Ion exchange membranes, filters and methods

Abstract

Described herein are ion exchange membranes comprising a porous polymer membrane and imidazolium ion exchange groups at the membrane surface; ion exchange membranes and filters containing the ion exchange membranes; and methods of separating charged biomolecules from liquids using the ion exchange membranes and filters.

Description

Technical Field

[0001] The following description relates to an ion exchange membrane comprising a porous polymer substrate and imidazolium ion exchange groups attached to the porous polymer substrate; an ion exchange membrane and a filter containing the ion exchange membrane; and a method of separating charged biomolecules (such as (but not limited to) plasmid DNA) from a liquid using the ion exchange membrane and the filter. Background Art

[0002] Purification of charged biomolecules, such as DNA, e.g., plasmid DNA ("pDNA"), is typically performed by ion exchange chromatography. However, chromatography resins can exhibit poor compatibility, slow mass transfer, and low binding capacity. Furthermore, filling, cleaning, and validating chromatography columns is labor-intensive and time-consuming. Summary of the invention

[0003] The following description relates to ion exchange membranes and related methods that address limitations of previous purification methods involving chromatographic resins by providing single-use (disposable), highly convective ion exchange membranes with pores large enough to adsorb charged biomolecules, such as DNA (e.g., plasmid DNA), proteins, nucleic acids, endotoxins, viruses, etc. The porous membranes have high surface area and high binding capacity for good accessibility of molecules to the membrane.

[0004] The present description relates to improved ion exchange membranes and related methods, wherein the membranes are effective, for example, in selectively adsorbing and subsequently releasing (desorbing) charged biomolecules based on pH. The membranes include ion exchange groups in the form of charged imidazole groups located on the surface of a porous polymer membrane. The ion exchange membranes are in the form of "sheets" or "films" that can be used and reused, or can be disposable. Disposable designs result in faster setup times, flexible handling, and ease of disposal. Optimizing membrane morphology (including specific surface area and pore size) enables more efficient separation and collection of biomolecules (e.g., plasmid DNA) from liquid media because of its enhanced exposure to available surface area and higher binding capacity for charged biological compounds as target molecules. Conventional chromatography resin bed technology cannot achieve the efficiency of the methods described herein.

[0005] As used herein, the term "target" molecule, etc. refers to a liquid molecule that is required to be separated from other molecules in the liquid. Biological fluids may contain a variety of different charged biomolecules, including plasmid DNA, RNA, lipopolysaccharide, protein, and genomic DNA. In a separation process or purification process, any of these may be a target molecule, and all other charged molecules are considered non-target molecules or "impurities". Based on various factors (such as the high commercial importance or value of the selected (target) charged biomolecule), relative to other charged biomolecules in the fluid, a specific charged biomolecule contained in the biological fluid (or other liquid) may be selected as a target molecule. In various examples, plasmid DNA may be selected as a target molecule in a biological fluid relative to non-target molecules that may include RNA, lipopolysaccharide, protein, and genomic DNA. According to other example methods described herein, different charged biomolecules may be selected as target molecules, such as RNA molecules, lipopolysaccharides, proteins, or genomic DNA.

[0006] Also according to the method, the charged biomolecule selected for adsorption onto the membrane may be a target molecule or may be a non-target molecule. The membrane may be used to selectively adsorb one or more charged biomolecules contained in the liquid from a mixture of multiple charged biomolecules present in the liquid, the mixture including both target molecules and non-target molecules. As a specific example, the membrane may be used to selectively bind (selectively adsorb) plasmid DNA contained in a biological liquid without adsorbing impurities, the biological liquid containing plasmid DNA in a mixture of various charged biomolecules (e.g., "impurities" such as RNA, lipopolysaccharide, and genomic DNA). Impurities pass through the membrane while the plasmid DNA is adsorbed on the membrane and may be subsequently desorbed (eluted) and collected. According to other examples, the membrane may be used to selectively bind (selectively adsorb) charged biomolecules other than plasmid DNA (e.g., binding "impurities" such as RNA, lipopolysaccharide, protein, and genomic DNA) without binding (adsorbing) plasmid DNA. Plasmid DNA passes through the membrane and may be collected.

[0007] The surface of the porous membrane includes charged imidazole ion exchange groups effective for selective adsorption and release of charged biomolecules. According to a single specific example, the exemplary membrane of the present disclosure can be effectively used for selective binding of plasmid DNA in biological fluids relative to "impurities" such as RNA, lipopolysaccharides, proteins and genomic DNA. Imidazole is a unique functional group with a pKa of about 5.2. The membrane has imidazole groups that exhibit pH dependence and transient surface charge density, such as exhibiting a positive charge at a pH below the 5.2 range and exhibiting a negative charge at a pH above the 5.2 range. The transient surface charge density of the imidazole functional group allows the desired separation of biomolecules from liquid media and the subsequent release and collection of the molecules when varying pH conditions are introduced. Under acidic conditions, the membrane surface is primarily positively charged and can bind charged biomolecules (e.g., plasmid DNA molecules) by means of electrostatic interactions. Under alkaline conditions, the imidazole group deprotonates and eliminates the electrostatic complex of the charged biomolecule (eg, plasmid DNA molecule) with the imidazole group, which results in the selective release of the charged biomolecule (eg, plasmid DNA molecule) at an ionic strength significantly lower than that of the chromatographic resin bed.

