Patent Application
20220097008
Multiblock polymer separators including an isoporous material, methods of preparing, and using to separate solutes through electrostatic interactions.
Despite major progress in understanding protein expression, structure, and function, purification of proteins from complex mixtures remains problematic. Target molecules, often in cell culture fluid, also contain a large variety of extraneous or even adventitious components, e.g. host proteins, cell debris, DNA, viruses, and endotoxins, which add to the complexities of separation. Target proteins are often highly sensitive to environmental conditions and may be easily denatured or otherwise damaged.
Upstream technology can result in product titers greater than 5 g/L, and are driving the need for high throughput purification. Traditionally, biomolecule purification has relied heavily on chromatography, including affinity chromatography and ion-exchange columns. However, these chromatographic techniques are throughput limited and contribute extensively to the high costs of downstream bioprocessing.
Ultrafiltration (UF) is frequently used in later stages of downstream bioprocessing for protein concentration and formulation. For example, there are commercially available UF membranes, which are either tracked-etched or phase-inverted. Track-etched membranes have very uniform pores, enabling selective separations, but suffer from low throughput due to low pore densities. On the other hand, phase-inverted membranes allow much higher fluxes, but have non-uniform pores and cannot achieve selective separations. Due to flux limitations, track-etched membranes are used almost exclusively for bench-scale research, while phase-inverted membranes are used extensively in downstream biopharmaceutical processes. Phase-inverted membranes empirically exhibit a broad log-normal pore size distribution, necessitating a 6-10× difference in protein molar mass for effective fractionation. This pore size variation causes uneven flow patterns across the membrane, broadening breakthrough curves and diminishing media capacity.
Deficiencies in membrane structure and chemical functionality have largely limited their use to concentration and formulation steps. Thus, there is a need to improve protein purification methods for both lab-scale-up verification, and of course commercial-scale manufacturing of medically relevant proteins. Applicants have found that combining the unique tuning of the physical structure of isoporous materials with chemical functionality to facilitate and separate similarly sized solutes through electrostatic interactions, enables the production of isoporous, charged multiblock polymers material, e.g., films, membranes, sheets and tubes, that result in high-throughput and high-resolution separation applications, that overcome the limitations of the prior art.
Other biomolecule separations from complex mixtures can also be especially difficult. Nucleic acids, for example, can be isolated from whole blood for diagnostics. The isolation of DNA from whole blood is an especially challenging separation due to solutes of various length scales including red blood cells, proteins, and salts. One method of DNA isolation is using electrostatic interactions to isolate the negatively charged DNA from complex mixtures. Nevertheless, no effective membrane technology has been commercialized to isolate DNA from whole blood due to the difficulty of the separation through existing membrane technology. Applicants have found that the herein described invention enables the separation and concentration of nucleic acids from whole blood.
Charged porous materials are effective for the prevention of biofilm growth. For example, in using such materials as dressings for wounds, porous materials enable the passage of gaseous phase chemicals like water vapor, nitrogen, and oxygen. However, when biofilms grow they can inhibit wound healing. The charged porous materials described herein may be used to inhibit biofilm growth. In addition, the charged porous materials may be further functionalized with antimicrobial chemistries to further enhance wound healing properties and aid in wound care applications.
Chromatography column have also been used extensively to separate species based on various physical and chemical features, including charge and size, however, chromatography is limited in throughput and often expensive. The charged porous materials described herein may be used to separate solutes based on charge and size. For example, species bound to the membrane through electrostatic interactions may be detached from the membrane by passing a gradient of eluent through the material. In this way, a gradient separation may be performed. Alternatively, a selection of species may be detached from the membrane by passing an eluent of fixed composition through the membrane. The fixed composition being suitable for detaching the desired selection of species only. In this way, an isocratic separation may be performed. Further a series of elutions may be performed with each elution being of fixed composition suitable for detaching additional selections of species. In this way, a step elution may be performed.
There has been a strong interest in using the combined effects of charge and size to fractionate solutes including biomolecules. However, a major limiting factor in the further development of this promising approach is the non-uniform pore sizes of existing membrane platforms.
