Charged isoporous material for electrostatic separation

Abstract

The present invention relates to isoporous multiblock polymeric materials, collectively filters / separators comprising the materials, methods for their preparation and their use for separating solutes, including biomolecules, via electrostatic interactions. [Solution] The present invention relates to a multi-block polymer material, its preparation method and use for separating proteins, nucleic acids, other biological or other biomolecules, compounds or solutes with high flux via electrostatic interactions, wherein the self-assembled block polymer material has at least one of macropores, mesopores or micropores, at least a portion of the pores are isoporous, and at least one polymer block has an electrostatically charged or reactive functional group to provide a large charged surface area in the isoporous structure.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority based on U.S. Provisional Patent Application No. 62 / 328,707, filed on April 28, 2016, the entire content of which is incorporated herein by reference.

[0002] This application relates to a multi - block polymer separator comprising an isoporous material for separating solutes via electrostatic interaction, a method for preparing the same, and its use.

Background Art

[0003] Despite major advances in understanding protein expression, structure, and function, problems remain in the purification of proteins from complex mixtures. Often, target molecules in cell culture supernatants include a wide variety of exogenous or adventitious components that increase the complexity of separation, such as host proteins, cell debris, DNA, viruses, and endotoxins. Target proteins are often very sensitive to environmental conditions and can be easily denatured or otherwise damaged.

[0004] Upstream technologies can provide product titers exceeding 5 g / L, driving the need for high - throughput purification. Conventionally, the purification of biomolecules has relied heavily on chromatography, including affinity chromatography and ion - exchange columns. However, these chromatography techniques have limitations in throughput and are a major contributor to the high cost of downstream bioprocesses.

[0005] Ultrafiltration (UF) is frequently used in the later stages of downstream bioprocesses for protein concentration and formulation. For example, there are commercially available UF membranes that are track-etched or phase-inverted. Track-etched membranes have very uniform pores that allow for selective separation, but suffer from low throughput due to low pore density. On the other hand, phase-inverted membranes allow for much larger fluxes, but have non-uniform pores and cannot achieve selective separation. Due to flux limitations, track-etched membranes are almost exclusively used for bench-scale research, while phase-inverted membranes are widely used in downstream biopharmaceutical processes. Phase-inverted membranes empirically exhibit a wide range of log-normal pore size distributions and require a 6- to 10-fold difference in protein molar mass for effective fractionation. This pore size variation causes non-uniform flow patterns across the membrane, broadens the breakthrough curve, and decreases media capacity.

[0006] Lack of membrane structure and chemical functionality greatly limits its use in concentration and formulation steps. Therefore, there is a need to improve protein purification methods, both for laboratory-scale up validation and, of course, for commercial-scale manufacturing of medically relevant proteins. The applicant has found that by combining unique tuning of the physical structure of isoporous materials with chemical functionality, high-throughput and high-resolution separation applications that overcome the limitations of the prior art can be achieved by promoting and separating solutes of similar size via electrostatic interactions, enabling the production of, for example, isoporous charged multi-block polymer materials such as films, membranes, sheets, and tubes.

[0007] The separation of other biomolecules from complex mixtures can also be particularly difficult. For example, nucleic acids can be isolated from whole blood for diagnostic purposes. The isolation of DNA from whole blood is a particularly difficult separation due to solutes of various length scales, including red blood cells, proteins, and salts. One method of DNA isolation is to use electrostatic interactions to isolate negatively charged DNA from a complex mixture. Nevertheless, an effective membrane technology for isolating DNA from whole blood has not been commercialized because separation via existing membrane technologies is difficult. The applicant has found that the invention described herein enables the separation and concentration of nucleic acids from whole blood.

[0008] Charged porous materials are effective in preventing biofilm development. For example, in using such materials as wound dressings, the porous materials allow the passage of gaseous chemical substances such as water vapor, nitrogen, and oxygen. However, the development of biofilms can prevent wound healing. The charged porous materials described herein can be used to prevent the development of biofilms. In addition, the charged porous materials can be further functionalized with antibacterial chemicals to further enhance wound healing properties and contribute to wound care applications.

[0009] Chromatography columns have also been widely used to separate species based on various physical and chemical characteristics, including charge and size. However, chromatography has limitations in throughput and is often expensive. The charged porous materials described herein can be used to separate solutes based on charge and size. For example, species bound to a membrane via electrostatic interactions can be detached from the membrane by passing a gradient of eluent through the material. In this way, gradient separation can be performed. Alternatively, by passing an eluent of a fixed composition through the membrane, the species of interest can be detached from the membrane. The fixed composition is suitable for detaching only the species of interest desired. In this way, separation at a uniform concentration can be performed. Furthermore, a series of elutions can be performed, each elution being of a fixed composition suitable for detaching further species of interest. In this way, step elution can be performed.

[0010] There has been strong interest in fractionating solutes, including biomolecules, using the combined effects of charge and size. However, a major limiting factor in the further development of this promising approach is the non-uniform pore sizes of existing membrane platforms. Figure 1 illustrates the problem due to pore size distribution. Regions of larger pore size (5) carry more fluid flow, as indicated by arrow (30) which represents a greater portion of the flow than that represented by arrow (35), than regions of smaller pore size (10). Charged sites are rapidly filled with bound species in regions of larger pore size, allowing such species to escape from the membrane, while regions of smaller pore size continue to bind. Thus, breakthrough of the species of interest for binding occurs at a lower throughput than would occur if all pores were of uniform size.

