description

The following terms, as used in the specification and claims of this application, have the definitions given below, unless otherwise indicated. It is noted that, as used in the specification and claims of this application, the singular terms "a," "an," and "the" are intended to cover one and more than one item, such as at least one, at least two, or at least three, but not to mean having only a single item. In addition, the use of the terms "comprising," "having," etc. in the claims means that the elements or components recited in the claims are combined, and that other elements or components not specified in the claims are not excluded. It should also be noted that the term "or" is also generally inclusive of "and/or" in a sense unless the context clearly indicates otherwise. The terms "about" or "substantially" as used in the specification and claims herein are intended to modify any slightly variable error without such slightly changing the nature thereof.

Fig. 4 shows an embodiment of the present invention, an adsorption chromatography column 10, wherein the column 10 comprises a hollow elongated tubular body having at least one fluid input 12 and at least one fluid output 14. In one embodiment, the tubular body is made of a material selected from the group consisting of stainless steel, titanium, quartz, glass, and hard plastics such as polypropylene, and is fabricated in the form of a cylindrical, rectangular, or polygonal tubular body in which the tubular string 10 is filled with a solid static phase 20. As the fluid mobile phase 30 flows through the column, molecules are separated by adsorptive interactions of various fluid molecules with the stationary phase 20. The adsorption chromatography may be of a type known in the relevant art including, but not limited to, ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography and reverse phase chromatography, although fluids also include liquids or gases. The term "stationary phase" as used herein may refer to an immobilized solid support through which the mobile phase 30 is allowed to flow during chromatography to enable retention of molecules by the stationary phase 20. Here, the static phase 20 comprises a population of porous microspheres 40 packed within the chromatographic column 10. The term "static phase medium" as used herein is intended to encompass porous microspheres 40 in a filled or unfilled state. Mobile phase 30 is fed from the top of column 10 and allowed to flow down to the bottom of column 10. As shown in fig. 4, molecules that are more readily attracted to the static 20 will remain in the column 10 for a longer period of time, while molecules that are less readily attracted to the static 20 will flow faster to the bottom of the column 10 and exit. As a result, molecules having different adsorption characteristics can be collected thereby, respectively. Adsorption chromatography according to the invention may be performed in a bind-and-elute mode, in which the target molecule is retained on the stationary phase medium and subsequently eluted with a suitable eluent, or in a flow-through mode, in which unwanted molecules and impurities are adsorbed by the stationary phase medium, allowing the target molecule to flow out.

FIGS. 5A-5C are electron microscope images of porous microspheres according to the present invention. It is apparent that these porous microspheres are substantially spherical and can be manufactured with monodispersity (narrowly distributed size) of substantially uniform particle size by manufacturing parameter control, hereinafter d microspheres are defined as median diameter of distribution. The size distribution of the porous microspheres can be measured using conventional laser scattering or diffraction techniques, such as using a laser diffraction particle size analyzer to make laser light incident on the microspheres suspended in a liquid phase. The porous microspheres are each formed with a plurality of spherical macropores stacked on each other inside, as indicated by solid circles in fig. 5C. The macro-bores are interconnected and in fluid communication via connecting bores, indicated by the dashed circles in connection Kong Rutu C. In individual porous microspheres, the interconnected macropores, the interconnected connecting pores, and a plurality of openings (indicated by arrows in FIG. 5C) at the surface of the microspheres together form a continuous three-dimensional porous network that is open to the environment and in fluid communication through the openings. In other words, the porous network communicates with the outside through n openings, and n+.2. Thus, the porous microspheres of the present invention allow molecules to enter the interior of the microsphere through one opening and leave the microsphere through another opening, as shown in FIG. 6. The equivalent diameter of the porous network, hereinafter abbreviated as d _{Holes and holes} May be measured by conventional porosimetry, including, but not limited to, for example, mercury porosimetry (mercury intrusion porosimetry), capillary flow porosimetry (capillary flow porometry), and electron microscopy (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 process.

