TWI824976B - Method for producing stationary phase medium of porous microspheres - 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

Method for manufacturing static phase media containing porous microspheres

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

With the Covid-19 global pandemic in recent years, the demand for the use of adsorption chromatography to purify biomolecules in vaccine development and production has increased rapidly. 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 form a network of tortuous micropores ranging from a few 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 Figure 1, these micropores are usually located close to 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 useful for separating small molecules, they perform poorly at separating large molecules because they cannot enter the small-sized pores. In other words, macromolecules can only bind to the surface of resin beads, resulting in low adsorption capacity. Slow separation rates are particularly detrimental for biomolecules that are susceptible to enzymatic degradation or other damaging conditions. Resin-based chromatography has other shortcomings Point, because diffusion within the beads is the rate-determining step in the adsorption process, resolution decreases with increasing flow rate, and insufficient convective flow between resin beads results in high pressure across the entire chromatography column. drop. All these disadvantages result in reduced separation efficiency and unsatisfactory molecular productivity. Generally speaking, chromatography processes using traditional resin beads as stationary phase media take several days to complete, making them very time-consuming and costly.

Figure 2 shows a conventional adsorption chromatography column, which includes a hollow elongated tube with a cover at the top and bottom openings, allowing the liquid mobile phase to flow through the column from top to bottom. The inside of the column is filled with porous material, which can be block-shaped porous monoliths, micro particulates or microspheres with a porous structure. The porous material filled inside the column has an adsorption effect on one or more substances, and has an upper limit for its adsorption amount. When the adsorption reaches saturation, it can no longer be adsorbed. The amount of these porous materials required to adsorb a specific sample to saturation per unit volume of filling material is called the binding capacity (expressed in mg/mL). When measuring the adsorption capacity, the mobile phase in which the specific sample is dissolved is usually flowed through the column from the upper hole, 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 the bottom of the column is detected by a UV detector. In the mobile phase that initially flows out, because the sample is adsorbed by the filling material, only a very low absorption value corresponding to a low sample concentration is observed at the outlet. When the adsorption capacity 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 capacity, so the sample concentration in the mobile phase at the outlet begins to increase. When the adsorption amount reaches the upper limit, the UV absorption value of the sample will eventually reach the maximum value (QB100). Through the rising curve, calculate the amount of sample injected when the UV absorption value at the outlet reaches 10% of the maximum value (QB10), which is called 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 , usually expressed in centimeters/hour). The pressure difference generated between the upper and lower openings of the column during the flow process is called back pressure ( Δp , usually expressed in MPa express).

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 that the chromatography column device or the internal packing material can bear, thereby increasing the operating flow rate and improving the efficiency of the application-end process purification. Second, it is hoped that in the purification application of chromatography columns, the DBC of the packing 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 put in a lot of effort to meet the above needs. Figure 3A shows the back pressure data of two representative conventional anion exchange columns. The CIMmultus ^TM QA column (purchased from BIA Separations, Slovenia) is filled with a polymethylmethacrylate-based and has The monolithic column with a pore size of 2 microns, while the Capto ^TM Q column (purchased from Danaher Corporation, USA) is filled with porous agarose microspheres, the surface of which has many single-end open and interconnected microspheres with a diameter of about 20-50 nm. Unconnected diffusion micropores. Figure 3A shows that the CIMmultus ^TM QA column (marked with the symbol ◆) filled with bulk monolithic column material will generate 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 flow rate. Increase (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 back pressure obtained by the Capto ^TM Q column (marked with the symbol ▲) filled with porous agar colloid microspheres rises slowly as the flow rate increases (slope is 8.3×10 ^-5 MPa hr cm ^-1 ) , reflecting better product stability. However, porous agar colloidal microspheres are limited by their weak structural strength, which may cause structural deformation and hole collapse due to pressure increase, thereby causing column blockage. As shown in Figure 3A, when the flow rate exceeds 500cm/hr, the back pressure of the Capto ^TM Q column will rise rapidly (see, for example, Nweke, MC et al. , Mechanical characterization of agarose-based chromatography resins for biopharmaceutical manufacture, J. Chromatogr.A , (2017), 1530: 129-137), which greatly limits the use range and efficiency of this column. Since the conventional chromatography columns mentioned above will cause irreversible damage due to increased back pressure, it is recommended in their product descriptions that the operating flow rate must be less than the recommended value (600cm/hr).