Applicants have resolved this issue by enhancing pore size uniformity to reduce the spread of the log-normal pore size distributions of historical membrane platforms. For example, in one embodiment of the invented membrane, as shown in
The invention relates to filters/separators with an isoporous multiblock polymer material, collectively material, methods of preparing, and using to separate solutes including biomolecules through electrostatic interactions.
The charged, self-assembled block polymer material of the invention contain at least one of macro, meso, or micro pores, at least some of which are isoporous, where at least one polymer block contains stationary electrostatic charges, or reactive functional groups. The material has major, minor and interstitial regions, partially or quantitatively functionalized by the charged moiety on the functional group to produce a stationary charge on the material, to provide a large surface area that is charged and an isoporous structure for facile separations of proteins, nucleic acids, other biological or other biomolecules or solutes, with high fluxes.
The isoporous, charged, multiblock polymer material of the inventions includes at least blocks A, B and C having at least one of macro, meso, or micro pores, at least some of which are isoporous, where at least one polymer block contains stationary electrostatic charge, or reactive functional groups, and defined by 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)
The multiblock polymer of the invention is generically defined as containing blocks A, B, C, (X)n, where n=0−7, and X can be the same or different and is selected from, A, B, C, D, E, F or G. A suitable block for one of A, B, or C, is a low Tg block (≤25° C.) with the proviso that when one of the blocks is a low Tg block, at least one other block is a high Tg block (>25° C.). At least one block must have stationary charge, or reactive functional groups which can react to generate stationary charge. A suitable block for D, E, F, or G includes but is not limited to: poly(butadiene), poly(isobutylene), poly(butylene), poly(isoprene) poly(ethylene), poly(styrene), poly(methyl acrylate), poly(butyl methacrylate), poly(ethersulfone), poly(methyl methacrylate), poly(n-butyl acrylate), poly(2-hydroxethyl 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 sulfonate), poly(allylamine hydrochloride), poly(pentafluorostyrene), poly(2-(perfluorohexyl)ethyl methacrylate).
The invention includes materials, such as, but is not limited to, supported or unsupported, in three-dimensional, membrane, film, sheet, tube, or helical or spiral configuration, where the material includes a functionalized isoporous block polymer with a charged moiety, and renders the material suitable for high through-put and charge-based separations.
The invention includes isoporous multiblock polymers possessing a stationary charge that facilitates solute separation based on both size and electrostatic characteristics of the solutes.
The invention includes functionalized separators with high fluxes for protein fractionation and isolation for analytical and industrial protein fractionation.
The invention relates to isoporous materials that: facilitate separation of similarly sized molecules, such as, proteins e.g. through electrostatic repulsion of one positively charge protein and one neutral protein, for protein production and analysis, biospecific molecules: including patient specific biomolecules, whether in the laboratory, scale-up or commercial environment.
The invention relates to isoporous materials that: facilitate separation of similarly sized proteins, methods of preparing and use, through electrostatic interactions; facilitate separation of nucleic acids through electrostatic interactions; facilitate separation of charged biomolecules through electrostatic interactions.
The invention relates to isoporous materials that: facilitate selective separation of matrix metalloproteinases through electrostatic interactions.
The invention relates to charged isoporous material of various configuration: three-dimensional, films, tubes, spirals, sheets, etc.
The charged isoporous material having selectivity of at least 10, and/or a permeability of 50 Lm−2hr−1bar−1 for electrostatically separating similarly size proteins with different charge states.
The invention includes materials of self-assembling multi-block polymers or self-assembling multi-block copolymers (MBP or BCP) with at least one chemically reactive functionalized polymer block that form hierarchically porous materials with high surface areas and uniform pore sizes, which are disclosed herein. The reactive regions of these isoporous materials are partially or quantitatively functionalized by the reaction of terminal or cross-linking agents with the reactive units to produce a stationary charge on the material, such that the degree of charge is at least 20% to 90%, e.g., at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of available reactive units.
Self-assembled multiblock copolymer charged material wherein the material has mesopores comprising a size of about 1-200 nm, and all unit sizes and ranges therebetween, and macropores comprising a size that is at least 50 nm or greater
Disclosed are self-assembled multi-block, isoporous charged materials described herein that effectively separate a charged solute or solutes at hydraulic permeabilities >50 Lm−2hr−1bar−1, >100 Lm−2hr−1bar−1, >200 Lm−2hr−1bar−1. The dual separation methodologies of size-exclusion and charge-interaction drive high-throughput bioprocessing of proteins, nucleic acids, therapeutic compounds, or other molecules or compounds of interest.