[0008] In one aspect, the present invention relates to a method for separating charged biomolecules from a liquid using a porous ion exchange membrane sheet containing imidazolium ion exchange groups. The method comprises contacting a liquid containing charged biomolecules with a porous ion exchange membrane comprising a porous polymer sheet and imidazolium ion exchange groups at the surface of the porous polymer sheet under adsorption conditions that allow the charged biomolecules to adsorb to the ion exchange membrane. Subsequent introduction of the membrane to an elevated pH value releases the charged biomolecules to allow the charged biomolecules to be collected in a concentrated form. In addition, the physical properties of the membrane can affect the contact and exposure of the biomolecules to the charged imidazolium ion exchange groups and enhance the attraction of the molecules to the membrane.

[0009] In another aspect, the present invention relates to a porous ion exchange membrane comprising imidazolium ion exchange groups at the surface of the membrane, wherein the charge of the imidazolium ion exchange groups is pH dependent and exhibits a positive charge at a pH below the 5.2 range and a negative charge at a pH above the 5.2 range.

[0010] In yet another aspect, the present invention relates to a method for preparing a porous ion exchange membrane comprising imidazolium ion exchange groups grafted to the surface of a polymer porous membrane substrate. The method comprises chemically bonding the imidazolium ion exchange groups to the surface of the porous polymer membrane.

[0011] In another aspect, the present invention relates to a method for preparing a porous ion exchange membrane, the porous ion exchange membrane comprising a polymer porous membrane substrate and a cross-linked polymer network containing imidazolium ion exchange groups on the substrate surface. The method comprises placing a coating composition at the substrate surface, the coating composition comprising vinyl imidazole and a cross-linking agent, and reacting the vinyl imidazole and the cross-linking agent to form a cross-linked polymer network on the surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1A and 1B Shows examples of described filter products and methods.

[0013] Figure 2 Table showing dye solution absorbance versus weight % dye, which is used in the method for measuring the charge density of the membrane. DETAILED DESCRIPTION

[0014] The following description relates to porous ion exchange membranes; products containing ion exchange membranes; and methods of using ion exchange membranes for, for example, separating charged biomolecules (eg, DNA, such as plasmid DNA) from liquids containing the charged biomolecules.

[0015] Porous ion exchange membrane contains a porous polymer film, which includes imidazolium ion exchange groups attached to the surface of the porous polymer film. Imidazolium ion exchange can be charged based on pH, for selective binding and debinding ("elution") of charged biomolecules. Imidazolium ion exchange groups are uniquely effective for purifying charged biomolecules, such as plasmid DNA, due to a pKa of about 5.2. Imidazolium ion exchange groups provide pH dependence and transient surface charge density. Under acidic conditions, imidazole groups mainly exhibit positive charge and can bind charged biogroups (e.g., plasmid DNA) by means of electrostatic interactions. On the other hand, imidazole groups deprotonate and subsequently release charged biomolecules (e.g., plasmid DNA) under alkaline conditions that lead to the destruction of electrostatic complexes, which have better yields and significantly lower ionic strengths than chromatographic resin beds.

[0016] The porous polymer membrane is preferably in the form of a "sheet" or "film" comprising two opposing major surfaces and a thickness between the two surfaces. The sheet is generally planar and has a generally uniform thickness when laid flat. A planar sheet membrane may be considered to extend in length and width dimensions to define two opposing major surfaces, which are separated by thickness in a third dimension. The sheet may preferably have a relatively uniform thickness in length and width dimensions. The "membrane" described is not in the form of particles (e.g., a collection of porous polymer resin particles), which are different forms of ion exchange media. The "membrane" described is also different from a "monolithic" chromatographic device, which is a different form of a porous chromatographic medium having a generally larger thickness dimension than a "membrane".

[0017] The membrane is composed of a polymer porous membrane substrate having physical properties that affect the separation and collection of charged biomolecules from liquids and can enhance the exposure time and contact of biomolecules to imidazolium ion exchange groups. The porous polymer membrane substrate can be selected to provide desired physical characteristics including porosity, pore size, and thickness.

[0018] A kind of property of described useful membrane is charge density, it can be measured by the technology of dye binding capacity, wherein various described technology is known to be used for measuring the amount (by mass, for example, expressed in grams) of specific functional groups per unit area of ​​membrane, for example, as the measurement of micrograms / square centimeter.The measurement technology is usually implemented by exposing the membrane to a dye solution containing a known amount of dye, and the dye is attracted to the ion exchange groups on the membrane surface.Negatively charged dye is bound to the positively charged ion exchange groups.Before and after the dye is bound to the ion exchange groups, the absorbance of the dye solution to the relevant ultraviolet wavelength is measured.The absorbance difference of the dye solution before and after being exposed to the membrane can be related to the amount of the ion exchange groups on the membrane surface.According to useful or preferred membrane, the charge density of membrane (as measured by dye binding technology) can be in the range of up to 300 micrograms / square centimeter, for example, 10 to 90 micrograms / square centimeter or 20 to 70 micrograms / square centimeter.