Figure 3B shows the dynamic adsorption capacity (DBC) data of the aforementioned two representative conventional anion exchange columns when used to separate molecules, in which thyroglobulin (TGY) is used as a representative molecule. As for the Capto ^TM Q column filled with porous agar colloid 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 forms by diffusion. ), that is, TGY diffuses from the porous structure on the surface of the microsphere and is adsorbed. Since the molecular size of TGY is larger than the diffusion pores on the microsphere surface, its diffusion efficiency into the microporous structure is significantly reduced (DBC for TGY is 1-2 mg/mL). Therefore, this type of column is not suitable for purification applications of biomolecules. On the other hand, the CIMmultus ^TM QA column filled with bulk monolithic column material (marked with the symbol ◆) has larger internal pores (about 2 microns in diameter), and because of its monolithic column structure, the mobile phase is completely It flows inside the structure, so TGY is transported in a convection manner and is adsorbed by the internal surface of the monolithic column. Compared with diffusion, it can directly contact the surface inside the monolithic column structure and be adsorbed. Therefore, for molecules The adsorption capacity is far better than that of microsphere products (DBC for TGY is 22mg/mL).

Therefore, relevant industries still need a static phase medium that is made into a form of polymer porous particles suitable for being filled in a chromatography column, has low back pressure and/or the structure will not be damaged under high operating flow rates of the mobile phase. to damage, and its DBC will not decrease as the flow rate of the mobile phase increases, and it can still maintain good adsorption capacity for molecules under high operating flow rates.

In order to overcome the aforementioned shortcomings, the present invention provides a static phase medium for adsorption chromatography, which is in the form of a group of porous microspheres suitable for being filled in a chromatography column. Each porous microsphere is made of cross-linked polymer material and has many interconnected macropores to form a porous network. The porous network provides a large specific surface area as an adsorption surface, making it easy for molecules to access and attach. More importantly, the porous microspheres in this case have a specific ratio of the diameter of the internal porous network to the particle size of the microspheres, and the porous network is connected to the outside world through multiple openings to allow convective transport of molecules through the microspheres. The porous network can achieve low back pressure values at high flow rates of the mobile phase, have a high adsorption capacity for molecules and remain consistent when the flow rate is increased, thereby overcoming long-standing industrial problems in related technical fields.

Therefore, the first aspect of this application provides a static phase medium for adsorption chromatography, which is particularly suitable for separating molecules. The static phase medium includes: a plurality of porous microspheres, each microsphere is formed with many spherical macropores, and the spherical macropores are connected to each other through connecting pores, thus forming an open porous network. The porous network is in fluid communication with the outside world through multiple openings located on the outer surface of the microspheres; wherein each porous microsphere satisfies the following formula (1): d _holes / d _microspheres ≧ (0.45/n)... ...............(1)

Where d _pores are the equivalent diameter of the porous network, d _microspheres are the particle size of the porous microspheres, and n is the number of openings in the microspheres connected to the outer surface of the porous network, and n≧2, n is an integer.

The second aspect of this case provides a method for manufacturing the aforementioned static phase medium, which includes the following steps: A) in the presence of a polymerization initiator and an emulsification stabilizer, combining at least one monomer and a cross-linking agent The continuous phase composition of the agent is emulsified with a dispersed phase composition including a solvent to obtain a first emulsion including a continuous phase and a dispersed phase dispersed in the continuous phase; B) the first emulsion The emulsion and a third phase immiscible with the first emulsion phase are mixed and a shearing device is used to apply shear force to prepare a first macroemulsion dispersed in the third phase, and then through a micro droplet The device further disperses the first macroemulsion into droplets evenly in the third phase to obtain a monodisperse high internal phase emulsion droplet containing the third phase and dispersed in the third phase. The second emulsion; and C) solidify the continuous phase, and remove the dispersed phase and the third phase to obtain a static phase medium in the form of porous microspheres.

In a preferred specific example, the d _holes of the porous microspheres are larger than 150 nanometers, more preferably larger than 300 nanometers, and even more preferably larger than 500 nanometers.

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

In a preferred specific example, the static phase medium is surface modified to have ion exchange functional groups. In a more preferred embodiment, the ion exchange functional group is selected from the group consisting of quaternary ammonium, diethylamine ethyl, sulfonyl and carboxymethyl.

In a preferred embodiment, the porous microspheres are made of cross-linked polymer materials. 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 A group composed of polysiloxane. In a more preferred embodiment, the cross-linked polymer material is selected from polymethacrylate.

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

The third aspect of this case provides a static phase medium made by the aforementioned method.

The fourth aspect of this case provides a chromatography column, which includes a hollow elongated tube filled with the aforementioned stationary phase medium.

