Separation of virus particles with ligating filter elements

Abstract

This present disclosure is directed to ligating monomers and polymers prepared therefrom are described. Filter elements including the ligating polymers are further described as are the uses thereof for separation of adeno-associated viruses containing a DNA payload ("full AAV") from adeno-associated viruses without a DNA payload ( "empty AAV).

Description

PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0SEPARATION OF VIRUS PARTICLES WITH LIGATING FILTER ELEMENTSNarendranath BhokishamEthan Laudermilch Semra Colak Atan Jerald K. Rasmussen Nisha Hollingsworth George W. Griesgraber Katie Fraass Wlaschin Logan Daniel Cruckson Bishop Patrick M. CrainErick I. Soto Cantu Lindsay Traeger Emily Julik Qiuge Zhang Shri Niwas Hector F. Espitia NavaarroCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application embodiments priority to U.S. Application No. 63 / 722,195, filed on November 19, 2024, the contents of which are hereby incorporated by reference in its entirety. FIELD OF THE INVENTION

[0002] The field of the invention relates generally to filter elements including ligating polymers and the uses thereof for separation.BACKGROUND

[0003] This background information is provided for the purpose of making information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should it be construed, that any of the information disclosed herein constitutes prior art against the present invention.

[0004] Adeno-associated viruses (AAVs) are small, non-enveloped, single-stranded DNA viruses belonging to the Parvoviridae family. AAVs are currently being used as therapeutic delivery vehicles for in vivo delivery of genetic material to target cells. AAVs can be engineered to lack viral genes and instead contain DNA sequences of interest for various therapeutic applications (the “DNA payload”), e.g., to treat systemic diseases such as hemophilia and muscular dystrophy. During AAV production, cells produce payload containing AAV viruses, referred as ‘full AAV’ as well as viruses with no DNA payload, referred as ‘empty AAV’. In addition, cells also produce viruses with partial DNA payloads of varying lengths, referred to as ‘partial AAV’, as well as payloads with lengths longer than thePCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0DNA payload, referred to as ‘extra-full AAV’. Current production techniques afford 5-30% AAVs that are filled (i.e., “full AAV”) with a DNA payload, 70-95% are empty AAVs, and a varying number of partially filled and extra full-AAVs.

[0005] Empty, partially-filled, and extra-filled AAVs are considered impurities since excessive injection of overall virus may increase the risk of adverse side effects. Thus, there is a need to eliminate, or at least reduce, the amount of empty, partially-filled, and extra-filled viruses to effectively treat complex systemic diseases via gene therapy. Ideally, only full AAV would be used for gene therapy treatments. Currently, there are no FDA guidelines on the full virus requirements; however, the industry is moving towards therapeutic treatments with 70- 90% full AAVs.

[0006] Separating full AAVs from empty AAVs is challenging since both virus scaffolds have identical shape and size. One physical difference between full and empty AAVs is that the DNA inside full viruses induces a small difference in the net surface charge. As a result, full viruses have a slightly lower isoelectric point, and this minor difference in charge can be exploited via ion-exchange chromatography. Anion-exchange chromatography with select quaternary ammonium ligands (commonly known as “Q chemistry”) is currently used to separate empty AAVs from full AAVs. Said select ligands are typically trimethylammonium (i.e., -N+(CH3)3) ligands. However, chromatography with current Q chemistries suffers from low salt tolerance leading to virus aggregation and lacks clear separation due to a narrow window of conductivity for separation (~l-2 mS / cm).

[0007] What is needed are ligands that can better separate full AAVs from empty AAVs so that safer gene-based therapies can be realized.

[0008] BRIEF DESCRIPTION OF THE FIGURES

[0009] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0010] FIG. 1A is a graph illustrating the salt tolerance of the filter elements of the present disclosure compared with other filter elements.PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0

[0011] FIG. 2A is a chromatogram illustrating the elution profiles for an empty / full AAV5 separation process across a gradient of salt concentrations / conductivities with a filter element of the present disclosure.

[0012] FIG. 2B is a chromatogram illustrating the elution profiles for an empty / full AAV5 separation process across a gradient of salt concentrations / conductivities with a filter element of the present disclosure.

[0013] DESCRIPTION

[0014] Summary

[0015] In one embodiment, a ligating monomer of Formula I is described.CH2=C(R1)-C(O)-W-R2-N+(R3)2-R4-Ar Z (I), wherein:R1is -H or a straight or branched Ci-4 alkyl,W is -O- or -N(R5)-,R2is a straight or branched C2-Ce alkylene, a straight or branched C4-Ci2alkoxyalkylene, or - R6-X-C(O)-Y-R7-, each R3is independently a straight or branched Ci-Ce alkyl,R4is a straight or branched Ci-Ce alkylene optionally substituted with oxo or oxa, or a straight or branched Ci-Ce alkylene, Ar is aryl,Z is a counterion,R5is -H or a straight or branched Ci-4 alkyl,R6is a straight or branched Ci-Ce alkylene, or a straight or branched C4-Ci2alkoxyalkylene, R7is a straight or branched Ci-Ce alkylene, or a straight or branched C4-Ci2alkoxyalkylene, X and Y are independently selected from -O-, or -N(R5)-, provided that at least one of X and Y is -O-, or -N(R5)-.

[0016] In one embodiment, a polymerizable composition is described. The polymerizable composition includes one or more ligating monomer of the present disclosure and optionally one or more co-monomer.

[0017] In one embodiment, a ligating polymer is described. The ligating polymer is derived from one or more ligating monomer of the present disclosure.

[0018] In one embodiment, a filter element is described. The filter element includes one or more porous substrate and a filter media. The filter media includes a ligating polymer of thePCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 present disclosure, wherein the ligating polymer is grafted to at least one of the one or more porous substrate.

[0019] In one embodiment, a filter element is described. The filter element includes one or more porous substrate and a filter media. The filter media includes a ligating polymer that is derived from one or more ligating monomer of the present disclosure, wherein the ligating polymer is grafted to at least one of the one or more porous substrate.

[0020] In one embodiment, a process for separating an adeno-associated virus containing a DNA payload (i.e., “full AVV”) from an adeno-associated virus without a DNA payload (“empty AVV”) is described. The process includes providing a filter element of the present disclosure and providing a loading solution having the full AVV and empty AAV. The process further includes contacting the loading solution to the filter element and separating the full AAV from the empty AAV.

[0021] In one embodiment, a process for preparing a filter element of the present disclosure is described. The process includes providing a polymerizable composition having one or more ligating monomer of the present disclosure, providing a porous substrate, and contacting the polymerizable composition to the porous substrate under conditions effective to polymerize the polymerizable composition onto the porous substrate.

[0022] In one embodiment, a process for preparing a filter element of the present disclosure is described. The process includes providing a ligating polymer of the present disclosure, providing a porous substrate, and contacting the ligating polymer to the porous substrate under conditions effective to graft the ligating polymer to the porous substrate.

[0023] In one embodiment, a kit is described. The kit includes a filter element of the present disclosure and a set of instructions for using the filter element for separating empty and full AAVs.

[0024] Detailed Description

[0025] The present disclosure is directed toward chromatographic separations of full vs empty AAVs with polymeric filter media prepared from ligating monomers that have a relatively hydrophobic quaternary ammonium group. The chemistries of the present disclosure have several advantages over current Q chemistries (e.g., Pall Mustang® Q, BIA CIMultus® Q monolith, Capto Q, and Emphaze™ filters), including, but not limited to, greater salt tolerance and a wider window of conductity for separation.

[0026] It is theorized that the ligating chemistries of the present disclosure bind preferably within the 5f pore site of the AAV virus and interact with the internal hydrophobic amino acidsPCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 of the virus. Conversely, current Q chemistries, having short alkyl chains bonded to the quadrivalent nitrogen, were observed to only interact with exterior regions of the 5f pore site via molecular docking computations. Without wishing to be bound by theory, the increased salt tolerance of the present chemistries compared to current Q chemistries may be explained by these differences in binding activity. In other words, the ligating chemistries of the present disclosure may bind more strongly to AAVs thereby requiring higher salt concentrations to release bound virus. In other words, the present ligating chemistries offer higher salt tolerances.