[0011] The applicant solved this problem by increasing the uniformity of the pore size and reducing the spread of the normal logarithmic pore size distribution of the historical membrane platform. For example, in one embodiment of the membrane of the present invention, as shown in FIG. 2, a single layer (15) of substantially the same pores is formed on the downstream surface of the membrane. This layer contributes to most of the resistance to the flow that evenly distributes the flow, as indicated by the flow arrows (20) and (25) of equal size, regardless of the pore size distribution in the upstream portion of the membrane.

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Non-Patent Documents

[0013]

Non-Patent Document 1

Non-Patent Document 11

Summary of the Invention

Problems to be Solved by the Invention

[0014] The present invention relates to an isoporous multi-block polymer material for separating a solute containing a biomolecule through electrostatic interaction, a filter / separator having the material as a whole, a method for preparing the same, and its use.

Means for Solving the Problems

[0015] The charged self-assembled block polymer material of the present invention has at least one of macropores, mesopores or micropores, at least a part of the pores is isoporous, and at least one polymer block has a static charge or a reactive functional group. The material has major, minor and interstitial regions that are partially or quantitatively functionalized by the charged portion on the functional group, resulting in a static charge on the material, and provides a charged large surface and an isoporous structure for the easy separation of proteins, nucleic acids, other biological or other biomolecules or solutes at high flow rates.

[0016] The isoporous charged multi-block polymer material of the present invention includes at least blocks A, B and C having at least one of macropores, mesopores or micropores, at least a part of the pores is isoporous, and at least one polymer block has a static charge or a reactive functional group, and further, the following formula A-B-C (I), A-B-C-B (II) A-B-C-D (III) A-B-C-B-A (IV) A-C-B-C-A (V) is defined by.

[0017] The multi-block polymer of the present invention is generally defined as having blocks A, B, C, (X)n, where n = 0 to 7, X may be the same or different, and is selected from A, B, C, D, E, F or G. One of the blocks suitable for A, B or C is a low Tg block (≤25 °C), but if one of the blocks is a low Tg block, at least one other block is a high Tg block (>25 °C) under the condition. At least one block must have a static charge or a reactive functional group that can react to generate a static charge. Blocks suitable for D, E, F or G include: poly(butadiene), poly(isobutylene), poly(butylene), poly(isoprene) poly(ethylene), poly(styrene), poly(methyl acrylate), poly(butyl methacrylate), poly(ether sulfone), poly(methyl methacrylate), poly(n-butyl acrylate), poly(2-hydroxyethyl methacrylate), poly(glycidyl methacrylate), poly(acrylic acid), poly(acrylamide), poly(sulfone), poly(vinylidene fluoride), poly(n,n-dimethylacrylamide), poly(2-vinylpyridine), poly(4-vinylpyridine), poly(ethylene glycol), poly(propylene glycol), poly(vinyl chloride), poly(tetrafluoroethylene), poly(siloxane), poly(ethylene oxide), poly(propylene oxide), poly(n-isopropylacrylamide), poly(dimethylaminoethyl methacrylate), poly(amic acid), poly(dimethylsiloxane), poly(lactic acid), poly(isocyanate), poly(ethyl cyanoacrylate), poly(ethylene glycol methyl ether methacrylate), poly(acrylonitrile), poly(hydroxystyrene), poly(α-methylstyrene), poly(ethyleneimine), poly(styrene sulfonic acid), poly(allylamine hydrochloride), poly(pentafluorostyrene), poly(2-(perfluorohexyl)ethyl methacrylate), but are not limited thereto.

[0018] The present invention includes, without limitation, materials such as three-dimensional membranes, films, sheets, tubes, or those in the form of helices or spirals, whether supported or unsupported, including materials containing a functionalized isoporous block polymer having charged moieties, and rendering the materials suitable for separation based on high-throughput charges.

[0019] The present invention includes an isoporous multi-block polymer having a stationary charge that promotes solute separation based on the electrostatic characteristics and size of the solute.

[0020] The present invention includes highly fluid functionalized separators for protein fractionation and isolation for analytical and industrial protein fractionation.

[0021] The present invention relates to an isoporous material, which, whether in a laboratory, scale-up, or commercial environment, promotes the separation of molecules of similar size, such as proteins, through, for example, the electrostatic repulsion between one positively charged protein and one neutral protein for protein production and analysis, and promotes the separation of molecules of similar size, such as biomolecule-specific molecules including patient-specific biomolecules.

[0022] The present invention relates to an isoporous material that promotes the separation of proteins of similar size through electrostatic interactions, a method for preparing the same, and its use. The isoporous material promotes the separation of nucleic acids through electrostatic interactions and promotes the separation of charged biomolecules through electrostatic interactions.

[0023] The present invention relates to an isoporous material that promotes the selective separation of matrix metalloproteinases through electrostatic interactions.

[0024] The present invention relates to charged isoporous materials of various configurations, such as three-dimensional films, tubes, spirals, sheets, etc.

[0025] The charged isoporous material has a selectivity of at least 10 and / or a permeability of 50 Lm for electrostatically separating similar-sized proteins having different charge states. -2 hr -1 bar -1 and has a permeability of.

[0026] The present invention includes a self-assembled multi-block polymer or self-assembled multi-block copolymer (MBP or BCP) material having at least one chemically reactive functionalized polymer block that forms a hierarchically porous material having a large surface and uniform pore size as disclosed herein. The reaction regions of these isoporous materials are partially or quantitatively functionalized by the reaction of reactive units with terminal or crosslinking agents such that the degree of charge generates a static charge on the material, for example, at least 20% to 90%, such as at least 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the available reactive units.

[0027] The charged material of the self-assembled multi-block copolymer has mesopores with sizes ranging from about 1 to 200 nm, the size of the entire unit, and sizes in between, and macropores with sizes of at least 50 nm or more.