Because of the monodispersity of the porous microspheres of the present invention, when packed in a chromatography column, they will tend to stack in a most closely packed form, i.e., adjacent microspheres are tangent to each other, the centers of any three pairwise tangent microspheres form an equilateral triangle, the coordination number of each microsphere is 12, and a plurality of approximately triangular voids (voids) remain between the microspheres. Preferably, at least 50% of the porous microspheres in the column, more preferably at least 60% of the porous microspheres, for example at least 75% of the porous microspheres, are arranged in a closest packed form. The closest packing includes, but is not limited to, three-dimensional hexagonal closest packing (hexagonal closest packing; hcp), three-dimensional face-centered cubic packing (face centered cubic packing; fcc), or combinations thereof. The pore system of the packed bed consisting of the closest packed microspheres mainly comprises the voids (voids) created by the microspheres and the stack of microspheres, and a three-dimensional porous network inside the microspheres. In this context, the diameter of the voids is simply referred to as d _{Void space} Which is in contact with the particle size (d) _{Microsphere(s)} ) There is the following relationship:

d _{void space} ＝0.225d _{Microsphere(s)} ……………(2)

So d _{Void space} Can be obtained by d _{Microsphere(s)} To represent. Further according to the Washburn' sequamation:

Pd＝-4γcosθ……………(3)

wherein P = pressure; d = hole diameter; γ = surface tension; θ=contact angle.

In the case where the mobile phase is unchanged from the type of porous microspheres, γ and θ are constants, where d is inversely proportional to P and d is represented by d _{Holes and holes} And d _{Microsphere(s)} Contribution. This shows the particle size (d) of the porous microspheres packed in the column _{Microsphere(s)} ) The larger, or equivalent diameter of the porous network (d _{Holes and holes} ) The larger the size, the lower the back pressure of the porous microsphere in the pipe column. The invention is characterized by endowing the porous microsphere with specific d _{Holes and holes} /d _{Microsphere(s)} Size ratio to achieve low back pressure value at high flow rate of mobile phase and higher adsorption to large biomoleculesThe loading is maintained consistent as the flow rate is increased.

Mass transfer (mass transfer) mechanisms in filler materials can be broadly divided into two types, diffusion and convection. As shown in FIG. 1, when the pores of the porous microspheres are small or the internal pores are not communicated, the mobile phase only goes outside the microspheres, and the substances in the mobile phase can only enter the microspheres for adsorption in a diffusion mode. As the flow rate of the mobile phase increases, the substances are not easy to diffuse into the internal structure of the porous microspheres, so that the surfaces of the microspheres contacted by the substances are reduced, and the adsorption capacity is reduced. In contrast, when the pores in the porous microspheres are larger and are in communication with each other, the mobile phase tends to flow through the microspheres in a convective manner, and the substances directly enter the microspheres through the mobile phase, so that the adsorption efficiency is better. As shown in fig. 6, the porous microspheres of the present invention are packed in a chromatographic column and a mobile phase can flow through the interstices between the microspheres and the three-dimensional porous network inside the microspheres.

According to Darcy's law (Darcy's law) for describing the flow of a liquid through a porous medium:

wherein eta: mobile phase viscosity; Δp: back pressure; q: a flow rate; a: the cross-sectional area of the flow channel; Δl: the length of the flow channel.