[0027] At higher salt tolerances, virus samples may be loaded onto filter elements in solutions of higher salt concentration. This is advantageous because virus samples tend to aggregate at lower salt concentrations. It is further advantageous because having to load virus samples at lower salt concentrations, as is the case with current Q chemistries, necessitates a salt removal step (e.g., by way of tangential flow filtration or dilution) prior to chromatographic separation. Said salt removal step is required due to preceding purification steps involving AAV affinity chromatography step, wherein the resulting virus-containing composition has a high salt concentration. Thus, the present chemistries avoid the cumbersome process of having to lower the salt concentration prior to separation. For direct comparison, loading of virus samples onto the chemistries of the present disclosure may be at a conductivity of 10-15 mS / cm compared to only 5 mS / cm for current Q chemistries.

[0028] Separation of full AAVs from empty AAVs occurs over a greater window of conductivities with the chemistries of the present disclosure compared to current Q chemistries. In other words, the conductivity range at which empty virus is eluted and the conductivity range at which full virus is eluted has much less overlap when chemistries of the present disclosure are used. Thus, the present filter elements allow for clear separation without having to maintain rigid control over buffer compositions. For direct comparison, the conductivity window for bound full AAV vs bound empty AAV with the chemistries of the present disclosure is ~2-4 mS / cm compared to ~l-2 mS / cm for current Q chemistries.

[0029] The filter elements of the present disclosure, having novel ligating polymeric filtering media, provide a promising avenue toward the realization of many therapeutics.

[0030] Definitions

[0031] For the purposes of promoting an understanding of the principles of the invention, reference will now be made to certain embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of thePCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 invention is thereby intended, and alterations and modifications in the illustrated invention, and further applications of the principles of the invention as illustrated therein are herein contemplated as would normally occur to one skilled in the art to which the invention relates.

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0033] For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated embodiments unless a contrary meaning is clearly intended (for example in the document where the term is originally used).

[0034] As used herein, “about” means ± 10 percent of a given value. For example, about 10 means 9 to 11.

[0035] As used herein, “average” refers to the mean.

[0036] As used herein, “acrylate” refers to a compound having at least one moiety represented by: -O(CO)-C(Rb)=C(Rc)2. The term “methacrylate” means Rbis -CH3. The term “(meth)acrylate” means Rbcan be -H or -CH3. The term alkyl (alk)acrylate means Ra-O(CO)-C(Rb)=C(Rc)2 wherein Raand Rbare each alkyl-based, e.g., alkyl, cycloalkyl, alkcycloalkyl, or the like.

[0037] As used herein, “alkaryl” means a bivalent alkylene group terminated with an aryl group. An alkaryl may be represented as follows: -alkylene-aryl. For example, a C7 alkaryl group may be benzyl, i.e., -CFfcPh.

[0038] As used herein, “alkaralkyl” means a bivalent alkylene group bonded to a bivalent arylene group, which in turn is bonded to a monovalent alkyl group. An alkaralkyl is represented as follows: -alkylene-arylene-alkyl.

[0039] As used herein, “alkcycloalkyl” means a bivalent alkylene group bonded to a cycloalkane. “Alkcycloalkylene” means a bivalent alkylene group bonded to a bivalent cycloalkyl group. “Alkcycloalkenylene” means a bivalent alkylene group bonded to a bivalent cycloalkene group.

[0040] As used herein, “alkenyl” means a monovalent unsaturated hydrocarbon chain having one or more alkene (i.e., -C(Ra)=C(Ra)2), wherein Rais -H, alkyl, alkenyl, or a substituent asPCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 indicated). The unsaturated hydrocarbon chain may be straight or branched as indicated. As used herein, “alkenylene” means a bivalent (i.e., -C(Ra)=C(Ra)-), unsaturated hydrocarbon chain having one or more alkene, straight or branched as indicated. As used herein, “olefin” is synonymous with alkene.

[0041] As used herein, “alkoxy” means a monovalent saturated hydrocarbon chain having one or more oxygen atoms intercepting the hydrocarbon chain. For example, a C3 alkoxy includes -CH2CH2-O-CH3, or the like; a C4 alkoxy includes -CH2CH2-O-CH2CH3, or the like; a C5 alkoxy includes -CH2CH2-O-CH2CH2CH3, -CH2CH2-O-CH2CH2-O-CH3, or the like. The term “alkoxy” does not encompass groups terminating with -OH - such groups are referred to herein as “alkoxylate,” e.g., -CH2CH2-O-CH2CH2-OH. The term “alkoxylene” or “alkoxyalkylene” means a bivalent alkoxy group, e.g., -CH2CH2-O-CH2-, -CH2CH2-O- CH2CH2-O-CH2CH2-, or the like.

[0042] As used herein, “alkyl” means a monovalent saturated hydrocarbon chain, straight or branched as indicated. For example, straight C1-6 alkyl includes Ci alkyl (i.e., methyl), a C2 alkyl (i.e., ethyl), C3 alkyl (i.e., propyl -(CH2)2CH3), C4 alkyl (e.g., butyl -(CTh^CHs), C5 alkyl (i.e., pentyl -(CH2)4CH3), or Ce (i.e., hexyl -(CTh^CHs). For example, a branched C3-6 alkyl includes C3 alkyl (i.e., isopropyl -CH(CH3)2), C4 alkyl (e.g., ec-butyl - CH(CH?)CH2CH3), C5 alkyl (e.g., neopentyl -CH2C(CH3)3). An “alkylene” means a bivalent saturated hydrocarbon chain, straight or branched as indicated. For example, a C2 alkylene is ethylene, i.e., -CH2CH2-; a C3 alkylene is propylene, i.e., -CH2CH2CH2- or isopropylene, i.e., -CH(CH3)CH2-.

[0043] As used herein, “aryl” describes an aromatic group that is free of heteroatoms (e.g., N, O, S) within the ring. An aromatic group is cyclic, planar, fully conjugated, and follows Hiickle’s Rule (i.e., having 4n + 2 ^-electrons, wherein n is an integer). For example, phenyl is a Ce aryl, naphthyl and azulenyl are C10 aryls, and anthracenyl and phenanthrenyl are C14 aryls. As used herein, “arylene” describes a bivalent aryl group. For example, a Ce arylene is phenylene, i.e., -Ph-, the connectivity may be ortho, meta, or para, unless otherwise as indicated. As used herein, “heteroaryl” is an aryl group having at least one heteroatom (e.g., N, O, S) within the ring.

[0044] As used herein, the expression “Cx-Cy” or “Cx” or “>CX”, wherein X, Y are integers, denotes the total number of carbon atoms or range of carbon atoms within a particular group.PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0

[0045] As used herein, “cycloalkyl” describes a monovalent saturated cycloaliphatic group. For example, a C5-6 cycloalkyl group includes C5 cycloalkyl (i.e., cyclopentyl -C5H9) and Ce cycloalkyl (i.e., cyclohexyl -CeHn). A “cycloalkylene” is a bivalent saturated cycloaliphatic group. For example, a C5-6 cycloalkylene group includes C5 cycloalkylene (i.e., cyclopentylene -CsHs-) and Ce cycloalkylene (i.e., cyclohexylene -CeHio-), including all modes of connectivity (e.g., 1,2-, 1,3-, or the like) unless otherwise indicated. As used herein, an “alkcycloalkyl” describes a bivalent alkylene group terminated with a cycloalkyl group.

[0046] As used herein, “cross-linked” means at least two compounds (e.g., polymers) are joined together by a connecting bond, a connecting atom, or connecting group of atoms.

[0047] As used herein, “exclude” means that the referenced substance is not present, i.e., 0 wt.%.