[0019] The membrane may have any thickness that allows the membrane to be performed according to the methods described herein. Useful or preferred thicknesses may be in the range of 5 to 300 microns, for example 10 or 20 to 50, 100 or 200 microns.

[0020] The porous membrane may have any porosity that will allow the membrane to effectively separate (selectively adsorb and release) charged biomolecules (e.g., DNA, e.g., plasmid DNA) from a liquid containing the charged biomolecules as described herein. Example porous membranes may have a porosity in the range of 20 to 95%, for example, a porosity in the range of 60 to 90% (volume). As used herein and in the art of porous membranes, the "porosity" (sometimes also referred to as the void fraction) of a porous membrane is a measure of the percentage of void (i.e., "empty") space in the membrane to the total volume of the membrane, and is calculated as the fraction of the void volume of the membrane divided by the total volume of the membrane. A body having a porosity of zero percent is completely solid.

[0021] Examples of useful porous membranes may have pores of a size (average pore size) that is considered a microporous membrane or a macroporous membrane. Example pore sizes (average pore size) may be in the range of 0.2 to 10 microns. Pore size is typically reported as the average pore size of the porous material, which can be measured by known techniques such as mercury intrusion porosimetry (MP), scanning electron microscopy (SEM), liquid-liquid displacement porosimetry (LLDP), or air-liquid displacement porosimetry (ALDP).

[0022] The membrane may have an open cell honeycomb structure, be filamentous, be a nonwoven (eg, electrospun nanofiber membrane), or other effectively porous structure.

[0023] The polymer of the porous polymer membrane substrate can be any polymer capable of having imidazolium ion exchange groups chemically bonded to the membrane surface. A variety of different polymers (i.e., "base polymers") can be utilized to form porous polymer filter membranes, some examples of which include non-fluorinated polymers such as polyolefins (e.g., polypropylene, polyethylene, polyhaloolefins), polyesters, polyimides, polyetherimides, polysulfones, polyethersulfones, polycarbonates; as well as fluoropolymers and other general and specific types of useful polymers.

[0024] Suitable polyolefins include, for example, polyethylene (e.g., ultra-high molecular weight polyethylene (UPE)), polypropylene, alpha-polyolefins, poly-3-methyl-1-butene, poly-4-methyl-1-butene, and copolymers of ethylene, propylene, 3-methyl-1-butene or 4-methyl-1-butene with each other or with small amounts of other olefins; example polyhaloolefins include polytetrafluoroethylene, polyvinylidene fluoride, and copolymers of said and other fluorinated or non-fluorinated monomers. Example polyesters include polyethylene terephthalate and polybutylene terephthalate and related copolymers.

[0025] The imidazolium ion exchange groups may be present at the surface of the porous polymer membrane in any effective form and may be placed at the surface by any effective method, such as a grafting technique for chemically bonding the imidazolium ion exchange groups to the base polymer of the porous membrane or by a technique for forming a cross-linked polymer network containing imidazolium ion exchange groups on the surface of the porous membrane.

[0026] According to the grafting technique, the imidazole group can be chemically attached (i.e., grafted, covalently chemically bonded) to the carbon atoms of the polymer chain of the polymer material forming the porous membrane, that is, attached to the base polymer constituting the membrane. For example, if the membrane is composed of a polyolefin (e.g., polyethylene), the imidazole ion exchange group is chemically bonded to the carbon atoms of the linear carbon backbone of the polyolefin (e.g., polyethylene). The imidazole group can be attached to the carbon atoms of the base polymer of the membrane by a chemical reaction, and the chemical reaction involves the UV-initiation reaction of the vinyl imidazole monomer with the base polymer and the polymerization of the vinyl imidazole monomer optionally in the presence of one or more comonomers, so as to chemically attach the vinyl imidazole monomer to the polymer material of the membrane at, for example, the polymer backbone. The vinyl imidazole monomer and the optional comonomer will form a side chain attached to the carbon atom of the base polymer. The side chain will include a divalent linking group between the carbon of the polymer backbone and the first imidazole group of the side chain. Preferably, the divalent linking group can be a linking group that causes imidazole to be stable under caustic conditions, such as a divalent alkylene group, such as a divalent methylene group, i.e., -CH2-.

[0027] The length of the side chain (ie, the number of vinylimidazole monomers and optional comonomers of the side chain) can be any useful length, such as from 5 to 1000 monomers, or from 10 to 100 or 500 monomers.

[0028] Examples of side chains and attachments to the main chain may have the structure of Formula 1:

[0029]

[0030] As illustrated, the side chains of Formula 1 are derived only from vinyl imidazole monomers. In other examples, the side chains may be derived from vinyl imidazole and one or more comonomers, which will become part of the side chains between the vinyl imidazole monomers. Examples of suitable comonomers include N-[2-(dimethylamino)ethyl]acrylamide and N-[2-(diethylamino)ethyl]acrylamide.

[0031] According to the technology of forming a cross-linked polymer network coating on the surface of a porous membrane, the polymer network is formed from monomers including vinyl imidazole and a cross-linking agent (e.g., a difunctional cross-linking molecule, such as a difunctional acrylate compound (e.g., methylene-bisacrylamide (MBAM))), which react in the presence of UV radiation and a UV initiator to form a polymerized cross-linked coating.