10: Pipe string

12: Fluid input end

14: Fluid output end

20: still phase

30:Moving phase

40: Porous microspheres

41: Spherical giant hole

42:Open porous network

43:Open your mouth

The above and other objects, features and effects of the present invention will become apparent with reference to the description of the following preferred specific examples together with the accompanying drawings, in which: Figure 1 is a schematic diagram of a conventional resin bead; Figure 2 is a conventional adsorption chromatography tube Schematic diagram of the column; Figure 3A is the back pressure curve of two representative conventional anion exchange columns at different linear flow rates; and Figure 3B is the dynamic adsorption capacity curve of two representative conventional anion exchange columns at different linear flow rates. ; Figure 4 is a schematic diagram of an adsorption chromatography column according to a specific example of the present invention; Figures 5A-5C are scanning electrographic images of porous particles according to a specific example of the present invention; Figure 6 is a schematic diagram showing the process of adsorption chromatography column according to the present invention. A specific example of material transport of porous particles; Figure 7 shows a flow chart of the manufacturing method of porous particles according to the present invention; Figure 8 shows the dynamic adsorption capacity of chromatography columns according to several specific examples of the present invention at different linear flow rates Curve; and Figure 9 shows the back pressure curves of chromatography columns according to several specific examples of the present invention at different linear flow rates.

Unless otherwise stated, the following terms used in the specification and claims have the definitions given below. Please note that the singular word "a" used in the specification and patent scope of this application is intended to cover one and more than one of the stated items, such as at least one, at least two or at least three, and does not mean that it only has A single matter contained. In addition, open connectives such as "comprising" and "having" used in the scope of the patent application indicate that the combination of elements or components described in the claim does not exclude other components or components not specified in the claim. It should also be noted that the term "or" generally also includes "and/or" unless the content clearly indicates otherwise. The terms "about" or "substantially" used in the specification and patent scope of this application are used to modify any error that may vary slightly, but such slight variations will not change its essence.

FIG. 4 shows an adsorption chromatography column 10 according to one embodiment of the present invention. The chromatography column 10 includes a hollow elongated tube with at least one fluid input end 12 and at least one fluid output end 14 . In a specific example, the tube body is made of a material selected from the group consisting of stainless steel, titanium, quartz, glass and hard plastic such as polypropylene, and is made into a cylindrical, rectangular or polygonal tube. In the form of a solid body, the column 10 is filled with a solid static phase 20. As the fluid mobile phase 30 flows through the column, molecules are separated by adsorbent interactions between various fluid molecules and the stationary phase 20 . The adsorption chromatography may be of a type known in the relevant technical fields, including but not limited to ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography and reverse phase chromatography. Of course, fluids also include liquid or gas. The term "stationary phase" used in this case can refer to a fixed solid phase carrier that allows the mobile phase 30 to flow through it during the chromatography process, so that the molecules can be retained by the stationary phase 20. Here, the static phase 20 includes a group of porous microspheres 40 filled in the chromatography column 10 . The term "static phase medium" as used in this case is intended to cover porous microspheres 40 in either a filled state or an unfilled 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 FIG. 4 , molecules that are more easily attracted to the stationary phase 20 will stay in the column 10 for a longer time, while molecules that are less likely to be adsorbed by the stationary phase 20 will flow to the bottom of the column 10 and leave faster. As a result, molecules with different adsorption properties can be collected separately. Adsorption chromatography in this case can be performed in bind-and-elute mode, in which the target molecules are retained on the stationary phase medium and then eluted with appropriate eluent, or in flow-through mode Performed in flow-through mode, in which unwanted molecules and impurities are adsorbed by the stationary phase medium, while 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 essentially spherical, and can be manufactured by controlling the manufacturing parameters to produce monodispersity (narrowly distributed size) with approximately uniform particle sizes. In the following, d _microspheres are defined as the median of the distribution. diameter. 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 incident laser light onto microspheres suspended in a liquid phase. Each of these porous microspheres has many mutually stacked spherical macropores formed inside, as indicated by the solid circles in Figure 5C . These giant pores are connected to each other and in fluid communication through connecting holes, which are marked by dotted circles in Figure 5C. In individual porous microspheres, these interconnected macropores, the connecting pores connected to them, and the many openings located on the surface of the microspheres (marked by arrows in Figure 5C), together constitute a continuous three-dimensional porous network. The porous network is open to the outside world and in fluid communication through these openings. In other words, the porous network is connected to the outside world through n openings, and n≧2. In this way, the porous microsphere in this case allows molecules to enter the interior of the microsphere through one opening and leave the microsphere through the other opening, as shown in schematic figure 6. The equivalent diameter of the porous network, hereinafter referred to as d _-holes , can be measured through commonly used porometry methods, including but not limited to, for example, mercury intrusion porosimetry and capillary flow porometry. and electron microscopy. As described later, 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 this case are monodisperse, when they are filled in a chromatography column, they will tend to be stacked in the most densely packed form, that is, adjacent microspheres are tangent to each other. The centers of the two tangent microspheres form an equilateral triangle. The coordination number of each microsphere is 12, and multiple approximately triangular voids are left between the microspheres. Preferably, there are at least 50% porous microspheres in the chromatography column, more preferably at least 60% of the porous microspheres, for example, at least 75% of the porous microspheres are arranged in the most densely packed form. The closest packing includes, but is not limited to, three-dimensional hexagonal closest packing (hcp), three-dimensional face-centered cubic packing (fcc), or combinations thereof. The pore system of a packed bed composed of the most densely packed microspheres mainly includes voids generated by the stacking of microspheres, and a three-dimensional porous network located inside the microspheres. In this article, the diameter of the void is referred to as d _void for short, and there is the following relationship between it and the microsphere particle size ( d _microsphere ): d _void = 0.225 d _microsphere ............. ..(2) Therefore, the d _void can be represented by d _microspheres . According to Washburn's equation: Pd =-4γ cosθ.............(3) where P = pressure; d = hole diameter; γ = surface tension; θ = contact angle. When the types of mobile phase and porous microspheres remain unchanged, γ and θ are constants. At this time, 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 ( d _microspheres ) filled in the column, or the larger the equivalent diameter of the porous network ( d _holes ), the greater the backlash caused by the porous microspheres in the column. Pressure drops. In this case, by giving porous microspheres a specific d _hole / d _microsphere size ratio, it is possible to achieve a low back pressure value at a high flow rate of the mobile phase, and have a high adsorption capacity for large biomolecules that can still be maintained when the flow rate is increased. consistent.