[0048] As used herein, “derived” or “derived from” is used to describe a reaction product in terms of its reactants. For example, a polymer derived from a stated monomer means that said polymer is a reaction product of at least the stated monomer that has undergone a polymerization reaction.

[0049] As used herein, “di(alk)acrylate monomer” means a compound having two (alk)acrylate units. Likewise, “tri(alk)acrylate monomer” means a compound having three (alk)acrylate units.

[0050] As used herein “ligand density” is a characterization of a filter element and refers to the millimoles of monomeric units per gram grafted to a substrate. The millimoles are calculated by dividing the mass gain by the molecular weight of the monomer and multiplying by 1000. The resulting value is then normalized by dividing by the original mass of the substrate. Ligand density is expressed as millimoles of monomeric units grafted per gram of substrate.

[0051] As used herein, “monomer” or “co-monomer” means a compound having at least one functional group (e.g., alkenes, alkynes, acryloyl, and the like) known to participate in radical polymerization reactions.

[0052] As used herein, “A-vinyl lactam monomer” refers to a lactam moiety having a vinyl group directly attached to a nitrogen (i.e., -N-CH=CH2) within a lactam ring system. A lactam is a cycloaliphatic system having an amide (i.e., -NR-C(O)-) within the ring atoms.

[0053] As used herein, the phrase “one or more of’ such as used in the phrase “one or more of A and B” or “one or more of at least one A and at least one B” means a composition may include at least one A, more than one A, at least one B, more than one B, at least one A and atPCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 least one B, more than one A and more than one B. In other words, the phrase does not mean the composition must have at least one of each of A and B.

[0054] As used herein, “octanol-water partition coefficient” (Log P) is a partition coefficient for the two-phase system of octanol and water. It serves as a measure of the relationship between lipophilicity and hydrophilicity of a substance. Lipophilic substances have greater solubility in the octanol portion of the two-phase system and are represented by greater Log P values. Substances that are more hydrophilic have smaller Log P values and negative Log P values. Octanol-water partition coefficients may be measured or may be estimated, for example, via quantitative structure-activity relationships (QSAR) or linear free energy relationships (LFER). Log P values for known monomers can be found in the literature.

[0055] As used herein, the phrase, “optionally substituted” when characterizing a chemical group, means that the chemical group may or may not be substituted with the subsequent group(s), i.e., a hydrogen is replaced with the listed group. It should be noted that the chemical groups identified herein are unsubstituted unless stated otherwise.

[0056] As used herein, “organic” defines a compound having carbon, whereas “inorganic” defines a compound without carbon.

[0057] As used herein, “polymerizable” describes a component or a collection of components (e.g., monomers) that can undergo polymerization reactions (e.g., radical polymerization reactions) to form a polymer. Thus, a “polymerizable composition” means a composition having at least one component that can undergo a polymerization reaction to form a polymer. Polymerizable compositions may include polymerizable components and non-polymerizable components.

[0058] Regarding ratios: the order of the ratio correlates to the order in which the components appear in the preceding text. For example, component A and component B are present in a weight ratio of about A:B to about A:B, never B: A.

[0059] As used herein, “soluble” refers to at least 1 g of a component A completely dissolved (no visible cloudiness, precipitate, or phase separation) in 30 mL or less component B at a temperature range of about 20 °C to about 23 °C and at atmospheric pressure (i.e., 760 mm / Hg), unless explicitly stated otherwise. It is to be understood that the conditions to determine solubility only include the component A and the component B, i.e., no added salts, or the like.

[0060] As used herein, “straight or branched,” when referring to an alkyl or alkylene group, or any such portion within a further functionalized group, means that the alkyl or alkylene may be linear or non-linear unless otherwise stated. For example, a “straight” C4 alkyl group wouldPCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 be / / -butyl (i.e., -CH2CH2CH2CH3), and a “branched” C4 alkyl group could be isobutyl, secbutyl, or tert-butyl.

[0061] As used herein, “ligating polymer” refers to a polymer derived from a ligating monomer.

[0062] As used herein, “ligating monomer” refers to a monomer comprising at least one functional group that can interact with (“ligate”) a target biomolecule (e.g., an AAV capsid).

[0063] As used herein, “full AAVs” refers to AAV capsids encapsulating a complete recombinant genome at or near the designed length, capable of transduction.

[0064] As used herein, “empty AAVs” refers to AAV capsids without encapsulated nucleic acid; structurally intact but non-functional for gene delivery.

[0065] As used herein, “partial AAVs” refers to AAV capsids containing incomplete or truncated genomes, shorter than the intended sequence; distinguished from empty AAVs by the presence of nucleic acid but insufficient for full activity.

[0066] As used herein, “extra-full AAVs” refers to AAV capsids with more than one genome copy or nucleic acid exceeding the designed length (concatemeric or oversized DNA); structurally distinct and potentially altered in density, stability, or transduction.

[0067] In practice the amount of full, empty, partial, and extra-full AAV in a sample can be estimated or quantified by a variety methods, such as, analytical ultracentrifugation (AUC), which separates AAV particles by sedimentation coefficient, allowing resolution of empty, partial, full, and extra-full capsids based on density, qPCR combined with ELISA to obtain a bulk genome-to-capsid ratio and mass spectrometry, including charge detection and mass photometry, which directly measures particle mass and thereby distinguishing empty, partial, full and extra-full AAV species.

[0068] Ligands of Ligating Polymers for Filter Elements

[0069] Ligating monomers of Formula I may be at least part of the chemistries attributed to the successful separation of full AVV and empty AAV described herein.

[0070] In various embodiments, a ligating monomer of Formula I is described: CH2=C(R1)-C(O)-W-R2-N+(R3)2-R4-Ar Z (I), wherein:R1is -H or a straight or branched C1-4 alkyl,W is -O- or -N(R5)-,R2is a straight or branched C2-C6 alkylene, a straight or branched C4-C12 alkoxyalkylene, or - R6-X-C(O)-Y-R7-,PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 each R3is independently a straight or branched Ci-Ce alkyl,R4is a straight or branched Ci-Ce alkylene optionally substituted with oxo or oxa, or a straight or branched Ci-Ce alkylene,Ar is aryl,Z is a counterion,R5is -H or a straight or branched C1-4 alkyl,R6is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene, R7is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene, X and Y are independently selected from -O-, or -N(R5)-, provided that at least one of X and Y is -O-, or -N(R5)-.

[0071] In some embodiments, W may be -O-.

[0072] In some embodiments, W is -N(R5)-.

[0073] In some embodiments, Ar may be a Ce, C9, or C10 aryl.

[0074] In some embodiments, Ar may be phenyl.

[0075] In some embodiments, Ar may be indenyl.

[0076] In some embodiments, Ar may be naphthyl.

[0077] In some embodiments, Z' may be a pharmaceutically acceptable counterion. As used herein, “pharmaceutically acceptable” means suitable for use in contact with tissues of humans and animals without undue toxicity, irritation, allergic response, or the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use within the scope of sound medical judgement. Pharmaceutically acceptable counterions are known in the art.

[0078] In some embodiments, Z may be selected from chloride, bromide, tosylate, or tritiate.

[0079] In some embodiments, Z' may be selected from chloride, bromide, and tosylate.

[0080] In some embodiments, Z' may be selected from chloride and bromide.

[0081] In some embodiments, Z may be bromide.

[0082] In some embodiments, Z' is not a sulfate (e.g., methyl sulfate, butyl sulfate, or the like).

[0083] In some embodiments, R1may be -H or -CH3.

[0084] In some embodiments, R2may be a straight or branched C2-C6 alkylene. In some embodiments, R2may be a straight or branched C2-C4 alkylene. In some embodiments, R2may be -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, or -CH2CH2CH2CH2-.