Disclosed are self-assembled multiblock, isoporous charged materials with stationary positive charges, to separate similarly sized proteins through electrostatic repulsion of one positively charge protein and one neutral protein.
Disclosed are self-assembled multiblock, isoporous charged materials with stationary negative charges, to separate similarly sized proteins through electrostatic repulsion of one positively charge protein and one neutral protein.
Disclosed are self-assembled multiblock, isoporous charged materials with stationary positive charges, to separate nucleic acids through charge interactions.
Disclosed are self-assembled multiblock, isoporous charged materials with stationary positive charges, to separate biomolecules through charge interactions.
Disclosed are self-assembled multiblock, isoporous charged materials with stationary negative charges, to separate biomolecules through charge interactions.
The isoporous nature of the 5-100 nm size pores, and all unit sizes and ranges therebetween is especially beneficial to charge separations since all species coming in contact with the isoporous separation layer are forced experience the same electrostatic environment. If the pores are too polydisperse in size, it is possible for solutes to experience different charge environments e.g. pass through the center of a large pore without any charge separation effects since electrostatic charge interactions dramatically drop off as a function of distance. The high porosity, and in some cases hierarchical structure, contribute to a high surface area and thus large amount of charge available for separations. In operation with a bind and elute mechanism, this also provides a large binding capacity.
In the following description, reference may be made to the accompanying drawings that form a part hereof, and in which is shown by way of illustration specific embodiments which may be practiced. These embodiments are described in detail to enable those skilled in the art to practice the invention, and it is to 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. The description of any example, embodiments or experimental procedures is, therefore, not to be taken in a limited sense.
The complexity of either or both the target molecules solutions and the target molecules themselves often require several steps and is a time consuming and expensive process, all exacerbated by the need to balance effective separation with high throughput. Two important performance parameters that will be improved through the chemical functionalization of isoporous membranes are the sieving coefficient, S is defined as:
where CF and CF are the concentration of the protein of interest in the permeate and feed, respectively, and the hydraulic permeability, LP, defined as:
where JV, is the volumetric filtrate flux and ΔP is the transmembrane pressure.
Separations involving nanoporous or ultrafiltration membranes have typically focused on size-selectivity, but electrostatic interactions have a significant impact on selectivity and permeability with protein solutes. The isoelectric point (pI) of a protein is the pH at which the biomolecule exhibits no net charge. In solutions below the pH of the protein pI, the molecule is positively charged, while at a pH above the protein pI, the molecule is negatively charged. Such charge characteristics have been exploited for protein filtration with nanoporous or ultrafiltration membranes by tuning solution pH as well as ionic strength, improving selectivity and throughput by up to an order of magnitude. Furthermore, there is a strong relationship between protein size and charge, with effective protein size increasing as ionic strength decreases due to decreased electrostatic shielding. More recently, combining processing conditions with charged nanoporous or ultrafiltration membranes has been shown to result in further improved protein selectivity.
One key differentiating feature of this invention over conventional charge-based membrane separations is the use of a focused pore distribution in isoporous membrane structure as opposed to nanoporous or ultrafiltration membranes with broad pore size distributions. This enables simplified processing conditions and higher operating pressures, which results in overall higher permeabilities.
Previous work on optimized charged ultrafiltration membrane separations in tangential flow configurations, in which the feed is passed parallel to the membrane surface, have resulted in average permeate fluxes of ˜15-200 L m−2 hr−1 (Zydney and Kuriyel, High-Performance Tangential Flow Filtration for Protein Separations. Downstream Processing of Proteins, Springer, 35-46, (2000)). In contrast, self-assembled membranes of the present invention, with narrow “focused” pore size distributions show permeate fluxes up to 850 L m−2 hr−1.9 While certain performance limiting factors are enhanced at high fluxes, e.g. concentration polarization and membrane fouling (Shukla and Thommes, Trends Biotechnol., 28, 253-261, (2010)), the combination of physical membrane characteristics combined with electrostatic properties enables performance that surpasses existing membrane-based protein separations.