The mass transfer mechanism in filling materials can be roughly divided into two types: diffusion and convection. As shown in Figure 1, when the pores of porous microspheres are small or the internal pores are not connected, the mobile phase will only flow to the outside of the microsphere, and the substances in the mobile phase can only diffuse into the interior of the microsphere for adsorption. . As the flow rate of the mobile phase increases, it will be difficult for substances to diffuse into the internal structure of the porous microspheres, resulting in a decrease in the surface of the microspheres that the substances can contact, resulting in a decrease in adsorption capacity. Relatively speaking, when the internal pores of porous microspheres are larger and When connected to each other, the mobile phase will tend to flow through the microspheres in a convection manner, and the substances will directly enter the interior of the microspheres through the mobile phase, resulting in better adsorption efficiency. As shown in Figure 6, the chromatography column is filled with porous microspheres 40 in this case. Each microsphere 40 is formed with many spherical macropores 41. The spherical macropores 41 are connected to each other through connecting holes, thereby forming a The open porous network 42 is in fluid communication with the outside world through a plurality of openings 43 located on the outer surface of the microspheres 40. The mobile phase can flow through the gaps between the microspheres 40 and the three-dimensional space inside the microspheres 40. Porous network 42.

其中η：流動相黏度；△p：背壓；Q：流量；A：流道截面積；△L：流道長度。在管柱、填充材料與流動相不變的情況下，流道截面積A與流量Q成正比。又假設多孔媒質的孔洞為圓形，則A=πr²。根據前述d _孔洞 的定義，則r

0.5d _孔洞 。因此流量會與d _孔洞 成正比關係。在本發明中，流動相流經一填充有多孔微球的管柱，其總流量為流經微球之間的空隙與微球內部的多孔網絡的總和，即Q_總合=Q_空隙+Q_孔洞，而流量與孔洞大小成正比。又依據式(2)，d _空隙 =0.225d _微球 ，則當尺寸比例d _孔洞 /d _微球 下降時，Q_空隙將會上升，使得物質傳輸傾向以擴散方式進行吸附。在此情況下，隨著流動相的流速提高，多孔微球的吸附量將會降低。反之，當尺寸比例d _孔洞 /d _微球 上升時，Q_孔洞將會上升，使得物質傳輸傾向以對流方式進行吸附。在此情況下，隨著流動相的流速提高，多孔微球的吸附量不會實質降低。又如前所述，本案個別微球的多孔網絡可以透過n個開口與外界連通，且n≧2，其中n為整 數。換言之，n為微球中多孔網絡連通外表面的開口的數目，其可透過掃描式電子顯微鏡拍攝多孔微球的影像，加以計算。因此，假設具有n/2個開口位於流動相的流入方向，另外n/2個開口位於流動相的流出方向，則(n/2)d _孔洞 ≧d _空隙 ，即d _孔洞 ≧(0.45/n)d _微球 。改以尺寸比例d _孔洞 /d _微球 表示：d _孔洞 /d _微球 ≧(0.45/n)..................(1)。本案多孔微粒滿足上式(1)，因而可以促使流動相以對流方式流經位於微球內部的多孔網絡，而減少流經微球之間的空隙，以達到高流速低背壓，而且具有高且穩定的吸附量。 According to Darcy's law, which is used to describe the flow of liquid through porous media:

Among them, eta : mobile phase viscosity; △ p : back pressure; Q: flow rate; A: flow channel cross-sectional area; △L: flow channel length. When the column, packing material and mobile phase remain unchanged, the cross-sectional area A of the flow channel is proportional to the flow rate Q. Assume that the holes of the porous medium are circular, then A=πr ² . According to the aforementioned definition of d _hole , then r