Under the condition that the pipe column, the filling material and the mobile phase are unchanged, the flow passage sectional area A is in direct proportion to the flow rate Q. Again assuming that the pores of the porous medium are circular, a=pi r ² . According to the foregoing d _{Holes and holes} Definition of (1), r.alpha.0.5 d _{Holes and holes} . Thus the flow rate will be equal to d _{Holes and holes} And in direct proportion. In the present invention, the mobile phase flows through a column filled with porous microspheres at a total flow rate of Q, which is the sum of the voids between the microspheres and the porous network inside the microspheres _{Summary of the invention} ＝Q _{Void space} +Q _{Holes and holes} And the flow is proportional to the hole size. Also according to (2), d _{Void space} ＝0.225d _{Microsphere(s)} When the dimension ratio d _{Holes and holes} /d _{Microsphere(s)} When falling, Q _{Void space} Will rise so that the mass transfer tends to adsorb in a diffuse manner. In this case, as the flow rate of the mobile phase increases, the adsorption amount of the porous microspheres will decrease. On the contrary, when the dimension ratio d _{Holes and holes} /d _{Microsphere(s)} When rising, Q _{Holes and holes} Will rise so that the mass transfer tendency will adsorb in a convective manner. In this case, as the flow rate of the mobile phase increases, the adsorption amount of the porous microspheres does not substantially decrease. As mentioned above, the porous network of the individual microspheres of the present invention can be connected to the outside through n openings, where n is an integer. In other words, n is the number of openings in the microsphere that are in communication with the outer surface of the porous network, which can be calculated by scanning electron microscopy images of the porous microsphere. Therefore, assuming that n/2 openings are located in the inflow direction of the mobile phase and n/2 openings are located in the outflow direction of the mobile phase, (n/2) d _{Holes and holes} ≧d _{Void space} I.e. d _{Holes and holes} ≧(0.45/n)d _{Microsphere(s)} . By changing the dimension ratio d _{Holes and holes} /d _{Microsphere(s)} The representation is:

d _{holes and holes} /d _{Microsphere(s)} ≧(0.45/n)………………(1)。

The porous particles of the present invention satisfy the above formula (1), and thus can induce the mobile phase to flow through the porous network located inside the microspheres in a countercurrent manner, thereby reducing the gaps between the flowing microspheres, achieving a high flow rate and a low back pressure, and having a high and stable adsorption capacity.

In a preferred embodiment, the porous microspheres of the invention are d _{Holes and holes} May be greater than 150 nanometers, more preferably greater than 300 nanometers, for example greater than 500 nanometers. In a preferred embodiment, the porous microspheres of the invention are d _{Microsphere(s)} May be less than 500 microns, more preferably less than 300 microns, for example less than 200 microns, in order to reduce d _{Void space} Is a numerical value of (2).

The microsphere disclosed by the invention has high porosity, and megaholes are uniformly distributed in the microsphere. The porosity (porosity) of a porous microsphere is defined as the percentage of pore volume relative to the total volume of the microsphere, 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 scanning electron microscopy of cross-sectional images of the porous microspheres followed by ImageJ software (national academy of sciences, bethesda, maryland, usa). In one embodiment, the microspheres have a porosity of at least greater than 50%, or greater than 70%, but not more than 90% in order to maintain the strength of the microspheres.

The porous microsphere is made of a cross-linked polymer material. Suitable polymeric materials for use in the present invention include, but are not limited to, polyacrylates, polymethacrylates, polyacrylamides, polystyrenes, polypyrroles, polyethylenes, polypropylenes, polyvinylchlorides, and polysilicones. In a preferred embodiment, the porous microspheres are made from polymethacrylates.

With rapid kinetic performance, high porosity, high mechanical properties, and low backpressure, the present static media can be used to isolate molecules of larger size, including molecules of sizes exceeding 15 nanometers, including but not limited to proteins (e.g., thyroglobulin (size about 17 nanometers), nucleic acids (mRNA, 100 nanometers; DNAosomes, 80-200 nanometers), viroids, viruses (e.g., adeno-associated viruses, AAV,20 nanometers; lentiviruses, 80-100 nanometers), viral vectors, virus-like particles (VLPs), extracellular vesicles (Extracellular Vesicles, EVs) (e.g., exosomes, 30-100 nanometers), and micro-liposomes.