[0085] In some embodiments, R2may be -R6-X-C(O)-Y-R7-. In some embodiments, X and Y may each be -N(R5)-. In some embodiments, X may be a bond and Y may be -O-. In some embodiments, X may be a bond and Y may be -N(R5)-. In some embodiments, X may be -O-PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 and Y may be a bond. In some embodiments, X may be -N(R5)- and Y may be a bond. In some embodiments, X may be -N(R5)- and Y may be -O-. In some embodiments, X may be - O- and Y may be -N(R5)-. In any of the said embodiments, R6and R7may each independently be a Ci-Ce alkylene, e.g., a Ci, C2, C3, C4, C5, Ce alkylene, or a range therebetween, e.g., C2-C4 alkylene.

[0086] In some embodiments, each R3may independently be a C1-C3 alkyl. In some embodiments, each R3may be -CH3.

[0087] In some embodiments, R4may be a straight or branched Ci-Ce alkylene. In some embodiments, R4may be a straight or branched C1-C4 alkylene. In some embodiments, R4may be -CH2-, -CH2CH2-, or -CH2CH2CH2-.

[0088] In some embodiments, R4may be a straight or branched C2-C6 alkylene substituted with oxo or oxa. In some embodiments, R4may be -(CH2)n-C(O)-(CH2)m-, wherein n and m may be an integer selected from 0-5, so long as n is not 0 and n+m does not exceed 5. In some embodiments, R4may be -(CH2)n-C(O)-, wherein n may be an integer selected from 1-3. In some embodiments, R4may be -(CH2)n-O-(CH2)m-, wherein n and m are defined above.

[0089] In some embodiments, R5may be -H or -CH3.

[0090] In some embodiments, the ligating monomer may be represented by the formula la:(la).

[0091] In some embodiments, the ligating monomer may be represented by formula la and Z may be chloride.

[0092] In some embodiments, the ligating monomer may be represented by the formula lb:

[0093] In some embodiments, the ligating monomer may be represented by formula lb and Z may be bromide.

[0094] Polymerizable Compositions with Ligating Monomers

[0095] In various embodiments, a polymerizable composition is described. The polymerizable composition may include one or more ligating monomer of Formula I above. In somePCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 embodiments, the polymerizable composition may include a ligating monomer of Formula la, a ligating monomer of Formula lb, or a combination thereof.

[0096] In some embodiments, the one or more ligating monomer of Formula I, Formula la, or Formula lb may be present within the polymerizable composition in an (total) amount of about 1 mol% to about 100 mol% with respect to the total molar amount of polymerizable components within the polymerizable composition. For example, the ligating monomer many be present in an amount, in mol%, of about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100, or a value within a range between any of the preceding values, e.g., between about 20 and about 80, between about 45 and about 95, or the like.

[0097] In some embodiments, the one or more ligating monomer of Formula I, Formula la, or Formula lb may be present within the polymerizable composition in an (total) amount of about 1 wt% to about 100 wt% with respect to the weight of polymerizable components within the polymerizable composition (i.e., not including non-polymerizable components such as solvent). For example, the ligating monomer many be present in an amount, in wt%, of about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100, or a value within a range between any of the preceding values, e.g., between about 20 and about 80, between about 45 and about 95, or the like.

[0098] In many embodiments, any of the polymerizable compositions may further include a photoinitiator, a catalyst, a chain transfer agent, a crosslinking agent, co-monomer(s), or a combination thereof. In some embodiments, any of the polymerizable compositions may also further include a solvent, e.g., de-ionized water.

[0099] In some embodiments, any of the polymerizable compositions may further include one or more co-monomer. The one or more co-monomer may be characterized by an octanol-water partition coefficient (i.e., Log P) of about -4 to about 4. For example, the one or more comonomer may be characterized by an octanol-water partition coefficient of about -4, -3.5, -3, -2.5, -2, -1.5, -1, -.5, 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4, or a value within a range between any of the preceding values, e.g., between about -2 and about 2, or the like. In some embodiments, the polymerizable composition may exclude co-monomers characterized by an octanol-water partition coefficient greater than 4. Modulating the hydrophilicity of the resulting ligating polymer with selected co-monomers may be important for adequate separation of AAVs. Hydrophilicity is generally imparted by the presence of hydrophilic groups such ether groups (e.g., polyalkylene glycol groups), ester groups, amide groups, hydroxyl groups, and aminoPCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 groups. Thus, in some embodiments, any co-monomer may include one or more of the described hydrophilic groups.

[0100] In some embodiments, any of the polymerizable compositions may further include one or more co-monomer characterized by a water solubility of at least 5 g per L at 25 °C. For example, one or more co-monomer may be characterized by a water solubility (g per L at 25 °C) of at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or at least about 100, or a value within a range between any of the preceding values, e.g., between about 5 and about 50, or the like. In some embodiments, the polymerizable compositions may exclude co-monomers characterized by a water solubility of less than 100 g per L at 25 °C. For example, the polymerizable compositions may exclude co-monomers characterized by a water solubility (g per L at 25 °C) of less than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or less than 5. In some embodiments, the polymerizable compositions may exclude co-monomers characterized by a water solubility of less than 5 g per L at 25 °C.

[0101] In some embodiments, any polymerizable composition may further include one more mono (meth)acrylate co-monomer, one or more di (meth)acrylate co-monomer, one or more acrylamide (e.g., acrylamide, alkyl acrylamide, dialkylacrylamide), a vinyl monomer having one or more hydrogen-bond accepting group (e.g., tri-substituted nitrogen) (e.g., an A-vinyl amide monomer), or a combination thereof. In some embodiments, the mono (meth)acrylate, di(meth)acrylate, or acrylamide may further include one or more hydrogen-bond donating group (e.g., -OH, -NHCH3, or -NH2), one or more hydrogen-bond accepting group (e.g., -O-, - C(O)-,-N(CH3)2), or a combination thereof. Example vinyl monomers having one or more hydrogenbond accepting group include A-vinyl pyrrolidone, -vinyl form am ide, A-vinyl acetamide, A- vinyl caprol actam .

[0102] In some embodiments, any polymerizable composition may further include one or more co-monomer having one or more hydroxyl group. In some embodiments, any polymerizable composition may further include one or more co-monomer having a glycerol group with at least one free hydroxyl group. In some embodiments, any polymerizable composition may further include one or more co-monomer having a glycol group with at least one free hydroxyl group. Example co-monomers having one or more hydroxyl group include, for example, hydroxyethyl (meth)acrylate, glycerol (meth)acrylate, glyceryl di(meth)acrylate, and hydroxypropyl (meth)acrylate.PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0

[0103] In some embodiments, the polymerizable composition may further include one or more co-monomer selected from hydroxyethyl methacrylate (“HEMA”), A -vinyl pyrrolidone (“NVP”), glycidyl methacrylate (“GMA”), glyceryl dimethacrylate (“GDMA”), di(ethyleneglycol) methyl ether methacrylate (“DEGMEMA”), acrylamide, and dimethylacrylamide. In some embodiments, the polymerizable composition may exclude comonomers other than HEMA, NVP, GMA, GDMA, DEGMEMA, acrylamide and dimethylacrylamide.

[0104] In embodiments further including a co-monomer described herein, the one or more comonomer may be present within the polymerizable composition in a (total) amount of about 5 mol% to about 95 mol% based on the total molar amount of polymerizable components within the polymerizable composition. For example, the one or more co-monomer may be present in an amount, in mol%, of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95, or a value within a range between any of the preceding values, e.g., between about 25 mol% and about 75 mol%, or the like. In other embodiments, the polymerizable composition may exclude co-monomers, i.e., 0 mol%.

[0105] In embodiments further including a co-monomer described herein, the one or more comonomer may be present within the polymerizable composition in a (total) amount of about 5 wt% to about 95 wt% based on the weight of the polymerizable components within the polymerizable composition. For example, the one or more co-monomer may be present in an amount, in wt%, of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95, or a value within a range between any of the preceding values, e.g., between about 25 wt% and about 75 wt%, or the like. In other embodiments, the polymerizable composition may exclude co-monomers, i.e., 0 wt%.