[0028] >50 Lm -2 hr -1 bar -1 and >100 Lm -2 hr -1 bar -1 and >200 Lm -2 hr -1 bar -1 Disclosed herein is the charged isoporous material of the self-assembled multi-blocks described herein that effectively separates one or more charged solutes with a water permeability of, which drives high-throughput bioprocesses of proteins, nucleic acids, therapeutic compounds or other molecules or compounds of interest. The dual separation methodology of size exclusion and charge interaction drives high-throughput bioprocesses of proteins, nucleic acids, therapeutic compounds or other molecules or compounds of interest.

[0029] To separate proteins of similar size via the electrostatic repulsion between one positively charged protein and one neutral protein, an isoporous charged material of self-assembled multi-blocks having a stationary positive charge is disclosed.

[0030] To separate proteins of similar size via the electrostatic repulsion between one positively charged protein and one neutral protein, an isoporous charged material of self-assembled multi-blocks having a stationary negative charge is disclosed.

[0031] An isoporous charged material of self-assembled multi-blocks having a stationary positive charge for separating nucleic acids via charge interaction is disclosed.

[0032] An isoporous charged material of self-assembled multi-blocks having a stationary positive charge for separating biomolecules via charge interaction is disclosed.

[0033] An isoporous charged material of self-assembled multi-blocks having a stationary negative charge for separating biomolecules via charge interaction is disclosed.

[0034] Since all species in contact with the isoporous separation layer are forced to experience the same electrostatic environment, the isoporous nature of pores with sizes ranging from 5 to 100 nm, the size of all units, and the sizes in between is particularly beneficial for charge separation. If the pore size is too polydisperse, solutes may experience different charge environments. For example, since electrostatic charge interactions decrease dramatically as a function of distance, there will be no effect of charge separation as they pass through the center of large pores. High porosity, and in some cases a hierarchical structure, contribute to a large surface area and thus to a large amount of charge available for separation. In an operation using binding and elution mechanisms, this also provides a large binding capacity.

Brief Description of the Drawings

[0035]

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Mode for Carrying Out the Invention

[0036] In the following description, reference can be made to the accompanying drawings that form a part of this specification, and in the drawings, specific embodiments that can be implemented are shown by way of example. These embodiments are described in detail so as to enable those skilled in the art to practice the present invention, and it should be understood that other embodiments may be utilized and that structural, logical, and electrical changes may be made without departing from the scope of the present invention. Accordingly, any description of examples, embodiments, or experimental procedures should not be construed in a limiting sense.

[0037] The complexity of either or both the target molecule solution and the target molecule itself often requires several steps, is time-consuming and expensive processes, all of which are exacerbated by the need to balance effective separation and high throughput. Two important performance parameters that will be improved through the chemical functionalization of the isoporous membrane are

[0038]

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[0039]

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[0040] Separation involving nanoporous membranes or ultrafiltration membranes has typically focused on size selectivity, but electrostatic interactions significantly impact the selectivity and permeability of protein solutes. The isoelectric point (pI) of a protein is the pH at which the biomolecule exhibits no net charge. Below the pH of the protein pI, the molecule is positively charged, while above the protein pI, the molecule is negatively charged. Such charge characteristics have been exploited in protein filtration using nanoporous or ultrafiltration membranes by adjusting the pH and ionic strength of the solution and improving selectivity and throughput by up to an order of magnitude. Furthermore, there is a strong relationship between the size and charge of proteins, and the effective protein size increases as the ionic strength decreases due to reduced electrostatic shielding. Recently, it has been shown that protein selectivity can be further improved by combining charged nanoporous or ultrafiltration membranes with processing conditions.

[0041] One key differentiating the features of the present invention from conventional charge-based membrane separations is the use of a concentrated pore distribution in the structure of the isoporous membrane as opposed to nanoporous membranes or ultrafiltration membranes with a broad pore size distribution. This enables simplified processing conditions and high operating pressures, resulting in overall higher permeability.

[0042] Previous studies on the separation of optimized charged ultrafiltration membranes in a tangential flow configuration where the feed solution passes parallel to the membrane surface have resulted in an average permeate flux of about 15 - 200 Lm -2 hr -1 . In contrast, the self-assembled membranes of the present invention with a narrow "concentrated" pore size distribution exhibit a permeate flux of up to 850 Lm -2 hr -1 . For example, although certain performance limiting factors such as concentration polarization and membrane fouling are enhanced at high fluxes (Non-Patent Document 2), the combination of physical membrane characteristics with electrostatic properties enables performance that surpasses protein separation based on existing membranes.

[0043] A suitable isoporous BCP material film for the present invention is self-assembling. Typically, the preparation of such an isoporous organic film involves dissolving a BCP (or MBP) polymer in one or more solvents that are at least partially volatile, casting the solution under conditions defined such that at least a portion of the solvent evaporates, and then contacting the resulting material, such as a membrane / film, with a phase-separating solvent system. Such procedures are generally described, for example, in Patent Documents 1, 2, 3, 4, 5, 6, 7, 8, Non-Patent Documents 3, 4, 5, and 6, each of which is incorporated herein by reference in its entirety.