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

The invention relates to the preparation of a static phase medium, which comprises the steps of emulsifying two immiscible phases to obtain a first emulsion, dispersing the first emulsion in a third phase through a sieve plate provided with holes to obtain microsphere-shaped high internal phase emulsion microdroplets which are suspended in the third phase and have uniform sizes, and solidifying the emulsion microdroplets to prepare the static phase medium in porous microspheres. Fig. 7 shows a flow chart of a method for manufacturing a static phase medium according to the invention, the method comprising the steps a: preparing a first emulsion; and (B) step (B): dispersing the first emulsion in a third phase to obtain a second emulsion comprising monodisperse high internal phase emulsion droplets; and C: solidifying the high internal phase emulsion droplets to obtain porous microspheres.

Step a involves preparing a first emulsion. The term "emulsion" as used herein means a mixture of a continuous phase (or external phase) and a dispersed phase (or internal phase) that is immiscible with the continuous phase. The term "continuous phase" as used herein refers to an interconnected phase of the same composition, and the term "dispersed phase" refers to a phase of a plurality of mutually isolated composition units distributed in the continuous phase, each isolated unit in the dispersed phase being surrounded by the continuous phase. According to the invention, the continuous phase is generally the phase in which the polymerization takes place, which may comprise at least one monomer and a crosslinking agent, optionally with an initiator and an emulsion stabilizer, while the dispersed phase may comprise a solvent and an electrolyte. In a preferred embodiment, the first emulsion is a water-in-oil emulsion.

The at least one monomer is intended to encompass any monomer (monomers) and oligomer(s) that can form a macromolecule by polymerization. In a preferred embodiment, the at least one monomer comprises at least one ethylenically unsaturated monomer (ethylenically unsaturated monomer) or acetylenically unsaturated monomer (acetylenically unsaturated monomer) suitable for free radical polymerization, i.e., an organic monomer having a carbon-carbon double bond or a dangling bond, including, but not limited to, acrylic acid and esters thereof, such as hydroxyethyl acrylate; methacrylic acid and 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; pyrrole; vinylpyridines, and combinations thereof.

The term "crosslinking agent" as used herein means an agent that forms a chemical bridge between polymeric backbones formed by polymerization of at least one of the aforementioned monomers. In a preferred embodiment, the "crosslinker" is a crosslinking monomer that is co-soluble in the continuous phase with the at least one monomer, typically having multiple functional groups, to form covalent bonds between polymeric backbones of the at least one monomer. Suitable cross-linking agents are well known in the art of the present invention and may be selected depending on the type of the at least one monomer, including but not limited to oil soluble cross-linking agents such as Ethylene Glycol Dimethacrylate (EGDMA), polyethylene glycol dimethacrylate (PEGDMA), ethylene Glycol Diacrylate (EGDA), triethylene glycol diacrylate (TriEGDA), divinylbenzene (DVB); and water-soluble cross-linking agents such as N, N-diallyl acrylamide, 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 monolith produced, i.e., the higher the degree of crosslinking, the higher the mechanical strength of the porous monolith. Preferably, the crosslinking agent comprises about 5 to 50 weight percent of the continuous phase, for example about 5 to 25 weight percent.

The continuous phase may optionally contain other materials in addition to the monomers and cross-linking agents to alter the physical and/or chemical properties of the resulting porous microstructure. Examples of such substances include, but are not limited to, magnetic metal particles, such as Fe ₃ O ₄ Particles; polysaccharides, such as cellulose, cyclodextrin (dextran), agarose, carrageenan, alginate; inorganic materials such as silica; and (3) graphene. For example, fe is added ₃ O ₄ The microparticles can increase the mechanical strength of the porous microstructure and impart ferromagnetism thereto.