Suitable isoporous BCP material membranes for this invention are those that self-assemble. Typically, the preparation of such an isoporous organic membrane involves dissolving the BCP (or MBP) polymer in solvent(s) that are at least partially volatile, casting the solution under defined conditions so that at least a portion of the solvents are evaporated, and then contacting the resulting material, e.g., membrane/film with a phase separation solvent system. Such procedures are generally described in, for example, U.S. Pat. No. 5,700,902; U.S. Pat. No. 6,565,782; U.S. Patent Pub. No. 20110240550; U.S. Patent Pub. No. 20130193075; International Patent Pub. No. WO2005082501; U.S. Patent Pub. No. 20090173694; International Patent Pub. No. WO 2012/151482; EP2705077A2; Clodt et al, Macromol. Rapid Commun., 34, 190-194 (2013), Nunes et al, Langmuir, DOI 10.1021/Ia20439p, web published 28 Jun. 2011; Phillip et al, Nano Letters, 11, 2892-2900 (2011); and Rangou et al, J. Membrane Sci., 451, 22662-2275 (2014), the entirety of each incorporated by reference.
Another method for the formation of the porous membranes from suitable block polymers is the “SIM2PLE” (spinodal decompostion-induced macro- and mesophase separation plus extraction by rinsing) process as described in Sai et al, Science, 341, 530 (2013). In this process, a suitable BCP is first prepared, mixed with a separate oligomeric polymer in a suitable organic solvent, casting the mixed solution to form a hierarchically porous membrane by partial solvent evaporation at an elevated temperature. At the final stage, 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 choice of solvent and polymer concentration, time and temperature of the solvent removal as well as other experimental factors. A suitable second oligomeric polymer for the “SIM2PLE” process is one that is water-soluble and can be removed from the BCP by washing with water. In one embodiment, poly(acrylic acid) or PAA is used as the second oligomeric polymer although other soluble oligomeric polymers such as poly(ethylene oxide) PEO could be used as well.
In one embodiment, the block copolymers that self-assemble comprise a polymeric block that has a low Tg (≤25° C.). This low Tg block provides mechanical toughness and eases processability and handleability of the material. Another block should have a Tg>25° C. which provides mechanical stability. At least one of the blocks should be a polymer that contains at least one functional group which can be chemically modified to introduce an electrostatic charge to the material, or is already charged.
In one embodiment, the functional polymer block contains an aromatic nitrogen heterocycle as the chemically modifiable functional group. In this embodiment, suitable aromatic heterocycles include, but are not limited to, pyridines, pyrazines, pyrimidines, pyridazines, quinolones, isoquinolines, quinoxalines, quinazolines, phenazines, isoxazoles, isothiazoles, imidazoles, benzimidazoles, triazoles, tetrazoles, and the like. Preferred aromatic nitrogen heterocycles are those that do not have an ionizable hydrogen on the nitrogen; that is, the nitrogen is fully part of an aromatic ring. Pyridine and its derivatives are particularly preferred heterocycles. Other examples of polymers that contain an aromatic heterocyclic group include, but are not limited to, poly(2-vinylpyridine) or poly(4-vinylpyridine) (P2VP or P4VP).
In one embodiment of the invention, the blocks used to form the self-assembled isoporous material, e.g., membrane, is an ISV, a triblock or terpolymer poly(isoprene-b-styrene-b-4-vinyl pyridine), see the
In this embodiment, the triblock terpolymer architecture contains approximately 0.30 volume fraction polyisoprene (PI), 0.55 volume fraction polystyrene (PS), and 0.15 volume fraction poly(4-vinyl pyridine) (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 material's architecture provides good mechanical properties while simultaneously allowing for small pore sizes. The ISV terpolymer starting material may be synthesized via anionic polymerization. In some embodiments, the isoporous (narrow pore size distribution) film has a surface layer (also referred to herein as a top layer) and a bulk layer. The suitable surface layers have a range of thicknesses. For example, the surface layer can have a thickness of from 5 nm to 500 nm, including all values to the nm and ranges therebetween. The surface layer has a plurality of pores extending thorough the depth of the surface layer. Pores size (e.g., diameter) in the surface layer ranges from 1 nm to 200 nm, including all values to the nm and ranges therebetween.