[0106] In some embodiments, the one or more ligating monomer of Formula I (e.g., Formula la or Formula lb) and the one or more co-monomer described herein may be present within the polymerizable composition at a molar ratio of about 1 :7 to about 1 :0. For example, the one or more ligating monomer and the one or more co-monomer may be present within the polymerizable composition in a molar ratio of about 1 :7, 1 :6.8, 1 :6.5, 1 :6.2, 1 :6, 1 :5.8, 1 :5.5, 1 :5.2, 1 :5, 1 :4.8, 1 :4.5, 1 :4.2, 1 :4, 1 :3.8, 1 :3.5, 1 :3.2, 1 :3, 1 :2.8, 1 :2.5, 1 :2.2, 1 :2, 1 : 1.8, 1 : 1.5, 1 : 1.2, 1 : 1, 1 :0.8, 1 :0.5, 1 :0.2, or 1 :0, or a value within a range between any of the preceding values, e.g., between 1 :5 and 1 :2, or the like.

[0107] In some embodiments, the polymerizable compositions may exclude co-monomers having a guanidine group.PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0

[0108] In some embodiments, the polymerizable compositions may exclude co-monomers having a quaternary ammonium trialkyl group. The quaternary ammonium trialkyl group may be represented by formula: -N+(Rq)3, wherein each Rqmay independently be a C1-C20 alkyl group.

[0109] The polymerizable compositions above may be used to prepare the ligating polymers described below. Consequently, the amounts and ratios described above may also pertain to the ligating polymers below. For example, a ligating polymer prepared from a polymerizable composition may include about 1 mol% to about 100 mol% (or any range or value selected therebetween) of one or more ligating monomer of Formula I (e.g., Formula la, Formula lb), about 5 mol% to about 95 mol% (or any range or value selected therebetween) of one or more co-monomer described herein, the one or more ligating monomer of Formula I and the one or more co-monomer present in a molar ratio of about 1 :7 to about 1 :0 (or any range or value selected therebetween), or a combination thereof.

[0110] Ligating Polymers

[0111] The ligating polymers of the present disclosure may be prepared from any of the polymerizable compositions described herein.

[0112] In many embodiments, the one or more ligating monomer of Formula I (e.g., Formula la, Formula lb) may be present within the ligating polymer in an amount of at least 5 mol% with respect to the molar amount of the ligating polymer. For example, the ligating monomer may be present within the ligating polymer in an amount (mol%) of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or a value within a range between any of the preceding values, e.g., between about 25 mol% and about 75 mol%, or the like.

[0113] In some embodiments, the ligating polymer may further be derived from one or more co-monomer, as described above within the polymerizable compositions. In such embodiments, the one or more co-monomer may be present within the ligating polymer in an amount of up to about 95 mol% with respect to the weight the ligating polymer. For example, one or more co-monomer may be present within the ligating polymer in an amount (mol%) of about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95, or a value within a range between any of the preceding values, e.g., between about 10 mol% and about 50 mol%, or the like.

[0114] In some embodiments, the ligating polymer may be derived from one or more ligating monomer of Formula I (e.g., Formula la, lb, or a combination thereof), and HEMA. The ligating monomer(s) of Formula I and HEMA may be present within the ligating polymer at aPCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 mol ratio of about 20: 1 to 1 :4. For example, the ligating monomer(s) of Formula I and HEMA maybe present within the ligating polymer at a mol ratio of about 20:1, 18:1, 15:1, 12:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3.8:1, 3.5:1, 3.2:1, 3:1, 2.8:1, 2.5:1, 2.2:1, 2:1, 1.8:1, 1.5:1, 1.2:1, 1:1, 1:1.2, 1:1.5, 1:1.8, 1:2, 1:2.2, 1:2.5, 1:2.8, 1:3, 1:3.2, 1:3.5, 1:3.8, or 1:4, or a value within a range between any of the preceding values, e.g., between about 1:2 to about 1:3, or the like. In some embodiments, the ligating polymer may be derived solely from the ligating monomer(s) of Formula I and HEMA. In other embodiments, the ligating polymer may be derived from the ligating monomer(s) of Formula I, HEMA, and GDMA and / or GMA. In such embodiments, GDMA, GMA, or the combination thereof may be present within the ligating polymer in a total amount of up to about 20 wt%, e.g., a weight percentage of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or a value within a range between any of the preceding values.

[0115] In some embodiments, the ligating polymer may be derived from one or more ligating monomer of Formula I (e.g., Formula la, lb, or a combination thereof) and NVP. The ligating monomer(s) of Formula I and NVP may be present within the ligating polymer at a mol ratio of about 20:1 to 1:4. For example, the ligating monomer(s) of Formula I and NVP may be present within the ligating polymer at a mol ratio of about 20:1, 18:1, 15:1, 12:1, 10:1,9:1,8:1, 7:1, 6:1, 5:1, 4:1, 3.8:1, 3.5:1, 3.2:1, 3:1, 2.8:1, 2.5:1, 2.2:1, 2:1, 1.8:1, 1.5:1, 1.2:1, 1:1, 1:1.2, 1:1.5, 1:1.8, 1:2, 1:2.2, 1:2.5, 1:2.8, 1:3, 1:3.2, 1:3.5, 1:3.8, or 1:4, or a value within a range between any of the preceding values, e.g., between about 1 :2 to about 1 :3, or the like. In some embodiments, the ligating polymer may be derived solely from the ligating monomer(s) of Formula I and NVP. In other embodiments, the ligating polymer may be derived from the ligating monomer(s) of Formula I, NVP, and GDMA and / or GMA. In such embodiments, GDMA, GMA, or the combination thereof may be present within the ligating polymer in a total amount of up to about 20 mol%, e.g., a molar percentage of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or a value within a range between any of the preceding values.

[0116] In some embodiments, the ligating polymer may be derived from one or more ligating monomer of Formula I (e.g., Formula la, lb, or a combination thereof) and DEGMEMA. The ligating monomer(s) of Formula lb and DEGMEMA may be present within the ligating polymer at a mol ratio of about 20: 1 to 1 :4. For example, the ligating monomer(s) of Formula I and DEGMEMA may be present within the ligating polymer at a mol ratio of about 20:1, 18:1, 15:1, 12:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3.8:1, 3.5:1, 3.2:1, 3:1, 2.8:1, 2.5:1, 2.2:1, 2:1, 1.8:1, 1.5:1, 1.2:1, 1:1, 1:1.2, 1:1.5, 1:1.8, 1:2, 1:2.2, 1:2.5, 1:2.8, 1:3, 1:3.2, 1:3.5, 1:3.8,PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 or 1 :4, or a value within a range between any of the preceding values, e.g., between about 1 :2 to about 1 :3, or the like. In some embodiments, the ligating polymer may be derived solely from the ligating monomer(s) of Formula I and DEGMEMA. In other embodiments, the ligating polymer may be derived from the ligating monomer(s) of Formula I, DEGMEMA, and GDMA and / or GMA. In such embodiments, GDMA, GMA, or the combination thereof may be present within the ligating polymer in a total amount of up to about 20 mol%, e.g., a molar percentage of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or a value within a range between any of the preceding values.

[0117] In some embodiments, the ligating monomer of Formula I (e.g., Formula la, Formula lb) may account for about 10 mol% to about 95 mol% (or a value or range therebetween) of the ligating polymer; and hydroxyethyl acrylate, A-vinyl pyrrolidone, di(ethylene glycol) methyl ether methacrylate, glycerol (meth)acrylate, glycerol di(meth)acrylate, or a combination thereof may account, in total, for about 5 mol% to 90 mol% of the ligating polymer.

[0118] Filter Elements with Ligating Polymers

[0119] In various embodiments, a filter element is described. The filter element may include one more porous substrate and a filter media. The filter media may include any ligating polymer of the present disclosure. The ligating polymer may be grafted to at least one of the one or more porous substrate.