The term "emulsion stabilizer" as used herein means a boundarySurfactants are useful for stabilizing high internal phase emulsions from merging with each other in dispersed phase units separated by a continuous phase in the emulsion. The emulsion stabilizer may be added to the continuous phase composition or the dispersed phase composition prior to preparing the emulsion. Emulsion stabilizers suitable for use in the present invention may be nonionic surfactants or anionic or cationic surfactants. In embodiments in which the high internal phase emulsion is a water-in-oil emulsion, the emulsion stabilizer preferably has a hydrophilic-lipophilic balance (HLB) of from 3 to 14, more preferably from 4 to 6. In preferred embodiments, nonionic surfactants are used as emulsion stabilizers in the present invention, and suitable types include, but are not limited to, polyoxyethylated alkylphenols, polyoxyethylated straight alkanols, polyoxyethylated polypropylene glycols, polyoxyethylated thiols, long chain carboxylic esters, alkanolamine condensates, quaternary acetylenic diols, polyoxyethylated polysiloxanes, N-alkylpyrrolidones, fluorocarbons liquids, and alkylpolyglycosides. Specific examples of emulsion stabilizers include, but are not limited to, sorbitan monolaurate (trade name 20 Sorbitan tristearate (trade name +)>65 Sorbitan monooleate (trade name +)>80 Glycerol monooleate, polyethylene glycol 200 dioleate, polyoxyethylene-polyoxypropylene block copolymers (e.g.)>F-68、/>F-127、/>L-121、P-123), castor oil, glyceryl monoricinoleate, distearyldimethylammonium chloride, and dioleyldimethylammonium chloride.

"initiator" means an agent capable of initiating polymerization and/or crosslinking of at least one of the foregoing monomers and/or crosslinkers. The initiator used in the present invention is preferably a thermal initiator, i.e., an initiator capable of initiating the aforementioned polymerization and/or crosslinking reaction upon heating. The initiator may be added to the continuous phase composition or the dispersed phase composition prior to preparing the high internal phase emulsion. Suitable initiators for addition to the continuous phase composition according to the present invention include, but are not limited to, azobisisobutyronitrile (AIBN), azobisisoheptonitrile (ABVN), azobisisovaleronitrile, 2-bis [4, 4-di (t-butylperoxy) cyclohexyl ] propane (BPO), and Lauroyl Peroxide (LPO), while suitable initiators for addition to the disperse 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 comprise a photoinitiator activated by ultraviolet light or visible light to initiate the polymerization and/or crosslinking reaction, and may even be substituted for the thermal initiator.

The dispersed phase comprises mainly solvent. The solvent may be any liquid that is immiscible with the continuous phase. In embodiments where the continuous phase has a high hydrophobicity, the solvents include, but are not limited to, water, fluorocarbon liquids (fluorocarbon liquids), and other organic solvents that are not miscible with the continuous phase. Preferably the solvent is water. In this case, the dispersed phase may further comprise 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 comprises alkali metal sulphates, such as potassium sulphate, and alkali and alkaline earth metal chloride salts, such as sodium chloride, calcium chloride and magnesium chloride.

The high internal phase emulsion may be supplemented with a polymerization accelerator. "accelerator" means an agent capable of accelerating 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 ' -tetramethyl ethylenediamine (TEMED), N, N, N ', N ", N" -pentamethyl diethylenetriamine (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 an initiator such as ammonium persulfate into free radicals, thereby accelerating the aforementioned polymerization and/or crosslinking reactions. The addition amount of the accelerator is preferably 10 to 100 mol% of the addition amount of the initiator.

The process of obtaining the first emulsion by emulsification is to uniformly mix components such as monomer and cross-linking agent to form a continuous phase composition and uniformly mix components such as solvent and electrolyte to form a disperse phase composition. The continuous phase composition and the disperse phase composition are then mixed in a predetermined ratio, for example, the continuous phase composition and the disperse phase composition are mixed at a ratio of 5:95 to 40:60, and is subjected to a disturbance to uniformly disperse the dispersed phase in the continuous phase. In one embodiment, the dispersed phase composition may be slowly added dropwise to the continuous phase composition while applying a vigorous agitation to make an emulsion. In another and preferred embodiment, the entire batch of the dispersed phase composition may be added directly to the continuous phase composition at a time while being subjected to vigorous agitation to form an emulsion. In the preferred embodiment of adding the dispersed phase composition in bulk, high shear force can be applied to the emulsion by vigorous stirring using a high speed homogenizer to make the dispersed phase size uniform for each spacer unit. As is well known to those skilled in the relevant art, the size and uniformity of each spacer unit in the dispersed phase can be adjusted by varying the volume ratio of the dispersed phase to the continuous phase in the emulsion, the rate of addition of the dispersed phase composition, the type and concentration of the emulsion stabilizer, and the rate and temperature of disturbance.