In an embodiment of the invention, the density of the surface pores of a membrane as described herein is at least 1014 pores/m2, or at least 1015 pores/m2. A narrow or focused pore size distribution (defined as the ratio of the maximum pore diameter to the minimum pore diameter (dmax/dmin)), is from 1 to 3, including all values to 0.1 and ranges therebetween.
In various examples, (dmax/dmin) is less than three, e.g., 1, 1.5, 2, 2.5, or 3, and all ranges thereof. For example, the film comprises a surface layer having nearly monodisperse mesopores. In an embodiment, the isoporous surface layer has a pore density of at least 1×1014 pores/m2 and a pore size distribution (dmax/dmin) of less than 3.
In some of the above embodiments, the P4VP material of the ISV terpolymer resides on the surfaces of the as-cast material matrix, as confirmed by both the pH dependent hydraulic permeability of the membranes that exhibit an inflection points near the pKa of P4VP, as well as stained transmission electron microscope images indicating nanometer-scale phase separation of each block.
The isoporous material, as described in some of the above embodiments (53-55), contains a polymer block with a nitrogen heterocycle that resides on the surfaces of the membrane and is accessible to quaternizing agents, which surfaces includes interstitial surfaces of the pores. For example, in the embodiment above where the copolymer contains P4VP (a weak base), the P4VP is quaternized using a variety of chemicals, resulting in a stationary positive charge on the membrane as shown in
The quaternization agent in this embodiment is chosen to optimize the purification of the particular solute being targeted. There are many characteristics of the agent that can be applied in a useful way. For example, the degree of the hydrophilicity/hydrophobicity of the charged material can be an important consideration for some proteins. In order to control the hydrophilicity/hydrophobicity, the quaternization agent can be selected, for example, according to a value or range of ClogP of the residue (a measurement that reflects the degree of hydrophilicity/hydrophobicity). For example, if a more hydrophilic environment is desirable, a quaternization agent such as iodoacetamide, iodoacetic acid, or crotonic acid which leaves a material with a relatively low ClogP may be chosen. If a more hydrophobic environment is desirable, then a quaternization agent such as dodecyl iodide that leaves a material with a relatively high ClogP may be chosen. There are other metrics of the degree of hydrophilicity/hydrophobicity other than ClogP that may be used. For example, there may be an optimum 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 useful functionality (e.g. pH response, thermal response, solvent resistance).
The quaternizing agent in the above embodiment is any suitable compound that reacts with the nitrogen heterocycle of the BCP to form a positively charged heterocycle. One feature of the invention is that the degree or amount of charge can be varied in order to control the amount of positive charge. This allows for optimization of the total charge for the protein that is being purified. The charge density (corresponding to the degree of quaternization) of the membrane after quaternization can be determined by FTIR and NMR.
One group of quaternizing agents of the above embodiment are compounds defined by the formula R—X, where R is a C1-C24, more preferably C1-C20 substituted or unsubstituted, straight-chain, cyclic or branched alkyl or alkenyl group and X is a leaving group such as halide (F—, Cl—, I—, Br—), or —OSO2CH3, etc. Typically, the leaving group would be a terminal group (i.e. located on the end) on the alkyl or alkylene group and attached to an unsubstituted sp3 carbon (i.e. —CH2—X) in order to maximize reactivity. The alkyl or alkylene group may have 1-24 carbon atoms in the backbone. Examples of straight-chain alkyl groups would include, but are not limited to methyl, ethyl, butyl, hexyl, octyl, decyl and dodecyl. One example of a cyclic alkyl group is —CH2-cyclohexane.
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 displacement reaction is preferred, it would be possible to quaternize the heterocycle by an addition reaction to a suitable olefin. For example, reaction with a H2C═CHQ compound where Q is an electron-withdrawing group would result in a quaternized heterocycle of the form Het+-CH2—CHQ, where Q is an electron-withdrawing group. Examples of this class of agent would include ethyl methacrylate or methyl acrylamide.
Another class of quaternizing agents of the above embodiment is defined by the formula R—W—(R)—CH2—X, where R is a C1-C24, more preferably C1-C12 substituted or unsubstituted alkyl, alkenyl or aryl group, W is a heteroatom such as oxygen, silicon, sulfur or nitrogen and X is a leaving group as above. Some examples of this class of quaternizing agent would be X—CH2—O—C2H5, X—CH2CH2—O—CH3 and X—CH2CH2-morpholine.