0.5 d _hole . Therefore, the flow rate will be directly proportional to the d _hole . In the present invention, the mobile phase flows through a column filled with porous microspheres, and the total flow rate is the sum of the gaps between the microspheres and the porous network inside the microspheres, that is, Q _total = Q _gaps + Q _hole , and the flow rate is proportional to the size of the hole. According to formula (2), d _void = 0.225 d _microsphere , then when the size ratio d _pore / d _microsphere decreases, Q _void will rise, making the material transport tend to be adsorbed by diffusion. In this case, as the flow rate of the mobile phase increases, the adsorption capacity of the porous microspheres will decrease. On the contrary, when the size ratio d _holes / d _microspheres rises, Q _holes will rise, making the material transport tend to be adsorbed in a convective manner. In this case, as the flow rate of the mobile phase increases, the adsorption capacity of the porous microspheres does not substantially decrease. As mentioned before, 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 microsphere that connect the porous network to the outer surface, which can be calculated by taking an image of the porous microsphere with a scanning electron microscope. Therefore, assuming that there are n/2 openings located in the inflow direction of the mobile phase, and n/2 openings located in the outflow direction of the mobile phase, then (n/2) d _holes ≧ d _voids , that is, d _holes ≧ (0.45/n) d _microspheres . Change to the size ratio d _holes / d _microspheres : d _holes / d _microspheres ≧(0.45/n).............(1). The porous particles in this case satisfy the above formula (1), so they can promote the mobile phase to flow through the porous network inside the microspheres in a convective manner, and reduce the flow through the gaps between the microspheres, so as to achieve high flow rate and low back pressure, and have high And stable adsorption capacity.

In a preferred embodiment, the d- _holes of the porous microspheres of this case can be larger than 150 nanometers, more preferably larger than 300 nanometers, for example, larger than 500 nanometers. In a preferred embodiment, the d _microspheres of the porous microspheres in this case can be less than 500 microns, more preferably less than 300 microns, such as less than 200 microns, in order to reduce the value of d _voids .

The microspheres disclosed in this case have high porosity, and macropores are evenly distributed in the microspheres. The porosity of porous microspheres is defined as the percentage of pore volume relative to the total volume of microspheres, which can be calculated by the following formula: 1-[(weight of porous microspheres/density of continuous phase)/porous microspheres Appearance volume] You can also use a scanning electron microscope to take cross-sectional images of porous microspheres, and then calculate the porosity through ImageJ software (National Academy of Sciences, Bethesda, MD, USA). In a specific example, 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. Polymer materials suitable for use in the present invention include, but are not limited to, polyacrylates, polymethacrylates, polyacrylamide, polystyrene, polypyrrole, polyethylene, polypropylene, polyvinyl chloride and polysiloxane. In a preferred embodiment, the porous microspheres are made of polymethacrylate.

With fast kinetic performance, high porosity, high mechanical properties and low back pressure, this stationary phase medium can be used to separate molecules with larger sizes, including molecules with a size exceeding 15 nm, including but not limited to proteins. (e.g., thyroglobulin (approximately 17 nm in size), nucleic acids (mRNA, 100 nm; DNA plasmid, 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) (such as exosomes, 30-100 nm) and liposomes.

In some embodiments, the stationary phase medium is chemically modified to have functional groups or ligands for adsorbing molecules. For example, in specific cases where a static phase 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. The exchange agent uses diethylamine ethyl (DEAE) as a weak anion exchanger, a sulfonyl group as a strong cation exchanger, and a carboxymethyl group as a weak cation exchanger.

The production of the static phase medium of the present invention involves emulsifying two immiscible phases to obtain a first emulsion, and then passing the first emulsion through a sieve plate provided with holes to disperse in the third phase to obtain a suspension suspended in the third phase. Microspherical high internal phase emulsion droplets with uniform size in three phases, and then these emulsion droplets are After solidification, a static phase medium in the form of porous microspheres is produced. Figure 7 shows a flow chart of a manufacturing method of a static phase medium according to the present invention. The method includes step A: preparing a first emulsion; step B: dispersing the first emulsion in the third phase to obtain an emulsion microdisperse containing high monodispersity internal phase. droplets of the second emulsion; 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" as used in the present invention refers to a phase that is connected to each other and is composed of the same composition, while the "dispersed phase" is a phase that is composed of many mutually isolated composition units distributed in the aforementioned continuous phase. Each isolated unit in the dispersed phase is surrounded by the continuous phase. According to the present invention, the continuous phase is usually the phase in which the polymerization reaction occurs, and may contain at least one monomer and cross-linking agent, and optionally an initiator and an emulsion stabilizer, while the dispersed phase may include 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 monomers and oligomers that can form polymers through polymerization reactions. In a preferred embodiment, the at least one monomer includes at least one ethylenically unsaturated monomer or acetylenically unsaturated monomer suitable for free radical polymerization, or That is, organic monomers with carbon-carbon double bonds or parabonds, 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, For example, chloromethylstyrene, divinylbenzene (DVB), styrene sulfonate; silanes, such as dichlorodimethylsilane; pyrrole; vinylpyridine, and combinations thereof.