In one embodiment, the first emulsion obtained by the emulsification described above is a high internal phase emulsion. The term "high internal phase emulsion" or "HIPE" as used herein means an emulsion having an internal phase fraction of more than 74.05%. According to the present invention, after the first emulsion is mixed with the third phase in the step B, the first emulsion is uniformly dispersed in the third phase by a microdroplet device, so as to obtain a second emulsion containing the third phase and monodisperse high internal phase emulsion microdroplets dispersed in the third phase.

The term "third phase" as used herein 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 contains primarily solvents including, but 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 comprise an electrolyte that substantially dissociates free ions in the solvent, including salts, acids, and bases that are soluble in the solvent. Preferably the electrolyte comprises alkali metal sulphates, such as potassium sulphate, and alkali and alkaline earth metal chloride salts, such as sodium chloride, calcium chloride and magnesium chloride. The third phase may also further comprise the emulsion stabilizer described above.

In a preferred embodiment, the first emulsion may be added to the third phase and subsequently prepared as a first macroemulsion dispersed in the third phase by applying shear force by a shearing device, which may be a mechanical stirring device or a 3D structure of pore arrangement. The first macroemulsion is then further dispersed uniformly in the third phase in droplets by a microdroplet device to obtain a second emulsion comprising the third phase and monodisperse high internal phase emulsion droplets dispersed in the third phase. The microdroplet device may be a screen structure with a plurality of fine channels (channels) that are not limited to straight lines, approximately straight lines, smooth curves, approximately smooth curves, or the microdroplet device may be a 3D structure with an array of pores for producing a plurality of monodisperse high internal phase emulsion droplets. The sieve plate with the pore channels can be made of any inert material which does not react with the first emulsion and the second emulsion physically and chemically, such as carbon fiber, ceramic, glass, quartz, silicon wafer or 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) and other plastic materials, or stainless steel, titanium, aluminum magnesium alloy and other metal materials.

The droplets of the high internal phase emulsion dispersed in the third phase spontaneously form into substantially spherical shapes due to their cohesive forces. The size of the high internal phase emulsion droplets can be adjusted by selecting the size of the orifice of the microdroplet device.

In step C, the high internal phase emulsion droplets may be further heated and/or subjected to light of a suitable wavelength, or an accelerator may be further added to allow the at least one monomer and/or crosslinking agent to complete the polymerization and/or crosslinking reaction, thereby curing and shaping. As used herein, "curing" means the process of converting a high internal phase emulsion into a structure having a stable free-standing configuration configuration. The dispersed phase and the third phase are then removed from the cured high internal phase emulsion droplets to produce a static phase medium in the form of porous microspheres. In the specific case where the first emulsion is a water-in-oil emulsion, the droplets of the cured high internal phase emulsion may be dried directly, preferably under vacuum, to assist in breaking the spacer units in the dispersed phase to create the communication holes. The size and uniformity of the macropores in the porous microspheres can be adjusted by changing the stirring rate and/or the stirring temperature during the preparation of the first emulsion, and 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 dispersion to the continuous phase.

In a preferred embodiment, the porous microspheres from step C are subjected to step D using a Taylor screen to exclude oversized, undersized or crushed microspheres in order to collect microspheres within a desired size range.

Table 1 shows the porous microsphere structures having four dimensions A, B, C, D, which were produced by the above-mentioned production method and satisfying the formula (1).