[0032] The cross-linked polymer network coating containing imidazole groups is formed on the membrane surface, and the coating or imidazole groups are not chemically bonded to the polymer of the membrane. However, the bond between the imidazole groups and the polymer of the cross-linked polymer network to which the imidazole groups are chemically attached is a direct bond between the imidazole groups and the carbon atoms of the polymer network. The structure of the cross-linked polymer network containing imidazole is as shown in Formula 2:

[0033]

[0034] Formula 2 shows only the polymer of the cross-linked polymer network coating. The base polymer of the porous membrane on which the illustrated cross-linked polymer network is coated is not illustrated.

[0035] Both the grafted polymer structure of Formula 1 and the imidazolium ion exchange groups of the polymer cross-linked network coating of Formula 2 exhibit useful or advantageous effectiveness and stability when used as part of the porous ion exchange membrane according to the method. The polymer structure can produce useful or advantageous charge density levels on the membrane surface.

[0036] Additionally and advantageously, the imidazolium ion exchange groups of Formula 1 and Formula 2 can exhibit relatively preferred stability during adsorption and desorption cycles of charged molecules in multiple use cycles, including caustic cleaning cycles. The imidazolium ion exchange groups of Formula 1 and Formula 2 can exhibit relatively high chemical stability during any process steps performed at alkaline (caustic, basic) pH. When exposed to basic conditions, the attachment of the imidazole group to the polymer (the base polymer of Formula 1 or the cross-linked polymer network coating of Formula 2) is substantially stable.

[0037] During the treatment of the membrane, alkaline conditions can be used for cleaning, for example, to remove charged molecules from the membrane surface so that the membrane can be used again for adsorbing charged molecules. This type of "cleaning" step is sometimes implemented by washing the membrane with a sodium hydroxide solution or another alkaline (caustic) solution to allow the membrane to be reused. When the membrane is cleaned in this way, the imidazolium ion exchange groups attached by a direct bond between the carbon atom of the polymer and the nitrogen atom of the imidazole group or another selection by means of an alkylene group (such as the methylene group) are substantially stable with respect to remaining on the membrane surface. For comparison, the imidazolium ion exchange groups attached by means of different divalent linking groups (such as linking esters (ROC (O) - (imidazole nitrogen)) (see Formula 3 below)) can be more easily removed when exposed to alkaline conditions, that is, "caustic stability" is much lower.

[0038] Formula 3 containing an ester (-OC(O)-) linkage between the polymer (designated R) and the imidazole group is less stable under harsh conditions, such as those experienced in harsh cleaning steps:

[0039]

[0040] The membrane can be used to selectively adsorb and optionally selectively desorb ("elute") one or a combination of target charged biomolecules, non-target charged biomolecules, or a combination of target and non-target charged biomolecules to remove target and non-target molecules from a liquid containing a combination of charged target molecules and other non-target molecules (e.g., "impurities"). After adsorption, the charged target or non-target molecules can be desorbed. If a combination of both target and non-target molecules is adsorbed, then the target or non-target molecules can be selectively desorbed.

[0041] According to one particular method, the membrane can be used to first selectively adsorb the desired target molecule without adsorbing non-target molecules. The adsorbed target molecules can then be eluted and collected in a concentrated form without the impurities found in the initial liquid.

[0042] By an alternative approach, a membrane can be used to selectively adsorb one or a combination of charged biomolecules that are non-target molecules, without adsorbing the desired target molecules. The adsorbed non-target charged biomolecules remain on the membrane surface, while the desired target molecules flow through the membrane and can be collected and recovered separately from the adsorbed non-target molecules. By this method, the membrane selectively removes only the non-target molecules, while the target molecules pass through the membrane.

[0043] By yet another approach, the membrane can be used to adsorb a combination of both target and non-target molecules, followed by selective desorption of either the target or non-target molecules, wherein the result is separation of the target molecules from the non-target molecules.After adsorption, selective desorption can be used to desorb either the target or non-target molecules.

[0044] Desorption of selected molecules from the membrane can be achieved by selecting the characteristics of the desorption solution, such as, in particular, the following chemical or physical characteristics: the composition of the desorption solution (e.g., by adding a surfactant or an ionic compound), salinity, pH, or conductivity. Desorption can also be controlled by the residence time of the desorption solution, which can be adjusted for biological compounds of different sizes (e.g., RNA versus DNA). By this method, the membrane selectively allows specific desorption of only target molecules or only non-target molecules to separate target molecules from non-target molecules. If only non-target molecules are desorbed, the target molecules remain on the membrane and can be subsequently desorbed in a separate desorption step after the step of desorbing non-target molecules.

[0045] According to the method, the amount of desired target molecules that can be recovered can be any useful amount. The useful amount may depend on the type of target molecule, in particular the value of the target molecule. The useful amount of the recovered target molecule can be measured as the amount of target molecules adsorbed on the membrane and collected by desorption (expressed as a percentage); the percentage of recovered target molecules is the amount of target molecules adsorbed on the membrane and then desorbed relative to the total amount of target molecules originally adsorbed on the membrane before desorption. On this basis, examples of useful amounts of recovered target molecules that can be achieved by the method can be at least 10, 40, 50, 70 or even 80 or 90% desorption of the total amount of target molecules initially adsorbed on the membrane.