Another class of quaternizing agents is acyl compounds of the formula R—(W)—COCH2—X where R is a C1-C24, more preferably C1-C10 alkyl or alkenyl, or C6-C10 aryl group, W is a heteroatom such as oxygen or nitrogen and X is a leaving group as above. Suitable examples of this class of acylation agents include X—CH2—CO—C6H5, X—CH2—CO2CH3, X—CH2—CONH2 and X—CH2CON(CH3)2.
Suitable quaternization compounds of the above embodiments include, but are not limited to quaternization, including: bromobutane, bromo-PEG, bromopropionic acid, bromovaleric acid, chloroacetamide, chlorobutane, chlorobutyric acid, crotonic acid, diiodobutane, iodobutane, iodoacetamide, and iodopropionic acid. Iodoacetamide is of particularly interest due to many desirable characteristics: low vapor pressure, relatively low hazard, inexpensive and readily available, water soluble, controllable quaternization, hydrophilicity, and preservation of the membrane's selective layer. These characteristics make this quaternization process easily scalable for large-scale production of electrostatically charged membranes. The final conditions used for quaternizing the membranes shown below were: 6.6 mM aqueous iodoacetamide, 60° C., 1 h. The nucleophilic substitution reaction yields the positively charged pyridinium, with a pendant acetamide group in the material, as shown below:
In the above embodiment, the iodoacetamide reacts with poly(4-vinylpyridine) and yields the stationary charge on the membrane. This reaction results in a terminal amide on the membrane and a persistent positive charge.
Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) was used to characterize the unquaternized and quaternized membranes. Characteristic peaks for the quaternization process were observed in the iodoacetamide-treated membranes, as seen in
Suitable bifunctional quaternizing agents of the above embodiment, which have two sites of reaction with the heterocycle and serves as a cross-linking agent, include cross-linking agents defined by the formulas X—R—X, X—CH2—CO—(W)—R—(W)—COCH2—X or X—CH2—(R)—W—(R)—CH2—X, where R is an alkyl, alkenyl or aryl group, W is a heteroatom such as oxygen or nitrogen and X is a leaving group as above. The cross-linking agent may not be symmetrical; that is, one reactive group may be different from the second reactive group. Examples of a bifunctional quaternizing group include X—(CH2)4—X, X—CH2CH2—O—CH2CH2—X, ethylene glycol di(meth)acrylate, derivatives of methylenebisacrylamide and derivatives of N-1-hydroxyl-2,2-dimethoxyethyl acrylamide. A cross-linking agent such as 1,4-diiodobutane is of particular interest due to the potential for increased membrane mechanical integrity and enhanced chemical resistivity.
Suitable quaternizing agents of the above embodiment also include additional functional groups that can bear positive or negative charges in order to adjust the overall charge of the material as well as the degree of the hydrophilicity/hydrophobicity. For example, the quaternization agent can be an anionic compound or a compound that ionizes to form an anion under the aqueous purification conditions. This would form a zwitterionic heterocycle that is neutral in overall charge and would reduce the positive charge of the material while still retaining highly polar characteristics. Examples of this kind of agent would include compounds with carboxylic or sulfonic acids groups such as —CH2CH2CO2H or —CH2CH2SO3H. Alternatively, the quaternization agent is a cationic compound or a compound that is protonated under the purification conditions. Examples of this kind would include —CH2CONHCH2CH2—NH(CH3)2+ or —CH2CH2—N(CH3)3+. This would increase the overall positive charge of the material. These kinds of charge control quaternization agents are used together with hydrophilicity/hydrophobicity control agents to control the membrane properties as required.
Since proteins and other kinds of biologic materials are often optically active, the functionalizing agent may be chiral. This would result in a material that has some degree of chirality associated with it. Examples of suitable optically active alkylation agents may be found in Houben-Weyl Methods of Organic Chemistry Vol. E 21a, 4th Edition Supplement, Stereoselective Synthesis, H. Ahlbrecht et al, Ed., Thieme, 1995. For example, suitable chiral quaternization agents of the above embodiment would include optically active primary alkyl halides or mesylates.