TABLE 1

Numbering device	d _{Holes and holes}	d _{Microsphere(s)}	d _{Holes and holes} /d _{Microsphere(s)}
				A	1.0 micron	50 micrometers	0.020
B	1.4 micrometers	50 micrometers	0.028
				C	1.8 micrometers	50 micrometers	0.036
D	2.2 micrometers	50 micrometers	0.044

The porous microspheres A, B, C and D prepared in table 1 were added to a 1% aqueous solution of tetraethylenepentamine, respectively, and heated at 70 ℃ for at least 5 hours. The porous microspheres were filtered off and added to a 1% aqueous solution of glycidyl trimethylammonium chloride and heated at 70 ℃ for at least 5 hours. The porous microspheres were washed with water to give four strong anion exchangers based on porous microspheres A, B, C and D.

1mL of the strong anion exchanger was packed in a polypropylene column having an inner diameter of 7.4 mm and a height of 3 mm, respectively.

Test results 1 dynamic adsorption capacity (Dynamic Binding Capacity)

The dynamic adsorption loading of Thyroglobulin (TGY) by the chromatography columns prepared from the A, B, C and D microspheres was tested. The mobile phase used in this example was 50mM Tris-HCl, pH 8.5, and 1mg/mL TGY was added to the mobile phase as analyte. Through a tool Pure chromatography system (Cytiva Sweden AB, uppsala, sweden) to detect dynamic adsorption loading. The results are shown in FIG. 8.

As can be seen from FIG. 8, the porous microspheres A, B, C and D have substantially the same particle size (D _{Microsphere(s)} ) On the premise of (d) porous microspheres a (d _{Holes and holes} =1.0 micrometers) has an adsorption capacity of 5-7mg/mL, and the adsorption capacity has a significant tendency to decrease as the flow rate of the mobile phase increases. This is illustrated when the size ratio d of the microspheres _{Holes and holes} /d _{Microsphere(s)} At smaller times, TGY tends to adsorb in a diffuse manner. As for the porous microspheres B (d) _{Holes and holes} =1.4 micrometers), C (d _{Holes and holes} =1.8 micrometers) and D (D _{Holes and holes} =2.2 micrometers), their adsorption capacities are 6-8mg/mL, 9-11mg/mL and 14-15mg/mL, respectively, whose adsorption capacities decrease with increasing flow rate of the mobile phase, and whose adsorption capacities can still maintain their trend at higher flow rates. Thereby, by adjusting the dimension ratio d of the porous microspheres _{Holes and holes} /d _{Microsphere(s)} The mode of mass transfer in the chromatography column can be adjusted. That is, the size ratio d of the microspheres is increased _{Holes and holes} /d _{Microsphere(s)} The substances can be transported in a convection manner in a flowing phase-to-phase manner to be absorbed The attaching behavior is less affected by the traffic.

Test results 2: backpressure test

FIG. 9 shows the backpressure curves of four chromatography columns made with A, B, C and D microspheres 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 that they do @ at high flow rates>600 cm/hr) is also significantly less than the effect of the flow rate on the back pressure value of the prior art of FIG. 3A, in this example, the slope of back pressure/flow rate is 7.5x10 even for sample A ^-5 MPa cm hr ^-1 Also much smaller than the 130 or 62x10 of the prior art ^-5 MPa cm hr ^-1 The advantages of the chromatographic column prepared by using the microspheres according to the embodiments of the present invention are fully revealed, and therefore the present invention further comprises a chromatographic column which is a hollow column and is filled with the porous microspheres, the column having at least one fluid input end and at least one fluid output end, and having a slope value of fluid back pressure relative to fluid flow rate of 50x10 or less ^-5 MPa cm hr ^-1 Or less than or equal to 30x10 ^-5 MPa cm hr ^-1 Or 10x10 or less ^-5 MPa cm hr ^-1 。

Claims

1. A static phase medium for adsorption chromatography comprising:

a plurality of porous microspheres, each microsphere having a plurality of spherical macropores formed therein, the spherical macropores being connected to each other via connecting pores, thereby forming an open porous network in fluid communication with the outside through a plurality of openings formed in the outer surface of the microsphere;

The individual porous microspheres satisfy the following formula (1):

d _{holes and holes} /d _{Microsphere(s)} ≧(0.45/n)………………(1)

2. The static phase medium of claim 1, wherein d of the porous microspheres _{Holes and holes} Greater than 150 nanometers.