[0046] The liquid containing charged biological compounds (e.g., target molecules and non-target molecules) can be a liquid derived from any source and containing at least one desired target biological molecule. Example liquids can be derived from bacterial cells containing biological molecules, such as by alkaline dissolution of bacterial cells. Alkaline dissolution methods can provide biological liquids containing a certain amount of target molecules and impurities (e.g., RNA and bacterial endotoxins). The methods described herein can selectively adsorb target biological compounds (which can usually be plasmid DNA) without adsorbing non-target molecules (e.g., RNA and endotoxins). The adsorbed target molecules can then be eluted in a purified and concentrated form. Alternatively, the method can adsorb non-plasmid DNA (or other non-target) molecules without adsorbing plasmid DNA. Unadsorbed plasmid DNA or other target molecules pass through the membrane without being adsorbed and can be collected. Alternatively, the method can adsorb a combination of charged molecules (including target molecules), and the desorption step can selectively desorb target or non-target molecules to separate target molecules from non-target molecules, as described herein.

[0047] In a useful or preferred method, a biological fluid containing target molecules and non-target impurities is contacted with an ion exchange membrane described herein under adsorption conditions (e.g., acidic conditions). The adsorption conditions may be a pH below about 6, for example in the range of about 4.5 to about 5.5. The adsorption conditions allow the charged biomolecules to adsorb to the membrane surface at the ion exchange groups. As desired, the target molecules, non-target molecules, or both are adsorbed to the ion exchange membrane by electrostatic attraction to the imidazolium ion exchange groups.

[0048] After adsorption, the membrane is removed from the remaining biological fluid and, particularly if the membrane contains adsorbed target molecules, is exposed to elution conditions that disrupt the electrostatic attraction between the target molecules and the imidazolium ion exchange groups to release the adsorbed charged biomolecules. Useful or preferred elution conditions can be achieved by contacting the membrane with a desorption solution having a pH above about 7 (e.g., a pH in the range of about 7.5 to about 9.5) while the membrane contains adsorbed charged molecules on its surface.

[0049] Optionally, to achieve selective desorption of target or non-target molecules, the desorption step can be controlled by selecting the characteristics of the desorption solution, such as chemical or physical characteristics including, among others: composition (e.g., by adding surfactants or ionic compounds), salinity, pH, conductivity. Selective desorption can also be achieved by selecting the residence time of the desorption solution, which can be adjusted for biological compounds of different sizes.

[0050] Certain prior art techniques for eluting adsorbed molecules from ionic membranes may also include or require a step of exposing the membrane to a desorption solution containing a salt or a salt mixture when the membrane contains adsorbed charged molecules, to thereby desorb the adsorbed charged molecules. In the presence of salt, the desorption solution exhibits increased conductivity. An example of the desorption solution may contain 50 mM Tris and 1.5 M sodium chloride or another salt or salt mixture so that the elution solution has a conductivity in the range of 40 to 120 milliSiemens / cm.

[0051] Although the methods described herein allow for and may benefit from the step of contacting the membrane (which contains adsorbed charged molecules) with a desorption solution containing a salt and having a conductivity within this range, the step of contacting the membrane with a desorption solution containing a salt and having an increased conductivity due to the presence of the salt is not required in the described example methods. According to certain example methods described herein, the step of contacting the membrane with a desorption solution containing a salt to achieve elution when the membrane contains adsorbed charged molecules is not required and may be specifically avoided or excluded.

[0052] The optional step of contacting the membrane with a desorption solution containing a salt and having an electrical conductivity that promotes elution is generally an additional step, separate from and in addition to the elution step of contacting the membrane with a low pH solution. If the additional step of desorption using a salt is avoided, the overall complexity and cost of the process for separating the desired target molecule from the liquid will be advantageously reduced.

[0053] The porous membrane may be included in a filter body, for example, a filter or filter element for a filtration system containing the membrane that is effective in adsorbing and desorbing (eluting) target molecules from biological fluids. The filtration system places the membrane, for example, as part of a filter body or filter element, in the flow path of the liquid, and causes the liquid to flow through the membrane under adsorption conditions, thereby causing the charged biomolecules in the liquid to adsorb onto the membrane. The structure of the filter body or filter element may include one or more of a variety of additional materials and structures that support the membrane within the filter so that the fluid flows from the filter inlet through the membrane and through the filter outlet, thereby passing through the membrane when passing through the filter body. The membrane supported by the filter body can be any useful shape, for example, a pleated cylinder, a cylindrical pad, a plurality of "stacked" membranes or more non-pleated (flat) cylindrical sheets, pleated sheets, etc.

[0054] An example of a membrane contained in a filter body is shown in Figure 1A and 1B As shown, the filter 100 includes a body 102 that defines an inlet 106, an outlet 108, and an interior containing a membrane 104. Liquid passing through the inlet 106 must pass through the membrane 104 before exiting the outlet 108.

[0055] In use, a biological liquid 110 containing target molecules and impurities enters the inlet 106 under adsorption conditions (e.g., acidic pH). As the liquid 110 passes through the membrane 104, the target molecules contained in the biological liquid are adsorbed on the surface of the membrane 104 at the imidazolium ion exchange groups. Non-target molecules in the liquid 110 are not adsorbed. The biological liquid 120 containing a reduced amount of target molecules leaves the body 102 by passing through the outlet 108. The liquid 110 passes through the filter 100 in an amount such that a desired amount of target molecules are adsorbed onto the membrane 104.