[0044] Another method of forming a porous membrane from a suitable block polymer is the "SIM" 2 PLE" (Spinodal decomposition-induced macrophase separation and mesophase separation plus extraction by rinsing) process as described in Non-Patent Document 7. In this process, a suitable BCP is first prepared, mixed with another oligomeric polymer in a suitable organic solvent, the mixed solution is cast, and a hierarchically porous membrane is formed by partial evaporation of the solvent at an elevated temperature. In the final step, the oligomeric polymer is removed from the resulting membrane (of the BCP) by rinsing. The thickness, pore size, and structure of the membrane can be adjusted as desired by the selection of the solvent and polymer concentrations, the time and temperature of solvent removal, and other experimental factors. "SIM" 2 A suitable second oligomeric polymer for the "PLE" process is water-soluble and can be removed from the BCP by washing with water. In one embodiment, other soluble oligomeric polymers such as poly(ethylene oxide) PEO can also be used, but poly(acrylic acid) or PAA is used as the second oligomeric polymer.

[0045] In one embodiment, the self-organizing block copolymer comprises a polymer block having a low Tg (≤ 25°C). This low-Tg block provides mechanical toughness and reduces the processability and handleability of the material. Another block should have a Tg > 25°C to provide mechanical stability. At least one of the blocks should be a polymer that can be chemically modified to introduce charge into the material or that already has at least one charged functional group.

[0046] In one embodiment, the polymer block having a functional group has an aromatic nitrogen heterocycle as a chemically modifiable functional group. In this embodiment, suitable aromatic heterocycles include, but are not limited to, pyridine, pyrazine, pyrimidine, pyridazine, quinoline, isoquinoline, quinoxaline, quinazoline, phenazine, isoxazole, isothiazole, imidazole, benzimidazole, triazole, tetrazole, and the like. Preferred aromatic nitrogen heterocycles do not have ionizable hydrogen on the nitrogen; that is, the nitrogen is a complete part of the aromatic ring. Pyridine and its derivatives are particularly preferred heterocycles. Other examples of polymers having an aromatic heterocyclic group include, but are not limited to, poly(2-vinylpyridine) or poly(4-vinylpyridine) (P2VP or P4VP).

[0047] In one embodiment of the present invention, the block used to form a self-organizing isoporous material such as a membrane, for example, is ISV, triblock or terpolymer poly(isoprene-b-styrene-b-4-vinylpyridine). Refer to Figure 3 showing the blocks (I, S, V corresponding to the inclusive structure ABC).

[0048] In this embodiment, the triblock terpolymer structure has about 0.30 volume fraction of polyisoprene (PI), 0.55 volume fraction of polystyrene (PS), and 0.15 volume fraction of poly(4-vinylpyridine) (P4VP). The volume fractions of PI, PS, and P4VP range from: 0.20 ≤ PI ≤ 0.40 (e.g., block A); 0.45 ≤ PS ≤ 0.65 (e.g., block B); 0.05 ≤ P4VP ≤ 0.35 (e.g., block C). The structure of this material provides good mechanical properties while allowing for small pore diameters. The starting materials for the ISV terpolymer may be synthesized via anionic polymerization. In some embodiments, an isoporous (narrow pore size distribution) film has a surface layer (also referred to herein as the top layer) and a bulk layer. A suitable surface layer has a certain range of thicknesses. For example, the surface layer may have a thickness ranging from 5 nm to 500 nm, including all values up to nm and the ranges therebetween. The surface layer has a plurality of pores extending across the depth of the surface layer. The pore diameter (e.g., diameter) in the surface layer ranges from 1 nm to 200 nm, including all values up to nm and the ranges therebetween.

[0049] In one embodiment of the present invention, the density of the surface pores of the film described herein is at least 10 14 pores / m 2 or at least 10 15 pores / m 2 . The narrow or concentrated pore size distribution (defined as the ratio of the maximum pore diameter to the minimum pore diameter (dmax / dmin)) is between 1 and 3, including all values up to 0.1 and the ranges therebetween.

[0050] In various examples, (dmax / dmin) is less than 3, such as 1, 1.5, 2, 2.5, or 3 and all ranges thereof. For example, the film includes a surface layer having substantially monodisperse mesopores. In one embodiment, the isoporous surface layer has a pore density of at least 1 × 10 14 pores / m 2 and a pore size distribution (dmax / dmin) of less than 3.

[0051] In some of the above embodiments, the P4VP material of the ISV terpolymer is present on the surface of the as-cast material matrix and is confirmed by both the pH-dependent water permeability of the membrane showing a flexion point close to the pKa of P4VP and the stained transmission electron microscope images showing the nanometer-scale phase separation of each block.

[0052] As described in some (53 - 55) of the above embodiments, the isoporous material is present on the surface of the membrane, has a polymer block having a nitrogen heterocycle available for the quaternizing agent, and the surface includes the surface of the pore gaps. For example, in the above embodiment where the copolymer has P4VP (weak base), P4VP is quaternized using various chemicals, and as shown in FIG. 4, stationary positive charges occur on the membrane.

[0053] The quaternizing agent in this embodiment is selected to optimize the purification of the specific solute targeted. There are many characteristics of agents that can be applied in useful methods. For example, the degree of hydrophilicity / hydrophobicity of the charged material can be an important consideration for some proteins. To control hydrophilicity / hydrophobicity, the quaternizing agent can be selected, for example, according to the ClogP value or range of the residue (a measurement value reflecting the degree of hydrophilicity / hydrophobicity). For example, if a more hydrophilic environment is desired, a quaternizing agent such as iodoacetamide, iodoacetic acid, or crotonic acid that leaves a relatively low ClogP in the material may be selected. If a more hydrophobic environment is desired, a quaternizing agent such as dodecyl iodide that leaves a relatively high ClogP in the material may be selected. There are other measurement criteria for the degree of hydrophilicity / hydrophobicity other than ClogP that can be used. For example, there may be an optimal value or range for the contact angle of the material after quaternization. The introduced functional groups (e.g., carboxylic acid, amide) may also be useful for subsequent chemical reactions or may have useful functionality (e.g., pH response, thermal response, solvent resistance).