The term "cross-linking agent" used in this case means an agent that forms chemical bridges between the polymer backbones formed by the polymerization reaction of at least one of the aforementioned monomers. In a preferred embodiment, the "cross-linking agent" is a cross-linking monomer, which can be co-soluble in the continuous phase with at least one of the aforementioned monomers, and usually has multiple functional groups, so that when the aforementioned at least one monomer is processed Covalent bonds are formed between polymerized polymer backbones. Suitable cross-linking agents are well known in the art and can 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, For example, N,N-diallylacrylamide and N,N'-methylenebisacrylamide (MBAA). As those with ordinary knowledge in the technical field of the present invention know, the amount of cross-linking agent is positively correlated with the mechanical strength of the porous monolithic column, that is, the higher the degree of cross-linking, the higher the mechanical strength of the porous monolithic column. . Preferably, the crosslinking agent accounts for about 5 to 50% by weight of the continuous phase, for example, about 5 to 25% by weight.

In addition to monomers and cross-linkers, 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, cyclodextrin (dextran), agarose, agarose, alginate; inorganic materials, such as oxidized Silicon; and graphene. For example, the addition of Fe ₃ O ₄ particles can improve the mechanical strength of the porous microstructure and impart ferromagnetic properties.

The term "emulsion stabilizer" as used in this case means a surfactant suitable for stabilizing high internal phase emulsions and preventing the dispersed phase units in the emulsion that are separated by the continuous phase from merging with each other. The emulsion stabilizer can be added to the continuous phase composition or dispersed phase composition before preparing the emulsion. Emulsion stabilizers suitable for use in the present invention may be nonionic surfactants, or anionic or cationic surfactants. In a specific example where the high internal phase emulsion is a water-in-oil emulsion, the emulsion stabilizer preferably has a hydrophilic-lipophilic balance (HLB) value of 3 to 14, and more preferably has an HLB value of 4 to 6. . In a preferred embodiment, the present invention uses nonionic surfactants as emulsion stabilizers. Applicable types include but are not limited to polyoxyethylene alkanols, polyoxyethylene linear alkanols, polyoxyethylene Polypropylene glycols, polyoxyethylene thiols, long-chain carboxylic acid esters, alkanolamine condensates, quaternary acetylenic glycols, polyoxyethylene polysiloxanes, N-alkylpyrrolidone , 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 Ester (trade name Span ^® 80), glyceryl monooleate, polyethylene glycol 200 dioleate, polyoxyethylene-polyoxypropylene block copolymer (such as Pluronic ^® F-68, Pluronic ^® F-127, Pluronic ^® L-121, Pluronic ^® P-123), castor oil, glyceryl monoricinoleate, distearyldimethylammonium chloride, and dioleyldimethylammonium chloride.

"Initiator" means a reagent capable of initiating the polymerization reaction and/or cross-linking reaction of at least one of the aforementioned monomers and/or cross-linking agents. Preferably, the initiator used in the present invention is a thermal initiator. That is, an initiator that can initiate the aforementioned polymerization reaction and/or cross-linking 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, starters suitable for addition to the continuous phase composition include, but are not limited to, azobisisobutyronitrile (AIBN), azobisisoheptanitrile (ABVN), azobisisovaleronitrile, 2,2- Bis[4,4-di(tert-butylperoxy)cyclohexyl]propane (BPO), and lauryl peroxide (LPO), and starters suitable for addition to the dispersed phase composition include but are not limited to Sulfates such as ammonium persulfate and potassium persulfate. The high internal phase emulsion in this case may also contain a photoinitiator activated by ultraviolet light or visible light to initiate the aforementioned polymerization reaction and/or crosslinking reaction, and the aforementioned thermal initiator may even be replaced by an appropriate photoinitiator.

The dispersed phase mainly contains solvent. The solvent can be any liquid that is immiscible with the continuous phase. In specific examples where the continuous phase is highly hydrophobic, the solvents include, but are not limited to, water, fluorocarbon liquids, and other organic solvents that are immiscible with the continuous phase. Preferably the solvent is water. In this example, the dispersed phase may further include an electrolyte that can substantially dissociate into free ions in the solvent, including salts, acids and bases that are soluble in the solvent. Preferably the electrolyte contains alkali metal sulfate salts, such as potassium sulfate, and alkali metal and alkaline earth metal chloride salts, such as sodium chloride, calcium chloride and magnesium chloride.