[0056] In subsequent steps, such as Figure 1B As shown, the adsorbed target molecules are eluted from the membrane 104 by passing a desorption solution 130 into the inlet 106 and passing the desorption solution through the membrane 104. The desorption solution 130 has a pH high enough to release the adsorbed target molecules from the membrane 104. The desorption solution 140 containing a certain amount of purified target molecules leaves the body 100 at the outlet 108.

[0057] pass Figure 1A and 1B In an alternative method, a liquid 110 containing target molecules and impurities (non-target molecules) enters the inlet 106 under adsorption conditions (e.g., acidic pH). As the liquid 110 passes through the membrane 104, the non-target molecules contained in the biological liquid are adsorbed on the surface of the membrane 104 at the imidazolium ion exchange groups. The target molecules in the liquid 110 are not adsorbed. The biological liquid 120 containing a reduced amount of non-target molecules leaves the body 102 by passing through the outlet 108. The target molecules contained in the biological liquid 120 can be collected by a useful method.

[0058] According to various methods, a liquid 110 containing target molecules and non-target molecules enters the inlet 106 under adsorption conditions (e.g., acidic pH). As the liquid 110 passes through the membrane 104, the target molecules and non-target molecules contained in the biological liquid are adsorbed on the surface of the membrane 104 at the imidazolium ion exchange groups. The biological liquid 120 containing reduced amounts of target molecules and reduced amounts of non-target molecules leaves the body 102 by passing through the outlet 108.

[0059] In subsequent steps, such as Figure 1B As shown, the adsorbed target molecules or adsorbed non-target molecules are eluted from the membrane 104 by passing a desorption solution 130 into the inlet 106 and passing the desorption solution through the membrane 104. The desorption solution 130 has a pH and other properties that may include a desired composition (e.g., the presence of a surfactant or an ionic compound), salinity, pH, conductivity, etc., and is in contact with the membrane 104 for a desired amount of time to cause selective desorption of the target or non-target molecules from the membrane 104 surface.

[0060] The features and advantages of the disclosed methods and membranes are more fully illustrated by the following non-limiting examples.

[0061] Examples

[0062] Example 1 : This example demonstrates the preparation of a surface modification solution containing monomers and a crosslinker for forming a coating.

[0063] In a representative experiment, a solution containing 0.2% Irgacure 2959, 5% methanol, 3% vinyl imidazole, 1% methylene bis acrylamide (MBAM) crosslinker, and 90.8% water was prepared at room temperature.

[0064] Example 2 : This example demonstrates the preparation of the initiator solution used in the grafting step.

[0065] To form a representative initiator solution, 0.2 grams of benzophenone (99%, Sigma-Aldrich) was dissolved in 39.8 grams of isopropyl alcohol (IPA) to obtain a 0.5 wt% benzophenone solution.

[0066] Example 3: This example demonstrates the preparation of monomer solutions (A) and (B) for grafting.

[0067] In a representative experiment, a solution (A) containing 5% vinylimidazole and 95% water was prepared at room temperature.

[0068] In a representative experiment, a solution (B) containing 4% vinylimidazole, 1% dimethylacrylamide and 95% water was prepared at room temperature.

[0069] Example 4 : This example shows how a polyethylene (PE) film can be surface modified with a coating of polymerized vinyl imidazole monomers having a positive charge.

[0070] In a representative test, a sheet of UPE membrane (1 micron nominal pore size rating) was wetted with an isopropyl alcohol (IPA) solution for 25 seconds. The membrane was rinsed with an exchange solution containing 100% water and the IPA was removed. The membrane sheet was then introduced into the surface modification solution described in Example 1. The membrane was soaked in the solution for 2 minutes. The membrane sheet was taken out and placed between 1 mil polyethylene sheets. When the polyethylene / membrane sheet / polyethylene sandwich was laid flat on the table, excess solution was removed by rolling a rubber roller on it. The polyethylene sandwich was then pasted on a conveying unit that transported the assembly through a Fusion Systems broadband UV exposure laboratory unit that emits at a wavelength of 200 to 600nm. The exposure time was controlled by the speed at which the assembly moved through the UV unit. In this example, the assembly moved through the UV chamber at 10 feet / minute. After coming out of the UV unit, the membrane was taken out from the sandwich and immediately placed in DI water, where the membrane was washed by soaking it for 5 minutes. Then, the treated membrane sample was washed in methanol for 5 minutes. After this washing procedure, the membrane was dried on a stand in an oven operated at 50°C for 10 min.

[0071] Example 5: This example illustrates how to determine the dye binding capacity (DBC) of a membrane modified according to Example 4.

[0072] Dye Binding Capacity (DBC) is an indirect measure of the surface charge density of a membrane. A higher DBC means a higher charge density on the membrane surface, i.e. a higher concentration of imidazole groups on the membrane surface.