[0054] The quaternizing agent in the above embodiment is any suitable compound that reacts with the nitrogen heterocycle of BCP to form a positively charged heterocycle. One feature of the present invention is that the degree or amount of charge can be varied to control the amount of positive charge. This enables optimization of the total charge on the purified protein. The charge density of the membrane after quaternization (corresponding to the degree of quaternization) can be determined by FTIR and NMR.

[0055] One group of quaternizing agents of the above embodiment is a compound defined by the formula R-X, where R is a C1-C 24 , more preferably a C1-C 20 substituted or unsubstituted linear, cyclic or branched alkyl or alkenyl group, and X is a halide (F-, Cl-, I-, Br-) or a leaving group such as --OSO2CH3. Typically, the leaving group is a terminal group (i.e., a group located at the end) on an alkyl or alkylene group and is bonded to an unsubstituted sp 3 carbon (i.e., -CH2-X) to maximize reactivity. The alkyl or alkylene group may have 1 to 24 carbon atoms in the backbone. Examples of linear alkyl groups include, but are not limited to, methyl, ethyl, butyl, hexyl, octyl, decyl and dodecyl. An example of a cyclic alkyl group is -CH2-cyclohexane.

[0056] Examples of branched alkyl groups of the above embodiment are -CH2-CH(CH3)2 and -CH2CH2-CH(CH3)2. An example of an alkenyl group would be geranyl (trans-3,7-dimethyl-2,6-octadien-1-X). An example of a substituted alkyl group would be benzyl (X-CH2-C6H5). Although quaternization by a substitution reaction is preferred, it would be possible to quaternize the heterocycle by an addition reaction to a suitable olefin. For example, by reaction with a compound of the formula H2C=CHQ where Q is an electron-withdrawing group, a quaternized heterocycle of the form Het + -CH2-CHQ is formed, where Q is an electron-withdrawing group. Examples of agents of this class would include ethyl methacrylate or methylacrylamide.

[0057] Another class of quaternizing agents in the above embodiments is defined by the formula R-W-(R)-CH2-X, where R is a C1-C 24 , more preferably C1-C 12 substituted or unsubstituted alkyl, alkenyl or aryl group, W is a heteroatom such as oxygen, silicon, sulfur or nitrogen, and X is the above leaving group. Some examples of this class of quaternizing agents would be X-CH2-O-C2H5, X-CH2CH2-O-CH3 and X-CH2CH2-morpholine.

[0058] Another class of quaternizing agents is an acyl compound of the formula R-(W)-COCH2-X, where R is a C1-C 24 , more preferably C1-C 10 alkyl or alkenyl, or a C6-C 10 aryl group, W is a heteroatom such as oxygen or nitrogen, and X is the above leaving group. Suitable examples of this class of acylating agents include X-CH2-CO-C6H5, X-CH2-CO2CH3, X-CH2-CONH2 and X-CH2CON(CH3)2.

[0059] Suitable quaternizing compounds in the above embodiments include, but are not limited to, quaternizations including bromobutane, bromo-PEG, bromopropionic acid, bromovaleric acid, chloroacetamide, chlorobutane, chlorobutyric acid, crotonic acid, diiodobutane, iodobutane, iodoacetamide and iodopropionic acid. Iodoacetamide is particularly interesting due to many desirable features such as low vapor pressure, relatively low hazard, inexpensive and readily available, water solubility, controllable quaternization, hydrophilicity, and preservation of selective layers of the membrane. These features make this quaternization process easily scalable for mass production of charged membranes. The final conditions used to quaternize the membranes shown below were: 6.6 mM water-soluble iodoacetamide, 60 °C, 1 hour. By nucleophilic substitution reaction, a positively charged pyridinium having a pendant acetamide group in the material is obtained as shown below.

[0060] In the above embodiment, iodoacetamide reacts with poly(4-vinylpyridine) to obtain a static charge on the membrane. This reaction results in terminal amides and persistent positive charges on the membrane.

[0061] Total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used to characterize the non-quaternized and quaternized membranes. As seen in Figure 8, characteristic peaks of the quaternization process were observed in the membranes treated with iodoacetamide. The characteristic increase at 1640 cm -1 indicates the conversion of the heterocyclic amine to the charged pyridinium. The appearance of the band at 1700 cm -1 is due to the amide C=O bond introduced during quaternization. 1 Measurements by H nuclear magnetic resonance (NMR) spectroscopy showed that 15.9% of the vinylpyridine protons did not change their chemical environment, meaning an 84.1% quaternization conversion using standardized conditions. It is noteworthy that similar experiments were conducted not only for the membranes treated with iodoacetamide but also for the other chemical groups mentioned above, varying the temperature, reagent concentration, and reaction time. By increasing the reaction time for iodoacetamide, the peak intensities at 1640 cm -1 and 1700 cm -1 increased, but a 1-hour reaction was sufficient to convert a significant proportion of the 4-vinylpyridine monomers and impart high-charge characteristics to the membrane.

[0062] Suitable bifunctional quaternizing agents of the above embodiments that have two reaction sites with a complex ring and serve as crosslinking agents include crosslinking agents defined by the formula X-R-X, X-CH2-CO-(W)-R-(W)-COCH2-X, or X-CH2-(R)-W-(R)-CH2-X, where R is an alkyl group, an alkenyl group, or an aryl group, W is a heteroatom such as oxygen or nitrogen, and further, X is the above-described leaving group. The crosslinking agent may not be symmetric; that is, one reactive group may be different from the second reactive group. Examples of bifunctional quaternizing groups include X-(CH2)4-X, X-CH2CH2-O-CH2CH2-X, ethylene glycol di(meth)acrylate, derivatives of methylene bisacrylamide, and derivatives of N-1-hydroxyl-2,2-dimethoxyethylacrylamide. Crosslinking agents such as 1,4-diiodobutane are of particular interest due to the potential for increased mechanical integrity and enhanced chemical resistance of the membrane.