3. The static phase medium of claim 2, wherein d of the porous microspheres _{Holes and holes} Greater than 300 nanometers.

4. A static phase medium according to claim 3, wherein d of said porous microspheres _{Holes and holes} Greater than 500 nanometers.

5. The static phase medium of claim 1, wherein d of the porous microspheres _{Microsphere(s)} Less than 500 microns.

6. The static phase medium of claim 5, wherein d of the porous microspheres _{Microsphere(s)} Less than 300 microns.

7. The static phase medium of claim 6, wherein d of the porous microspheres _{Microsphere(s)} Less than 200 microns.

8. The static media of claim 1, wherein the static media is surface modified to have ion exchange functionality.

9. The static phase medium of claim 8, wherein the ion exchange functionality is selected from the group consisting of quaternary ammonium, diethylaminoethyl, sulfonyl, and carboxymethyl.

10. The static phase medium according to claim 9, wherein the porous microspheres are made of a crosslinked polymeric material.

11. The static phase medium according to claim 10, wherein the crosslinked polymeric material is selected from the group consisting of polyacrylates, polymethacrylates, polyacrylamides, polystyrenes, polypyrroles, polyethylenes, polypropylenes, polyvinylchlorides, and polysilicones.

12. A static phase medium according to claim 11, wherein the cross-linked polymeric material is selected from the group consisting of polymethacrylates.

13. The static phase medium of claim 12, wherein the porous microspheres have a monodispersity and a porosity of 70% to 90%.

14. A method for producing a static phase medium, comprising the steps of:

c) Solidifying the continuous phase of the second emulsion and removing the dispersed phase and the third phase to obtain a static phase medium in the form of porous microspheres;

wherein, a plurality of spherical macro holes are formed in each porous microsphere, and the spherical macro holes are connected with each other through connecting holes, thus forming an open porous network which is in fluid communication with the outside through a plurality of openings positioned on the outer surface of the microsphere;

the individual porous microspheres satisfy the following formula (1):

d _{holes and holes} /d _{Microsphere(s)} ≧(0.45/n)………………(1)

15. The method of claim 14, wherein preparing the first macroemulsion dispersed in the third phase comprises arranging the 3D structure with a mechanical stirring device or voids to provide the shear force.

16. The method of claim 14, wherein the microdroplet device is a screen plate structure having a plurality of fine cells or the microdroplet device is a 3D structure having an array of cells.

17. The method of claim 14, further comprising a step D after step C, wherein the porous microspheres obtained in step C are screened through a taylor screen to exclude oversized, undersized or crushed microspheres.

18. A static media for adsorption chromatography comprising a static media made by the method of claim 14.

19. A chromatographic column, characterized in that it is a hollow column and is filled with a plurality of porous microspheres, the column has at least one fluid input end and at least one fluid output end, a plurality of spherical macro-holes are formed in each microsphere, the spherical macro-holes are connected with each other through connecting holes, thereby forming an open porous network, and the porous network is in fluid communication with the outside through a plurality of openings positioned on the outer surface of the microsphere;

the individual porous microspheres satisfy the following formula (1):

d _{holes and holes} /d _{Microsphere(s)} ≧(0.45/n)………………(1)

20. A chromatography column according to claim 19, having a slope of fluid back pressure relative to fluid flow rate of 50x10 or less ^-5 MPa cm hr ^-1 。

21. A chromatography column according to claim 19, having a slope of fluid back pressure versus fluid flow rate of 30x10 or less ^-5 MPa cm hr ^-1 。

22. A chromatography column according to claim 19, having a slope of fluid back pressure relative to fluid flow rate of 10x10 or less ^-5 MPa cm hr ^-1 。

23. The chromatography column of claim 19, wherein at least 50% of the porous microspheres in the column are arranged in a closest packed form.