[0073] A dried 47 mm disc of membrane prepared according to Example 4 was placed in a beaker containing 0.002 wt % Ponceau-S dye (Sigma). The beaker was covered and the membrane was soaked for 5 minutes at room temperature with continuous stirring. The membrane disc was then removed and the absorbance of the dye solution was measured using a Cary spectrophotometer (Agilent Technologies) operating at 512 nm. The measurement was compared to the absorbance of the starting solution (before membrane soaking). Using Figure 2 The calibration curve shown in the figure converts the absorbance data of the dye solution into weight % and finally into the mass of dye bound per unit area of ​​the membrane. The dye binding capacity of the membrane prepared according to this procedure is 42 μg / cm 2 .

[0074] Example 6 : This example shows how PE membranes can be surface modified using graft polymerized vinyl imidazole monomers with positive charges.

[0075] In a representative test, a sheet of UPE membrane (0.2 micron nominal pore size rating) was wetted with an initiator solution prepared according to Example 2 for 25 seconds. An exchange solution containing 100% water was used to rinse the membrane and precipitate the initiator on the membrane surface. The membrane sheet was then introduced into the surface modification solution (A) described in Example 3. The membrane was soaked in the solution for 2 minutes. The membrane sheet was removed and placed between 1 mil polyethylene sheets. Excess solution was removed by rolling a rubber roller over the polyethylene / membrane sheet / polyethylene sandwich while it was lying flat on a table. The polyethylene sandwich was then taped to a transport unit that transported the assembly through a Fusion Systems broadband UV exposure laboratory unit emitting at a wavelength of 200 to 600 nm. The exposure time was controlled by the speed at which the assembly moved through the UV unit. In this example, the assembly moved through the UV chamber at 10 feet / minute. After exiting the UV unit, the membrane was removed from the sandwich and immediately placed in DI water, where the membrane was washed by soaking for 5 minutes. The treated membrane sample was then washed in methanol for 5 minutes. After this washing procedure, the membrane was dried on a stand in an oven operated at 50°C for 10 min.

[0076] The dye binding capacity of the membrane prepared according to this procedure was 10 μg / cm 2 The assay was performed as explained in Example 5.

[0077] Example 7 :This example shows how PE membrane can be surface modified using graft polymerized vinyl imidazole and N,N-dimethylacrylamide monomers with positive charges.

[0078] In a representative test, a sheet of UPE membrane (0.2 micron nominal pore size rating) was wetted with an initiator solution prepared according to Example 2 for 25 seconds. An exchange solution containing 100% water was used to rinse the membrane and precipitate the initiator on the membrane surface. The membrane sheet was then introduced into the surface modification solution (B) described in Example 3. The membrane was soaked in the solution for 2 minutes. The membrane sheet was removed and placed between 1 mil polyethylene sheets. Excess solution was removed by rolling a rubber roller over the polyethylene / membrane sheet / polyethylene sandwich while it lay flat on a table. The polyethylene sandwich was then taped to a transport unit that transported the assembly through a Fusion Systems broadband UV exposure laboratory unit that emits at wavelengths of 200 to 600 nm. The exposure time was controlled by the speed at which the assembly moved through the UV unit. In this example, the assembly moved through the UV chamber at 10 feet / minute. After exiting the UV unit, the membrane was removed from the sandwich and immediately placed in DI water, where the membrane was washed by soaking for 5 minutes. The treated membrane sample was then washed in methanol for 5 minutes. After this washing procedure, the membrane was dried on a stand in an oven operated at 50°C for 10 min.

[0079] Example 8 : This example demonstrates how a polyethylene (PE) film can be surface modified using a polymerized cross-linked coating containing vinyl imidazole monomers and N-[2-(dimethylamino)ethyl]acrylamide monomers.

[0080] In a representative test, a sheet of UPE membrane (1 micron nominal pore size rating) was wetted with an isopropyl alcohol (IPA) solution for 25 seconds. The membrane was rinsed with an exchange solution containing 100% water and the IPA was removed. The membrane sheet was then introduced into a surface modification solution containing 4% N-[2-(dimethylamino)ethyl]acrylamide, 4% vinylimidazole, 2% methylenebisacrylamide, 0.4% Irgacure 2959, 10% methanol, and 79.6% deionized water. The membrane was soaked in the surface modification solution for 2 minutes. The membrane sheet was taken out and placed between two polyethylene sheets. When the polyethylene / membrane sheet / polyethylene sandwich was laid flat on a table, excess solution was removed by rolling a rubber roller thereon. The polyethylene sandwich was then pasted on a conveying unit that transported the assembly through a Fusion Systems broadband UV exposure laboratory unit that emitted at a wavelength of 200 to 600nm. The exposure time was controlled by the speed at which the assembly moved through the UV unit. In this example, the assembly was moved through the UV chamber at 9 feet per minute. After exiting the UV unit, the membrane was removed from the sandwich and immediately placed in DI water, where the membrane was washed by soaking for 5 minutes. The treated membrane sample was then washed in methanol for 5 minutes. After this washing procedure, the membrane was dried on a rack in an oven operated at 50°C for 10 min.

[0081] Comparative Example 9 : This example demonstrates how a polyethylene (PE) film can be surface modified using a polymerized, cross-linked coating containing N-[2-(dimethylamino)ethyl]acrylamide monomers.