[0063] Suitable quaternizing agents of the above embodiments also include additional functional groups that can have a positive or negative charge to adjust the overall charge of the material as well as the degree of hydrophilicity / hydrophobicity. For example, the quaternizing agent can be an anionic compound or a compound that ionizes under aqueous purification conditions to form an anion. This will form a zwitterionic complex ring with a neutral overall charge and reduce the positive charge of the material while still retaining highly polar characteristics. Examples of this type of agent include compounds having a carboxylic acid group or a sulfonic acid group such as -CH2CH2CO2H or -CH2CH2SO3H. Alternatively, the quaternizing agent can be a cationic compound or a compound that is protonated under purification conditions. Examples of this type include -CH2CONHCH2CH2-NH(CH3)2 + or -CH2CH2-N(CH3)3 + which would increase the overall positive charge of the material. These types of charge-control quaternizing agents are used in combination with hydrophilicity / hydrophobicity control agents to control membrane properties as needed.

[0064] Since proteins and other types of biological materials are often optically active, the functionalizing agent may be chiral. This will result in materials that have some degree of chirality associated with them. Examples of suitable optically active alkylating agents can be found in Non-Patent Document 8. For example, suitable chiral quaternizing agents of the above embodiments will include optically active primary alkyl halides or mesylates.

[0065] Modification of the material to introduce charge is done by immersing the as-prepared membrane in a solution having the modifying agent. In some embodiments, the charge introduction is quantitative, being >20%, >30%, >40%, >50%, >60%, >70%, >80% or >90% of all available reactive sites, but in other embodiments, the degree of quaternization may be sub-stoichiometric. A suitable solvent for quaternization is water. However, since some of the agents may have limited solubility in water, a combination of water and an organic co-solvent (such as an alcohol) is used. In some cases, it may be necessary to use a non-aqueous organic solvent or a mixture of organic solvents. Typical conditions for this process would be to dissolve the quaternizing agent in a suitable solvent system and treat the membrane at reagent concentrations of 2 mM and 100 mM with a reaction time of 1 to 24 hours at a temperature of 20 to 30 °C. In some cases, for less reactive agents, longer reaction times and higher temperatures may be required. The progress of this process can be monitored using Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR).

[0066] In some embodiments, as high a charge density as possible is desirable for the most selective separation of charged solutes. However, even in these cases, the membrane must retain a high flux of the feed solution at various pHs. The performance of the membrane can be measured using a pressurized dead-end stirred cell apparatus. This apparatus can use a feed reservoir of 10 to 50 mL and a membrane area of about 4 cm 2 to about 13 cm 2 of membrane area.

[0067] The type or kind of protein or other biologically-based material to be purified or concentrated by the charged isoporous membrane of the present invention is not limited. In particular, the feedstock may be a protein derived from a mammalian, bacterial, insect, viral or fungal cell line, and is typically used in an aqueous feedstock that may already be partially purified by another process. It is also possible to use a fermentation broth as the feedstock. The feedstock may be whole blood, diluted blood, plasma, or serum. The feedstock may also have nucleic acids that can be separated using the above materials.

[0068] Three model proteins of interest include myoglobin (Mgb), lysozyme (Lys), and cytochrome C (CytC). The typical molar masses and isoelectric points of these proteins are shown in Table 1, and they are representative of proteins that may be present in cell culture media. The flow of the feed solution with protein solutes can be measured using the same method as above, and protein rejection can be quantitatively measured using ultraviolet-visible spectroscopy and aqueous high-performance liquid chromatography (HPLC) for the feed and permeate solutions. The calculated rejection R is related to the sieving coefficient as shown in the following equation

[0069]

Equation

[0070]

Table 1

[0071] However, in some embodiments of the present invention, the feedstock may have a binary (or even more complex) solute mixture, such as the protein Mgb / Lys or Mgb / CytC. One key performance parameter for such mixtures is described by Non-Patent Document 9

[0072]

Number

[0073] In this example, the components of Mgb / Lys and Mgb / CytC are very similar in size and cannot be separated by simple ultrafiltration. When using the membrane of the present invention with any of the mixtures at neutral pH, Mgb is charged neutrally (due to its isoelectric point) and passes through the membrane with the selectivity corresponding to the case where Mgb is not present in the mixture. Lys or CytC is expected to be positively charged at neutral pH (due to its isoelectric point) and will be rejected by the similarly charged membrane. The membrane of the present invention is designed to achieve a selectivity of at least 10 such membrane pairs in a dead-end system at a flow rate exceeding 50 LMH / bar.

[0074]

Table 2

[0075] In another embodiment, a charged isoporous material is used to isolate nucleic acids. As in the above embodiment, after quaternizing the P4VP of the ISV isoporous material, the isoporous material has a positive charge. When exposed to a mixture of DNA fragments, negatively charged DNA binds to the membrane. Thereafter, a small amount of elution buffer can be used to elute the DNA from the membrane. The recovery rates for 200 and 400 base pairs (bp) in four different membranes are shown in FIG. 10. The yield of the eluted DNA was quantified using gel electrophoresis, and the yields of 200 and 400 bp fragments were quantified for four quaternized membranes. For 200 bp, the yield varied from 71% to 85%, and the coefficient of variation (SD / mean) was 8.8%. The yields of 400 bp were very similar, varying from 67% to 86%, and the coefficient of variation was 10.6%. Compared to commercially available kits having a relative standard deviation ranging from about 10% to 49% in the best case for other commercially available kits (Non-Patent Document 10; Non-Patent Document 11), these results demonstrate very consistent high DNA recovery using a very rapid process with a charged polymeric material of isoporous.