[0120] In many embodiments, the filter element may be characterized by binding to full adeno- associated viruses at conductivities of at least about 10 mS / cm. In some embodiments, the filter element may be characterized by binding to full adeno-associated viruses of up to about 21 mS / cm. For example, the filter element may be characterized by a binding to full adeno- associated viruses at conductivities (mS / cm) of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or a value within a range between any of the preceding values, e.g., between 10 mS / cm and 15 mS / cm, or the like. In other words, at conductivities of 10 mS / cm to 21 mS / cm, full AAV (e.g., full AAV5) may be bound to the filter element allowing for flow-through of other substances (e.g., empty AAV, other viruses, bacteria, etc.) that are not bound at the stated conductivity values. For example, empty AAVs typically bind at a conductivity of 2-4 mS / cm less than the conductivity of full AAVs. For comparison, filter elements based on current Q chemistry (i.e., ligating monomers having a trialkyl quaternary ammonium group) typically bind to full AVVs up to only 10 mS / cm, whereas binding for empty AAVs is only 1-2 mS / cm less than that.PCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT0

[0121] Further details regarding features of the filter element are provided below.

[0122] Porous Substrate

[0123] In some embodiments, one or more of, or each of, the porous substrate(s) may be characterized by an average pore size of about 0.1 pm to about 10 pm. For example, a porous substrate may be characterized by an average pore size, in pm, of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.8, 2.0, 2.2, 2.5, 2.8, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10, or a value within a range between any of the preceding values, e.g., between about 0.5 pm and about 3 pm, between about 0.8 pm and about 2 pm, or the like. Pore size may minimize size exclusion separations and diffusion constraints and may also maximize surface area and separation of full AAVs from empty AAVs and other biomaterials. The pressure within the filter elements and acceptable flow rates may further be determined by selected pore size. Pore size for membranes is measured by BET nitrogen adsorption or can be estimated by bubble point measurement.

[0124] In some embodiments, one or more porous substrate may be in the form of a membrane. In some embodiments, each of the one or more porous substrates may be a membrane material.

[0125] In some embodiments, one or more porous substrate may be in the form of a nonwoven. In some embodiments, each of the one or more porous substrate may be a nonwoven material. Porosity of nonwoven substrates is typically characterized by one or more of fiber diameter, basis weight, and web loft (rather than pore size). In some embodiments, a nonwoven substrate may be characterized by fibers having a fiber diameter of about 0.5 micrometers to about 15 micrometers, as calculated according to the method set forth in Davies, C.N., “The Separation of Airborne Dust and Particles,” Institution of Medical Engineers, London, Proceedings IB, 1952. For example, the nonwoven substrate(s) may be characterized by fibers of a diameter (micrometers) of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, or 15, or a value within a range between any of the preceding values, e.g., between about 1 micrometer and about 8 micrometers, or the like. In some embodiments, a nonwoven substrate may be characterized by a basis weight of about 5 g / m2to about 800 g / m2. For example, the nonwoven substrate(s) may be characterized by a basis weight (g / m2) of about 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, or 800, or a value within a range between any of the preceding values, e.g., between about 100 g / m2and about 300 g / m2, or the like.

[0126] In some embodiments, the filter element may include a combination of membrane substrate(s) and nonwoven substrate(s). Membranes can have smaller and more narrowlyPCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 distributed pore sizes than nonwovens, whereas nonwovens can have greater mechanical strength compared to membranes.

[0127] In some embodiments, the one or more porous substrate may include any thermoplastic polymeric material. Suitable polymeric materials include polyolefins, polyisoprenes, polybutadienes, fluorinated polymers, chlorinated polymers, polyamides, polyimides, poly ethers, poly ether sulfones, poly sulfones, polyvinyl acetates, polyesters (e.g., polylactic acid), copolymers of vinyl acetate (e.g., polyethylene-co-polyvinyl alcohol), polyphosphazenes, polyvinyl esters, polyvinyl ethers, polyvinyl alcohols, polycarbonates, and a combination thereof. In some embodiments, the one or more porous substrate may include, or otherwise be constructed from, nylon, polypropylene, polyethersulphone, PVDF, cellulose, or a combination thereof.

[0128] In some embodiments, the one or more porous substrate may be a nylon membrane. In some embodiments, the one or more porous substrate may be a nylon membrane characterized by an average pore size of between about 0.1 pm to 5 pm, e.g., between about 0.1 pm and 1 pm.

[0129] In some embodiments, the one or more porous substrate may be a polypropylene non woven.

[0130] In many embodiments, the filter element may include a number and size of porous substrates based on capacity (i.e., scale of separation needed). In some embodiments, the filter element may include 1 to 10 porous substrates described herein. For example, the filter element may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 porous substrates within the filter element, or a value within a range between any of the preceding values, e.g., between 1 and 4 porous substrates, or the like. In some embodiments, the number of porous substrates selected may depend upon the overall thickness of the combined porous substrates. The thickness of each porous substrate is defined as the distance between a first major surface and a second major surface, and the overall thickness defined as the distance between a first major surface and the second major surface of the outermost porous substrates. In some embodiments, the one or more porous substrates may be characterized together by an overall thickness of about 0.2 mm to about 10 mm. An overall thickness of about 0.2 mm to about 10 mm may be suitable for lab scale; however, production scale may involve a much greater in thickness. For example, the one or more porous substrates may be characterized by an overall thickness of about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or a value within a range between any of the preceding values, e.g., between 0.2mm and 0.4 mm.PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0

[0131] In some embodiments, the ligating polymer may be present on at least one of the one or more porous substrates. In many embodiments, the ligating polymer may be present on each of the one or more porous substrates.

[0132] Filter Media

[0133] In some embodiments, the filter media may be characterized by a weight gain of about 10% to about 200%. Weight gain is calculated as follows: ((Final mass of dry grafted substrate - Initial mass of the porous substrate(s)) / (Initial mass of the porous substrate(s))) x 100. For example, the filter media may be characterized by a weight gain % of about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200, or a value within a range between any of the preceding values, e.g., between about 50 and about 90, or the like.

[0134] In many embodiments, the filter media may be characterized by an amount of the one or more ligating monomer (e.g., Formulae I, la, lb) on a porous substrate(s) in terms of ligand density. In some embodiments, the filter media may be characterized by a ligand density of about 0.05 mmol to about 2.0 mmol of the one or more ligating monomer per gram of porous substrate. For example, the one or more ligating monomer may be present on a porous substrate at a ligand density, in mmol per g of porous substrate, of about0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 1.0, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, or 2.0, or a value within a range between any of the preceding values, e.g., between about 0.35 mmol to about 1.55 mmol ligating monomer per g of porous substrate, or the like. In some embodiments, each of the one or more porous substrates may include the one or more ligating monomer present thereon at any of such ligand densities. In some embodiments, one or more of the porous substrates may include a ligating polymer present thereon at a ligand density of about 0.25 mmol to about 2.0 mmol per gram of porous substrate.

[0135] In some embodiments, the filter media may exclude polymers other than the ligating polymers described herein.

[0136] In some embodiments, the filter media may exclude polymers derived from monomers having a quaternary ammonium trialkyl groups. For example, the quaternary ammonium trialkyl group may be represented by -N+(Rq)3 wherein each Rqis independently a C1-C20 alkyl group.

[0137] In some embodiments, the filter media may exclude polymers derived from monomers having a guanidine group.PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0

[0138] Separation Processes with Filter Elements having Ligating Polymers

[0139] In various embodiments, a process for separating an adeno-associated virus containing a DNA payload from an adeno-associated virus without a DNA payload is described. The process may include providing a filter element of the present disclosure and a loading solution having the adeno-associated virus containing a DNA payload and the adeno-associated virus without a DNA payload. The process may further include contacting the loading solution to the filter element and separating the adeno-associated virus containing a DNA payload from the adeno-associated virus without a DNA payload.