[0082] In a representative test, a sheet of UPE membrane (1 micron nominal pore size rating) was wetted with an isopropyl alcohol (IPA) solution for 25 seconds. The membrane was rinsed with an exchange solution containing 100% water and the IPA was removed. The membrane sheet was then introduced into a surface modification solution containing 8% N-[2-(dimethylamino)ethyl]acrylamide, 2% methylenebisacrylamide, 0.4% Irgacure 2959, 10% methanol and 79.6% deionized water. The membrane was soaked in the surface modification solution for 2 minutes. The membrane sheet was taken out and placed between two polyethylene sheets. When the polyethylene / membrane sheet / polyethylene sandwich was laid flat on the table, excess solution was removed by rolling a rubber roller thereon. The polyethylene sandwich was then pasted on a conveying unit that transported the assembly through a Fusion Systems broadband UV exposure laboratory unit that emitted at a wavelength of 200 to 600nm. The exposure time was controlled by the speed at which the assembly moved through the UV unit. In this example, the assembly was moved through the UV chamber at 9 feet per minute. After exiting the UV unit, the membrane was removed from the sandwich and immediately placed in DI water, where the membrane was washed by soaking for 5 minutes. The treated membrane sample was then washed in methanol for 5 minutes. After this washing procedure, the membrane was dried on a rack in an oven operated at 50°C for 10 min.

[0083] Example 10 : The three membranes of Example 4 were stacked together and challenged with 12 mL of 125 ng / uL plasmid DNA lysate in 50 mM potassium acetate, 630 mM NaCl pH 5. The membranes were then washed with 50 mM potassium acetate, 630 mM NaCl pH 5 and then eluted with 50 mM Tris pH 9. The binding capacity was 4.6 mg / mL with a yield of 93%.

[0084] Example 11 : The three membranes of Example 4 were stacked together and challenged with 12 mL of a 41 ng / uL plasmid DNA solution in 0.875 M potassium acetate pH 5. In one case, elution was performed using 50 mM Tris, 1.5 M NaCl pH 9, yielding 353 ug DNA. In the second case, elution was performed using 50 mM Tris, 1.5 M NaCl pH 9, yielding 343 ug. The yields were similar with and without salt in the elution buffer.

[0085] Example 12: Two membranes of Example 8 were stacked together and challenged with 8 mL of 30 ng / uL plasmid DNA lysate and RNase in 50 mM potassium acetate, 630 mM NaCl pH 5. The membrane was then washed with 50 mM potassium acetate, 630 mM NaCl pH 5 and then eluted with 50 mM Tris, 1.5 M NaCl pH 9. The binding capacity was 3.3 mg / mL with a yield of 64%.

[0086] Comparative Example 13 : Two membranes of Comparative Example 9 were stacked together and challenged with 8 mL of 30 ng / uL plasmid DNA lysate and RNase in 50 mM potassium acetate, 630 mM NaCl pH 5. The membrane was then washed with 50 mM potassium acetate, 630 mM NaCl pH 5 and then eluted with 50 mM Tris, 1.5 M NaCl pH 9. The binding capacity was 2.3 mg / mL with a yield of 84%.

[0087] Although various methods and films have been disclosed herein with reference to illustrative embodiments and features, it should be understood that the embodiments and features described above are not intended to limit the scope of the present disclosure, and based on the disclosure herein, other changes, modifications, and other embodiments may be presented to those skilled in the art. Therefore, the present disclosure encompasses all such changes, modifications, and alternative embodiments within the spirit and scope of the claims set forth below.

Claims

1. A method for separating charged biomolecules from a liquid using a porous ion exchange membrane, the method comprising contacting a liquid containing the charged biomolecules with a porous ion exchange membrane under adsorption conditions such that the charged biomolecules adsorb to the porous ion exchange membrane, the porous ion exchange membrane comprising a polymer porous membrane substrate and imidazolium ion exchange groups at the surface of the polymer porous membrane substrate, wherein: The charged biomolecule is plasmid DNA, The pH of the adsorption condition is lower than 6, The method comprises eluting the adsorbed charged biomolecules from the porous ion exchange membrane under elution conditions, wherein the pH of the elution conditions is above 7, The charge of the imidazolium ion exchange group is pH dependent and exhibits a positive charge at a pH range below 5.2 and a negative charge at a pH range above 5.2, The polymer porous membrane substrate comprises a base polymer, and The imidazolium ion exchange groups are grafted onto the surface of the polymer porous membrane substrate by exposing the vinyl imidazole monomer to ultraviolet radiation to form a grafted polymer structure of Formula 1, or the imidazolium ion exchange groups are formed on the surface of the polymer porous membrane substrate by exposing the vinyl imidazole monomer and methylene-bisacrylamide to ultraviolet radiation to form a cross-linked polymer network structure containing imidazole of Formula 2: The base polymer comprises one or more of polyolefin, polysulfone, cellulose acetate or nylon.

2. The method according to claim 1, wherein the porous ion exchange membrane has a porosity of 20 to 95% by volume.

3. The method of claim 1, wherein the porous ion exchange membrane has a thickness in the range of 5 to 300 microns.

4. The method of claim 1, wherein the porous ion exchange membrane has pores with an average pore size in the range of 0.2 to 10 microns.

5. The method of claim 1, wherein the base polymer comprises ultra high molecular weight polyethylene.

6. The method of claim 1, wherein the polyolefin comprises polyvinylidene fluoride, polypropylene or polytetrafluoroethylene.

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