Modification of the material to introduce charge is performed by immersing the as-prepared membranes in solutions containing the modification agent. In some embodiments, the charge introduction will be quantitative, >20%, >30%, >40%, >50%, >60%, >70%, >80%, or >90% of all available reaction sites, but in other embodiments, the degree of quaternization may be less than quantitative. A suitable solvent for the quaternization is water. However, some of the agents may have limited solubility in water, so combinations of water and an organic auxiliary solvent (such as an alcohol) are used. In some cases, it may be necessary to use a non-aqueous organic solvent or mixtures of organic solvents. Typical conditions for this process would be to dissolve the quaternization agent in an appropriate solvent system and treat the membrane at reagent concentrations of 2 and 100 mM, with a reaction time of 1 to 24 hours at a temperature of 20 to 30° C. In some cases with less reactive agents, it might be necessary to use longer reaction times and elevated temperatures. The progression of this process can be monitored using fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR).
In some embodiments, the highest charge densities possible are desirable for the most selective charge-based separations of charged solutes. However, even in these cases, the membranes must retain high flux of the feed solutions at various pHs. Membrane performance can be measured utilizing a pressurized dead-end stirred-cell apparatus. This apparatus may use a 10 to 50 mL feed reservoir and membrane areas of ˜4 cm2 to ˜13 cm2.
The type or kinds of proteins or other biologically based materials to be purified or concentrated by the charged isoporous membrane of the invention is not limited. In particular, the feedstocks may be proteins derived from mammalian, bacterial, insect, viral or fungal cell lines. They are typically used in an aqueous feedstock, which may already been partially purified by another process. It is also possible to use fermentation broths as the feedstock. The feedstock may be whole blood, diluted blood, plasma, serum. The feedstock may contain nucleic acids, which may be separated using the material.
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 found in cell culture fluids. Flux with feeds containing protein solute can be measured using the same methods described above, while protein rejection can be quantitatively measured using UV-vis spectroscopy and aqueous high performance liquid chromatography (HPLC) on feed and permeate solutions. The calculated rejection, R, is related to the sieving coefficient as:
UV-Vis detection with HPLC is a simple and rapid method for separating protein mixtures and quantitatively determining protein concentrations.
However, in some embodiments of the invention, the feedstock may contain binary (or even more complex) solute mixtures, for example, the proteins Mgb/Lys or Mgb/CytC. One key performance parameter for such mixtures is the selectivity, Ψ, defined by van Reis and Saksena:
where S1 and S1 are the sieving coefficients for lesser- and greater retained protein (van Reis and Saksena, J. Membrane Sci. 129, 19-29 (1997)). This parameter allows a simple and direct comparison of the relevant performance property between the charged and untreated membrane.
In this example, the Mgb/Lys and Mgb/CytC components are very similar in size and cannot be separated with simple ultrafiltration. Using the inventive membrane with either mixture at neutral pH, the Mgb is neutrally charged (due to its isoelectric point) and passes through the membrane with a selectivity corresponding to when Mgb is not in a mixture. The Lys or CytC are expected to be positively charged at neutral pH (due to their isoelectric points) and will be rejected by the like-charged membrane. The inventive membrane is designed to achieve selectivity between such membrane pairs of at least 10 in a dead-end system at flow rates of over 50 LMH/bar.
In some embodiments, the purification process using the charged isoporous membranes involves using pressurized buffered feed streams containing the biological material at, below, and above the isoelectric point of the desired material.
In another embodiment, the charged isoporous material is used to isolate nucleic acids. After quaternization of P4VP of an ISV isoporous material, as in the above embodiments, the isoporous material has a positive charge. When exposed to a mixture of DNA fragments, the negatively charged DNA binds to the membrane. Subsequently, the DNA can be eluted from the membrane using a small amount of elution buffer solution. The recovery yields for 200 and 400 base pairs (bp) for four different membranes are shown in
In this embodiment, size-selective elution of DNA fragments is also possible. Different dilutions of elution buffer (0.6×, 0.3×) were used to elute.
In another embodiment, the block copolymer is poly(styrene-b-isoprene-b-styrene-b-4vinylpyridine), corresponding to generic structure A-B-A-C. The P4VP is quaternized to generate stationary charge as in 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.