[0076] In this embodiment, size-selective elution of DNA fragments is also possible. Elution buffers of different dilutions (0.6x, 0.3x) were used for elution. FIG. 11 shows the relative yields of 0.6x buffer elution of 200 bp and 600 bp fragments. The yield of the larger 600 bp decreased to less than 1 / 4, and all bands larger than 600 bp were not detectable, demonstrating that the charged isoporous membrane system can be used for DNA extraction, concentration, and size selection. At a buffer concentration of 0.3x, even 200 bp fragments were not eluted. These results show the strong advantage of the charged membrane that targets a very specific DNA fragment size and enables a single product to be used for a number of DNA isolation applications simply by adjusting the elution conditions.

[0077] In another embodiment, the block copolymer is poly(styrene-b-isoprene-b-styrene-b-4-vinylpyridine) corresponding to the inclusive structure A-B-A-C. P4VP is quaternized to generate a static charge as in the above embodiments. In some embodiments, the isoporous charged material is formed into a two-dimensional structure. In other embodiments, the isoporous charged material is formed into a three-dimensional structure.

[0078] In one embodiment, the reaction that brings about a static charge on the isoporous material changes the hydrophilicity of the material. In another embodiment, the reaction that brings about a static charge on the isoporous material introduces a functional group that can be used for further chemical reactions or the functionality of the material into the material.

[0079] In some embodiments, the static charge on the isoporous charged material is positive, such as, for example, a quaternized pyridine group, a quaternized diethylaminoethyl group, a quaternized dimethylaminoethyl group, etc. In other embodiments, the static charge on the isoporous charged material is negative, such as, for example, a sulfonic acid group. In some embodiments, the isoporous material has an amine group (such as pyridine, diethylaminoethyl, dimethylaminoethyl, etc.) that can be quaternized using a chemical substance to generate a positive charge on the material. In one embodiment, the amine is quaternized using a monofunctional alkylating agent having one halogen selected from iodine, chlorine, and bromine, such as, for example, 2-iodoacetamide, 2-bromoethanol, etc. In one embodiment, the monofunctional alkylating agent has the formula: R-X, where R is a C1-C 24 substituted or unsubstituted linear, cyclic, or branched alkyl or alkenyl group, and X is a halogen group.

[0080] In another embodiment, the monofunctional alkylating agent has the formula: R-W-(R)-CH2-X, where R is a C1-C 24 alkyl, alkenyl, or aryl group, W is a heteroatom such as oxygen, silicon, sulfur, or nitrogen, and X is a halogen group. In another embodiment, the monofunctional alkylating agent has the formula: R-(W)-COCH2-X, where R is a C1-C24 is an alkyl, alkenyl or aryl group, W is a heteroatom such as oxygen or nitrogen, and X is a halogen group.

[0081] In another embodiment, a plurality of amines are quaternized using a polyfunctional alkylating agent having two or more halogens selected from iodine, chlorine, and bromine, such as 1,4-diiodobutane.

[0082] In another embodiment, the amine is quaternized using an alkylating agent having at least one reactive double bond, such as crotonic acid.

[0083] In some embodiments, two or more of the above amine-alkylating agent-containing substances are used to introduce charges onto an isoporous material.

[0084] In some embodiments, the degree of charge on the charged isoporous material is at least 20% to 90%. In other embodiments, the degree of charge on the charged isoporous material is ≦90% of all available units.

[0085] In one embodiment, the charged isoporous material is further treated or functionalized with an antibacterial agent.

[0086] In some embodiments, the geometric shape and area of the isoporous material having a static charge are controlled. In some embodiments, the geometric shape and area of the charged region are patterned by lithography, or achieved by attaching some or all of the charged modified material to an unmodified material or another substrate, or patterned on some or all of the unmodified material through printing or extrusion.

[0087] In some embodiments, the charged isoporous material is used as a separation medium. In some embodiments, the charged isoporous material is used to separate biomolecules from other biomolecules. In one embodiment, the target biomolecule is a protein. In another embodiment, the target biomolecule is a nucleic acid.

[0088] In some embodiments, the separation mechanism using a charged isoporous material as a separation medium includes a binding and elution mechanism in which the target species binds to the material by electrostatic interaction and is isolated, and the target species can dissociate from the material to recover the target species.

[0089] In some embodiments, the separation mechanism using a charged isoporous material as a separation medium includes a charge repulsion mechanism, a size selection mechanism, and / or a concentration mechanism.

[0090] In some embodiments, the separation mechanism using a charged isoporous material as a separation medium having a binding and elution mechanism also includes chemical-selective or size-selective elution.

[0091] In some embodiments, the charged isoporous material is used for gradient separation, isocratic separation, or step separation.

[0092] In some embodiments, the charged isoporous material is used as a wound dressing or adhesive plaster, or is used to regulate the development of biofilms.

[0093] In some embodiments, the charged isoporous material is combined with two or more charged materials and incorporated into a device, or the charged isoporous material is combined with an uncharged isoporous material and incorporated into a device as a unit.

[0094] In some embodiments, the charged material is incorporated into a pleated pack, a cross-flow cassette, a hollow fiber module, a syringe filter, a capsule, a pipette tip, a centrifuge tube, a spiral module, or a sensor device.