[0140] In many embodiments, the separating of adeno-associated virus containing a DNA payload from the adeno-associated virus without a DNA payload may occur over an eluting conductivity window of about 2 mS / cm to about 4 mS / cm on filter elements of the present disclosure. In other words, full AAV may elute at conductivities that are 2 mS / cm to about 4 mS / cm greater than the conductivities at which empty AAV elutes. In some embodiments, at least 35% of the adeno-associated virus containing the DNA payload (i.e., full AAV) from the loading solution may be recovered, e.g., 35, 40, 45, 50, 55, 60, 65, 70, 75, 78, 80, 82, 85, 88, 90, 92, 95, 98, or 100% recovery of full AAV, or a value within a range between any of the preceding values, e.g., between about 40% and 55%, or the like. In some embodiments, the adeno-associated virus containing a DNA payload may be recovered at an enrichment of at least 50%, e.g., 50, 52, 55, 58, 60, 62, 65, 68, 70, 72, 75, 78, 80, 82, 85, 88, 90, 92, 95, 98, or 100% enrichment of full AAV, or a value within a range between any of the preceding values, e.g., between about 75% and about 90%, or the like. In some embodiments, the adeno- associated virus containing the DNA payload may be recovered at an enrichment of at least 75%. In some embodiments, the adeno-associated virus containing a DNA payload may be recovered in an amount of at least 35% at an enrichment of at least 75%.

[0141] AAVs

[0142] In some embodiments, each of the adeno-associated virus containing a DNA payload and the adeno-associated virus without a DNA payload may be an adeno-associated virus of serotype 2 or serotype 5 or serotype 6.

[0143] In some embodiments, the DNA payload may be a sequence effective to treat hemophilia A, hemophilia B, muscular dystrophy, AADC deficiency, lipoprotein lipase deficiency, retinal dystrophy and any other inherited genetic disease.

[0144] In some embodiments, the DNA payload may be selected from any protein coding gene fragment or a regulatory gene fragment.PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0

[0145] Loading Solutions

[0146] In many embodiments, the loading solution may further include one or more (in)organic salt that ionizes in water. In some embodiments, the loading solution may include an inorganic salt having a monovalent cation (+1 oxidation state). In other embodiments, the loading solution may include an inorganic salt having a divalent cation (+2 oxidation state). In some embodiments, the loading solution may include a monovalent cation salt and a divalent cation salt. In some embodiments, the loading solution may include one or more salt selected from selected from sodium chloride, ammonium chloride, magnesium chloride, sodium acetate, choline chloride, tetramethyl ammonium chloride, tetramethyl ammonium acetate, tetraethyl ammonium chloride, tetraethyl ammonium acetate, and a combination thereof. In some embodiments, the loading solution may exclude sulfate salts, phosphate salts, or a combination thereof.

[0147] In some embodiments, the loading solution may be characterized by a salt concentration (e.g., total solubilized inorganic salts) of about 50 mM to about 150 mM. For example, the loading solution may include solubilized salts at a concentration (mM) of about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, or a value within a range between any of the preceding values, e.g., between about 50 mM to about 100 mM, or the like.

[0148] In many embodiments, the loading solution may be characterized by a conductivity value of about 5 mS / cm to about 15 mS / cm. For example, the adeno-associated virus containing a DNA payload and the adeno-associated virus without a DNA payload may be loaded onto the filter element in a loading solution characterized by a conductivity value of about 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15, or a value within a range between any of the preceding values, e.g., between about 11 mS / cm and 15 mS / cm, or the like.

[0149] In many embodiments, the loading solution may further include one or more buffering agent. Suitable buffering agents may include tris-acetate, bis-tris propane, or a combination thereof. Buffering agents may contribute to the overall conductivity of the loading solution. In some embodiments, the buffering agent may be present in a concentration of about 5 mM to about 100 mM . For example, the buffering agent (e.g., tris-acetate) may be present in a concentration (mM) of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or a value within a range between any of the preceding values, e.g., between about 30 and about 60, or the like.PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0

[0150] In some embodiments, the loading solution may be characterized by a pH of about 8.5 to about 10. For example, the loading solution may be characterized by a pH of about 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, or a value within a range between any of the preceding values, e.g., between about 8.7 and about 9.2, or the like. In many embodiments, the loading solution may be characterized by a pH of about 9.

[0151] In some embodiments, the adeno-associated virus containing a DNA payload and the adeno-associated virus without a DNA payload may be present in the loading solution at a combined concentration of about 1011to about 4*1014AAV capsids in total. For example, the loading solution may have a total AAV concentration of about 1011, 1012, 1013, 1014, 2*1014, 3*1014, 4*1014AAV capsids, or a value within a range between any of the preceding values, e.g., between about 1014AAV capsids and about 2*1014AAV capsids, or the like.

[0152] In some embodiments, an amount of loading solution to contact the filter element may include a total amount of AAV of no greater than 9*1014AAV per ml of filter media.

[0153] In many embodiments, the loading solution has not undergone a salt removal step prior to the contacting of the loading solution to the filter element. In other words, any prepurification steps involving salt precipitations may be used directly in the processes described herein.

[0154] Conditions for Separation

[0155] In many embodiments, the separating of the adeno-associated virus containing a DNA payload (full AAV) from the adeno-associated virus without a DNA payload (empty AAV) may be under conditions involving:1. Gradient Separation: Loading a sample onto a filter element in a loading solution (e.g., a loading solution described herein) characterized by a conductivity such that both the empty AAV and the full AAV bind to the filter media, subsequently contacting the filter element with one or more eluting solution (e.g., an eluting solution described below) characterized by a conductivity such that the empty AAV elutes from the filter media while the full AAV remains bound to the filter media, and then further contacting the filter element with one or more eluting solution (e.g., an eluting solution described herein) characterized by a conductivity such that the full AAV elutes from the filter media, or

[0156] 2. Non-gradient Separation: Loading a sample onto a filter element in a loading solution (e.g., a loading solution described herein) characterized by a conductivity such that full AAV binds to the filter media whereas empty AAV flows through the filter media at saidPCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 conductivity. Subsequently, contacted an eluting solution to the filter element such that the full AAV elutes from the filter media.

[0157] Gradient Separation. In many embodiments, conditions for a gradient separation process may involve contacting the filter element with one or more eluting solution and collecting solution fractions. The solution fractions may be monitored for the presence of empty AAV, full AAV, and other contaminants. In some embodiments, a plurality of eluting solutions may be passed through the filter element in succession wherein each successive eluting solution may be characterized by an increased conductivity relative to preceding eluting solutions. In other embodiments, a continuous flow eluting solution may be passed through the filter element wherein the eluting solution increases in conductivity over time.

[0158] An elution profile may be designed to selectively release certain component s) from the filter media. In some embodiments, the eluting solution(s) may increase in conductivity by about 1 mS / cm to about 5 mS / cm per unit volume passing through the filter element. For example, the eluting solution(s) may increase in conductivity (mS / cm) by about 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5, 4.8, or 5 for each solution, or a value within a range between any of the preceding values, e.g., between about 1 and about 2.5, or the like. In some embodiments, this increase in conductivity may occur per 0.1 column volume units to about 20 column volume units. For example, the eluting solution may increase in conductivity of about 1 mS / cm to about 5 mS / cm (or any range therein between), per column volume unit, every two column volume units, every three column volume units, or the like.

[0159] In some embodiments, the eluting solutions may pass through the filter element at a flow rate of about 0.5 CV / min to about 20 CV / min. As used herein, “CV” refers to the column volume used with the filter element. For example, the eluting solutions may pass through filter element at a flow rate, in CV / min, of about 0.5, 0.8, 1.0, 1.2, 1.5, 1.8, 2.0, 2.2, 2.5, 2.8, 3.0, 3.2, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or a value within a range between any of the preceding values, e.g., between about 3 CV / min and about 12 CV / min, or the like. The flow rates may be combined with any elution profile, such as those described above.

[0160] Non-gradient separation. In many embodiments, the conditions for a non-gradient separation process may involve allowing the loading solution (containing the full AAV and the empty AAV) to pass through the filter element and collecting the empty AAV while the full AAV remains bound to the filter media.PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0

[0161] In other embodiments, the conditions for a non-gradient separation process may include centrifuging the filter element with the loading solution and collecting a solution enriched in the adeno-associated virus without the DNA payload. Centrifuging the filter element may not be required or feasible on larger scale.