In one embodiment, the reaction yielding stationary charge on the isoporous material changes the hydrophilicity of the material. In another embodiment, the reaction yielding the stationary charge on the isoporous material introduces a functional group to the material that can be used for further chemical reactions or material functionality.
In some embodiments, the stationary charge on the isoporous charged material is positive e.g. quaternized pyridine group, quaternized diethylaminoethyl group, quaternized dimethylaminoethyl group. In other embodiments, the stationary charge on the isoporous charged material is negative e.g. sulfonate group. In some embodiments, the isoporous material contains an amine group (e.g. pyridine, diethylaminoethyl, dimethylamino ethyl) which can be quaternized with a chemical agent to generate positive charge on the material. In one embodiment, amines are quaternized with a monofunctional alkylating agent, with one halogen selected from iodine, chlorine, and bromine e.g. 2-iodoacetamide, 2-bromoethanol. In one embodiment, the monofunctional alkylating agent has the formula: R—X where R is a C1-C24 substituted or unsubstituted, straight-chain, cyclic or branched alkyl or alkenyl group and X is a halogen group.
In another embodiment, the monofunctional alkylating agent has the formula: R—W—(R)—CH2—X where R is a C1-C24 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 R is a C1-C24 alkyl, alkenyl or aryl group, W is a heteroatom such as oxygen or nitrogen and X is a halogen group.
In another embodiment, multiple amines are quaternized with a multifunctional alkylating agent with two or more halogens selected from iodine, chlorine, and bromine e.g. 1,4-diiodobutane.
In another embodiment, amines are quaternized with an alkylating agent containing at least one reactive double bond e.g. crotonic acid.
In some embodiments, more than one of the aforementioned amine-alkylating agents containing is used to introduce charge on the isoporous material.
In some embodiments, the degree of charge on the isoporous charged material is at least 20% to 90%. In other embodiments, the degree of charge on the isoporous charged material is ≤90% of all available units.
In one embodiment, the charged isoporous material is further treated or functionalized with an antimicrobial agent.
In some embodiments, the geometry and area of the isoporous material that contains a stationary charge is controlled. In some embodiments, the geometry and area of the charged region is patterned lithographically, or achieved by attaching a portion or portions of charge modified material to an unmodified material or another substrate, or patterned onto a portion or portions of the unmodified material through printing or extrusion.
In some embodiments, the isoporous charged material is used as a separation media. In some embodiments, the isoporous charged material is used to separate biomolecules from other biomolecules. In an embodiment, the target biomolecule is a protein. In another embodiment, the target biomolecule is a nucleic acid.
In some embodiments, the separation mechanism using the charged isoporous material as separation media includes a bind and elute mechanism where the target species is bound to the material by electrostatic interactions to isolate it, wherein the target species can be unbound from the material to recover the target species.
In some embodiments, the separation mechanism using the charged isoporous material as separation media includes a charge repulsion mechanism, a size selection mechanism, and/or a concentration mechanism.
In some embodiments, the separation mechanism using the charged isoporous material as separation media with a bind and elute mechanism also includes a chemistry selective or size selective elution.
In some embodiments, the charged isoporous material is used for gradient separation, isocratic separation, or step separation.
In some embodiments, the charged isoporous material is used as a dressing or bandage for a wound, or is used to modulate the growth a biofilm.
In some embodiments, the charged isoporous material is combined with more than one charged material and packaged in a device, or charged isoporous material is combined with uncharged isoporous material and packaged as a device.
In some embodiments, the charged material is packaged in a pleated pack, crossflow cassette, hollow fiber module, syringe filter, capsule, pipette tip, centrifuge tube, spiral wound module, or sensor device.
In another embodiment, the charged material is packaged as a flat sheet.
In some embodiments, the charged isoporous material is immobilized on a support material or integrated directly with a textile.
In one embodiment, more than one charged isoporous material is packaged together as a kit.
In another embodiment, more than one device incorporating the charged isoporous material is packaged together as a kit.
In another embodiment, at least one charged isoporous material is packaged with one or more chemical solutions to elute bound target species after binding.
This application claims the benefit of U.S. provisional application No. 62/328,707, filed Apr. 28, 2016, the contents of which are incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62328707 | Apr 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15538600 | Jun 2017 | US |
| Child | 17545902 | US |