[0095] In another embodiment, the charged materials are grouped together as a flat sheet.

[0096] In some embodiments, the charged isoporous material is immobilized on a support material or is directly integrated with a fabric.

[0097] In one embodiment, two or more charged isoporous materials are grouped together as a kit.

[0098] In another embodiment, two or more devices incorporating the charged isoporous material are grouped together as a kit.

[0099] In another embodiment, at least one charged isoporous material is grouped together with one or more chemical solutions to elute the bound target species after binding.

[0100] Table of features identified in the figure 5 Region of large pore size 10 Region of small pore size 15 Layer of substantially the same pore size 20 Arrow indicating uniform flow 25 Arrow indicating uniform flow 30 Arrow indicating higher flow rate 35 Arrow indicating lower flow rate 40 Functionalization reaction for introducing charge 45 Functional group introduced from the functionalization reaction 50 Positively charged material 55 Uncharged solute / solvent molecules 60 Large solutes such as cell debris 65 Negatively charged nucleic acid solutes 70 Positively charged solutes

Claims

**Claim 1** A charged self - assembled A - B - C triblock copolymer material having a surface layer comprising a plurality of mesopores for nucleic acid isolation, wherein the mesopores are isoporous and have a pore size distribution of 1 to 3, the pore size distribution being defined as the ratio of the largest pore diameter to the smallest pore diameter, and the A - B - C triblock copolymer is a poly(isoprene - b - styrene - b - 4 - vinylpyridine) copolymer, wherein the polyisoprene block of the triblock copolymer has a Tg of ≤ 25 °C, wherein the poly(styrene) block of the triblock copolymer has a Tg of > 25 °C, wherein the poly(4 - vinylpyridine) block of the triblock copolymer contains an aromatic heterocycle quaternized to provide a positive static charge to the material, and the aromatic heterocycle is selected from the group consisting of pyridine, pyrazine, pyrimidine, pyridazine, quinoline, isoquinoline, quinoxaline, quinazoline, phenazine, isoxazole, isothiazole, imidazole, benzimidazole, triazole, and tetrazole, wherein the polyisoprene block has a volume fraction of 0.20 to 0.40, wherein the poly(styrene) block has a volume fraction of 0.45 to 0.60, wherein the poly(4 - vinylpyridine) block has a volume fraction of 0.05 to about 0.35, and the total volume fraction is 1.0, the material. **Claim 2** The material has a water permeability of 50 to 200 Lm －２ hr －１ bar －１ at a pH of 7.0, the material according to claim 1. **Claim 3** The aromatic heterocycle is quaternized by reacting with a quaternizing agent defined by the formula R-X, where R is a substituted or unsubstituted linear, cyclic or branched alkyl or alkenyl group of C １ to C ２４ and X is a leaving group. The material according to any one of claims 1 to 2 **Claim 4** The aromatic heterocyclic ring is quaternized by reacting with a quaternizing agent having the formula R-W-(R)-CH ２ -X, where R is a substituted or unsubstituted alkyl, alkenyl, or aryl group having from C １ to C ２４ , W is a heteroatom, and X is a leaving group. The material according to any one of claims 1 to 2. **Claim 5** The aromatic heterocyclic ring is quaternized by reacting with a quaternizing agent having the formula R-(W)-COCH ２ -X, where R is C １ to C ２４ alkyl or alkenyl, or a C ６ to C １０ aryl group, W is a heteroatom, and X is a leaving group. The material according to any one of claims 1 to 2. **Claim 6** R is C １ ~C １２ The material according to claim 3, which is a substituted or unsubstituted linear, cyclic or branched alkyl or alkenyl group. **Claim 7** X is a halide or -OSO ２ CH ３ The material according to claim 3, wherein it is **Claim 8** R is C １ ~C １２ The material according to claim 4, which is a substituted or unsubstituted alkyl, alkenyl, or aryl group. **Claim 9** The material according to claim 4, wherein W is oxygen, silicon, sulfur, or nitrogen. **Claim 10** X is a halide or -OSO ２ CH ３ The material according to claim 4, wherein the material is such. **Claim 11** The quaternizing agent is X-CH ２ -O-C ２ H ５ , X-CH ２ CH ２ -O-CH ３ , or X-CH ２ CH ２ -morpholine, the material according to claim 4. **Claim 12** R is C １ ~C １０ The material according to claim 5, which is an alkyl or alkenyl group of **Claim 13** The material according to claim 5, wherein W is oxygen or nitrogen. **Claim 14** X is a halide or -OSO ２ CH ３ The material according to claim 5, wherein **Claim 15** The acyl compound is X-CH ２ -CO-C ６ H ５ , X-CH ２ -CO ２ CH ３ , X-CH ２ -CONH ２ , or X-CH ２ CON(CH ３ ) ２ The material according to claim 5. **Claim 16** The density of the mesopores on the surface layer is at least 10 １４ pores / m ２ The material according to claim 1, wherein the material is such that the density of the mesopores on the surface layer is at least 10 pores / m ２ . **Claim 17** The material according to claim 1, wherein the mesopores have a size of 5 nm to 200 nm. **Claim 18** A method of forming a charged isoporous A - B - C triblock copolymer material according to claim 1, the method comprising contacting the A - B - C triblock copolymer with a chemical substance that reacts with the aromatic heterocycle to introduce the positive static charge thereon. **Claim 19** A process for separating a nucleic acid, which is a target biomolecule, from a mixture of other solutes, the process comprising the step of contacting the material according to any one of claims 1 to 17 with the mixture, and the step of separating and / or removing the target biomolecule from the mixture.

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