Polymerization accelerators can be added to high internal phase emulsions. "Accelerator" means an agent capable of accelerating the polymerization reaction and/or cross-linking reaction of the at least one monomer and/or cross-linking agent. Typical examples of accelerators include, but are not limited to, N,N,N',N'-tetramethylethylenediamine (TEMED), N,N,N',N",N"-pentamethyldiethylenetriamine Amine (PMDTA), tris(2-dimethylamino)ethylamine, 1,1,4,7,10,10-hexamethyltriethylenetetramine, 1,4,8,11-tetramethyl -1,4,8,11-tetraaza Cyclotetradecane promotes the decomposition of initiators such as ammonium persulfate into free radicals, thereby accelerating the aforementioned polymerization reaction and/or cross-linking reaction. The added amount of accelerator is preferably 10-100 mol% of the added amount of starter.

The process of obtaining the first emulsion through emulsification is to first uniformly mix components such as monomers and cross-linking agents to form a continuous phase composition, and then uniformly mix components such as solvents and electrolytes to form a dispersed phase composition. Then, a predetermined ratio of the continuous phase composition and the dispersed phase composition is mixed, for example, the continuous phase composition and the dispersed phase composition are mixed at a volume ratio of 5:95 to 40:60, and disturbance is applied to make the dispersed phase Evenly dispersed in the continuous phase. In a specific example, the dispersed phase composition can be slowly added dropwise to the continuous phase composition while being vigorously disturbed to form an emulsion. In another and preferred specific example, the entire batch of the dispersed phase composition can be directly added to the continuous phase composition at one time while being violently disturbed to form an emulsion. In a preferred embodiment of adding the dispersed phase composition in a whole batch, a high-speed homogenizer can be used to stir vigorously and apply high shear force to the emulsion, thereby making the size of the dispersed phase in each isolation unit uniform. As is well known to those with ordinary knowledge in the relevant technical fields, it can be achieved by changing the volume ratio of the dispersed phase relative to the continuous phase in the emulsion, the addition rate of the dispersed phase components, the type and concentration of the emulsion stabilizer, and the rate and temperature of the disturbance. Parameters to adjust the size and uniformity of individual isolation units in the dispersed phase.

In a specific example, the first emulsion obtained through the aforementioned emulsification is a high internal phase emulsion. The term "high internal phase emulsion" or simply "HIPE" used in the present invention means an emulsion in which the volume fraction of the internal phase exceeds 74.05%. According to the present invention, in step B, the first emulsion can be mixed with the third phase, and then the first emulsion can be evenly dispersed in the third phase through a microdroplet device, thereby obtaining a mixture containing the third phase and dispersed in the third phase. A second emulsion of monodisperse highly internal phase emulsion droplets within the third phase.

The term “third phase” 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 primarily contains 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 can substantially dissociate into free ions in the solvent, including salts, acids and bases soluble in the solvent. Preferably the electrolyte contains alkali metal sulfate salts, such as potassium sulfate, and alkali metal and alkaline earth metal chloride salts, such as sodium chloride, calcium chloride and magnesium chloride. The third phase may also include the emulsifying stabilizer described above.

In a preferred embodiment, the first emulsion can be added to the third phase first, and then a shearing device is used to apply shearing force to prepare the first macroemulsion dispersed in the third phase. The shearing force The device can be a mechanical stirring device or a pore-arranged 3D structure. Then, the first macroemulsion is further dispersed in the third phase into microdroplets through a microdroplet device, so as to obtain a highly monodispersed emulsion containing the third phase and dispersed in the third phase. The second emulsion of the internal phase emulsion droplets. The droplet device may be a sieve plate structure with a plurality of fine channels (the channels are not limited to straight lines, approximately straight lines, smooth curves, or approximately smooth curves), or the droplet device may be a 3D structure with pores arranged for A large number of monodisperse high internal phase emulsion droplets are produced. The sieve plate provided with 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, ceramics, glass, quartz, silicon wafer or polyvinyl chloride ( PVC), polyoxymethylene (POM), polycarbonate (PC), polyphenylene ether (PPO), PA6/66 nylon plastic, polycarbonate/acrylonitrile-butadiene-styrene copolymer (PC/ABS) composite Plastic, polyethylene terephthalate (PET), polyethylenimine (PEI), polymethacrylic acid Plastic materials such as methyl ester (PMMA), polyphenylene sulfide (PPS), polyethylene (PE), polypropylene (PP), polystyrene (PS), ethylene/vinyl acetate copolymer (EVA), or stainless steel , titanium, aluminum, aluminum-magnesium alloy and other metal materials.

The high internal phase emulsion droplets dispersed in the third phase will spontaneously form into a substantial spherical shape due to their own cohesion. The size of the high internal phase emulsion droplets can be adjusted by selecting the pore 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 wavelengths, or a promoter can be further added to allow the aforementioned at least one monomer and/or cross-linking agent to complete the polymerization reaction and/or cross-linking reaction to solidify. The term "curing" here refers to the process of converting a 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, thereby producing a static phase medium in the form of porous microspheres. In a specific example where the first emulsion is a water-in-oil emulsion, the solidified high internal phase emulsion droplets can be dried directly, preferably under vacuum, which helps to break the isolation units in the dispersed phase. Create connected 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 process of the first emulsion, and the size of the connected pores, and the porous network formed in the porous microspheres, etc. The effective diameter 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 sieved through step D using a Taylor sieve to exclude microspheres that are too large, too small or broken, so as to collect microspheres within the desired size range. ball.

Table 1 shows the porous microsphere structure produced by the aforementioned manufacturing method and satisfying formula (1), which has four sizes: A, B, C, and D.

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°C for at least 5 hours. The porous microspheres are filtered out, then added to a 1% aqueous solution of glycidyltrimethylammonium chloride, and heated at 70°C for at least 5 hours. The porous microspheres were washed with water to obtain four strong anion exchangers with porous microspheres A, B, C and D as the matrix.

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

It can be seen from Figure 8 that under the premise that porous microspheres A, B, C and D have substantially the same particle size ( d _microsphere ), porous microsphere A ( d _pores = 1.0 micron) has the smallest porous network diameter. The adsorption capacity is 5-7mg/mL, and its adsorption capacity has an obvious downward trend as the flow rate of the mobile phase increases. This shows that when the size ratio of microspheres d _holes / d _microspheres is smaller, TGY tends to adsorb in a diffusion manner. As for the porous microspheres B ( d _pores =1.4 microns), C ( d _pores =1.8 microns) and D ( d _pores =2.2 microns) with relatively large porous network diameters, their adsorption capacities are 6-8mg/mL respectively. , 9-11 mg/mL and 14-15 mg/mL, the decrease in adsorption capacity slows down as the flow rate of the mobile phase increases, and the adsorption capacity trend can still be maintained at higher flow rates. Thereby, the material transport mode in the chromatography column can be adjusted by adjusting the size ratio of porous microspheres d _holes / d _microspheres . That is to say, increasing the size ratio of microspheres d _holes / d _microspheres can make the material tend to be transported in a convective manner between the mobile phases, making the adsorption behavior less affected by the flow rate.

Test result 2: Back pressure test

Figure 9 shows the back pressure curves of four chromatography columns made of A, B, C and D microspheres at different linear flow rates. The results show that the four chromatography 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 (>600cm/hr) are also much smaller than those shown in Figure 3A The effect of the flow rate on the back pressure value of the known technology. In this embodiment, even for sample A, the back pressure/flow rate slope of 7.5x10 ^-5 MPa cm hr ^-1 is much smaller than the 130 or 62 x10 ^- of the conventional technology. ⁵ MPa cm hr ^-1 , fully highlighting the advantages of the chromatography column made using the microspheres of the embodiments 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 the slope value of the fluid back pressure relative to the fluid flow rate is 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 above preferred specific examples, it should be recognized that the preferred specific examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and changes may be made that are obvious to those skilled in the relevant art. without departing from the spirit and scope of the invention.

Claims (4)

A method for manufacturing a static phase medium, which includes the following steps: A) in the presence of a polymerization initiator and an emulsion stabilizer, a continuous phase composition containing at least one monomer and a cross-linking agent, emulsify with a dispersed phase composition containing a solvent to obtain a first emulsion containing a continuous phase and a dispersed phase dispersed in the continuous phase; B) combine the first emulsion and a dispersion phase that is not mixed with the first emulsion. An emulsion-phase miscible third phase is mixed and a shearing device is used to apply shearing force to prepare a first macro-drop emulsion dispersed in the third phase, and then the first macro-droplet is dispersed through a micro-droplet device. The emulsion is further dispersed evenly in the third phase into droplets to obtain a second emulsion including the third phase and monodisperse high internal phase emulsion droplets dispersed in the third phase; and C ) Solidify the continuous phase of the second emulsion, and remove the dispersed phase and the third phase to obtain a static phase medium composed of porous microspheres; wherein each porous microsphere is formed with many spherical macropores, and the spheres -shaped macropores are connected to each other through connecting holes, thereby forming an open porous network. The porous network is in fluid communication with the outside world through multiple openings located on the outer surface of the microsphere; each of the porous microspheres satisfies In the following formula (1): d _holes / d _microspheres ≧(0.45/n).............(1) where d _holes are the equivalent of the porous network Diameter, d _microsphere is the particle size of the porous microsphere, and n is the number of openings in the microsphere that connect the porous network to the outer surface, and n≧2, n is an integer. The method of claim 1, wherein the method of preparing the first macroemulsion dispersed in the third phase includes arranging a 3D structure with a mechanical stirring device or pores to provide shear force. The method of claim 1, wherein the droplet device can be a sieve plate structure with a plurality of fine channels (channels) or the droplet device can be a pore-arranged 3D structure. The method of claim 1, further comprising a step D after step C, in which the porous microspheres obtained in step C are sieved through a Taylor screen to exclude microspheres that are too large, too small or broken.