[0162] After collecting the adeno-associated virus without the DNA payload, the adeno- associated virus with the DNA payload may be removed from the filter element. In some embodiments, the conditions may further include contacting the filter element with an eluting solution and collecting a solution enriched in the adeno-associated virus with the DNA payload.

[0163] In some embodiments, a combination process may involve separating adeno-associated virus without a DNA payload (i.e., empty AAV) via a non-gradient separation process, followed by a gradient separation process.

[0164] Eluting solutions are described in greater detail below.

[0165] Eluting Solutions

[0166] As used herein, “eluting solution” is a solution characterized by a conductivity that is greater than the conductivity of the loading solution. In some embodiments, an eluting solution may be characterized by a conductivity value of greater than 14 mS / cm. For example, an eluting solution may be characterized by a conductivity value, in mS / cm, of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, or more, or value within a range between any of the preceding values, e.g., between about 19 mS / cm and 22 mS / cm, or the like.

[0167] In many embodiments, the eluting solution may include the same or different conductive components as the loading solutions described above. For example, the eluting solutions may include salt(s) such as sodium chloride, ammonium chloride, magnesium chloride, sodium acetate, choline chloride, tetramethyl ammonium chloride, tetramethyl ammonium acetate, tetraethyl ammonium chloride, tetraethyl ammonium acetate or a combination thereof; however, in some embodiments, the eluting solutions may exclude magnesium chloride or only include magnesium chloride in an amount of up to 10 mM. In some embodiments, the eluting solutions may exclude sulfate salts, phosphate salts, or a combination thereof.

[0168] In many embodiments, the eluting solutions may be characterized by a pH of about 8.8 to about 9.5, or any range therebetween, e.g., pH of 9.

[0169] Processes for Preparing Filter Elements

[0170] In various embodiments, a process for preparing a filter element of the present disclosure is described. The process may include providing a polymerizable compositionPCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 described herein, providing a porous substrate, and contacting the polymerizable composition to the porous substrate under conditions effective to polymerize the polymerizable composition onto the porous substrate.

[0171] In various embodiments, a process for preparing a filter element of the present disclosure is described. The process may include providing a ligating polymer described herein, providing porous substrate, and grafting the ligating polymer to the to the porous substrate.

[0172] In many embodiments, the conditions to polymerize the polymerizable composition and / or the conditions to graft the ligating polymer may include e-beam irradiation (e.g., at a dose of about 6-10 MRad), UV irradiation, or a combination thereof.

[0173] Kits

[0174] In various embodiments, a kit is described. The kit may include a filter element described herein and a set of instructions directing a user to separate full adeno-associated viruses from empty adeno-associated viruses.

[0175] In some embodiments, any of the kits may further include instructions for separating adeno-associated virus containing a DNA payload (i.e., full AAV) from adeno-associated virus within a DNA payload (i.e., empty AAV).

[0176] LIST OF EMBODIMENTS1. A compound represented by Formula I:CH2=C(R1)-C(O)-W-R2-N+(R3)2-R4-Ar Z (I) wherein:R1is -H or a straight or branched Ci-4 alkyl,W is -O- or -N(R5)-,R2is a straight or branched C2-Ce alkylene, or a straight or branched C4-Ci2alkoxyalkylene, or -R6-X-C(O)-Y-R7-, each R3is independently a straight or branched Ci-Ce alkyl,R4is a straight or branched Ci-Ce alkylene optionally substituted with oxo or oxa, or a straight or branched Ci-Ce alkylene,Ar is aryl,Z is a counterion,R5is -H or a straight or branched Ci-4 alkyl,R6is a straight or branched Ci-Ce alkylene, or a straight or branched C4-Ci2alkoxyalkylene,PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0R7is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene, andX and Y are independently selected from -O-, or -N(R5)-, provided that at least one of X and Y is -O-, or -N(R5)-, and with the proviso that when R4is -CH2- and R3is methyl, Ar is not phenyl.2. The compound of embodiment 1, wherein W is -O-.3. The compound of embodiment 1, wherein W is -N(R5)-.4. The compound of any one of the preceding embodiments, wherein each R3is methyl.5. The compound of any one of the preceding embodiments, wherein R4is a straight or branched Ci-Ce alkylene.6. The compound of any one of the preceding embodiments, wherein R4is a straight or branched C2-C6 alkylene substituted with oxo.7. The compound of any one of the preceding embodiments, wherein Ar is a Ce, C9, or C10 aryl.8. The compound of any one of the preceding embodiments, wherein Ar is phenyl.9. The compound of embodiment 1, represented by the formula:10. The compound of embodiment 1, represented by the formula:11. The compound of embodiment 1, wherein each of R3is methyl, and Ar is phenyl.12. The compound of any one of the preceding embodiments, wherein Z' is selected from chloride, bromide, tritiate, and tosylate.13. A ligating monomer represented by Formula I: CH2=C(R1)-C(O)-W-R2-N+(R3)2-R4-Ar Z (I) wherein:R1is -H or a straight or branched C1-4 alkyl,PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0W is -O- or -N(R5)-,R2is a straight or branched C2-C6 alkylene, or a straight or branched C4-C12 alkoxyalkylene, or -R6-X-C(O)-Y-R7-, each R3is independently a straight or branched Ci-Ce alkyl,R4is a straight or branched Ci-Ce alkylene optionally substituted with oxo or oxa, or a straight or branched Ci-Ce alkylene,Ar is aryl,Z is a counterion,R5is -H or a straight or branched C1-4 alkyl,R6is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene, R7is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene, andX and Y are independently selected from -O-, or -N(R5)-, provided that at least one of X and Y is -O-, or -N(R5)-, and with the proviso that when R4is -CH2- and R3is methyl, Ar is not phenyl.14. The ligating monomer of embodiment 13, wherein W is -O-.15. The ligating monomer of embodiment 13, wherein W is -N(R5)-.16. The ligating monomer of any one of the preceding embodiments, wherein each R3is methyl.17. The ligating monomer of any one of the preceding embodiments, wherein R4is a straight or branched Ci-Ce alkylene.18. The ligating monomer of any one of the preceding embodiments, wherein R4is a straight or branched C2-C6 alkylene substituted with oxo.19. The ligating monomer of any one of the preceding embodiments, wherein Aris a Ce, C9, or C10 aryl.20. The ligating monomer of any one of the preceding embodiments, wherein Ar is phenyl.21. The ligating monomer of embodiment 13, represented by the formula:22. The ligating monomer of embodiment 13, represented by the formula:PCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT023. The ligating monomer of embodiment 13, wherein each of R3is methyl, and Ar is phenyl.24. The ligating monomer of any one of the preceding embodiments, wherein Z' is selected from chloride, bromide, tritiate, and tosylate.25. A polymerizable composition comprising: one or more ligating monomer of any one of embodiments 13-24, and optionally one or more co-monomer.26. A ligating polymer derived from: one or more ligating monomer of any one of embodiments 13-24.27. A ligating polymer derived from a polymerizable composition of embodiment 26.28. The ligating polymer of any one of embodiments 26-27, further being derived from one or more co-monomer characterized by octanol-water partition coefficient of about -4 to about 4.29. The ligating polymer of any one of embodiments 26-28, further being derived from one or more co-monomer characterized by a water solubility of at least 5 g per L at 25 °C.30. The ligating polymer of any one of embodiments 26-29, further being derived from one or more co-monomer, the one or more co-monomer selected from a (meth)acrylate co-monomer, a di(meth)acrylate co-monomer, an acrylamide co-monomer, or a combination thereof.31. The ligating polymer of any one of embodiments 26-30, further being derived from one or more co-monomer, the one or more co-monomer being a (meth)acrylate co-monomer or a di(meth)acrylate co-monomer, each having at least one hydrogen bond donating group, at least one hydrogen-bond accepting group, or a combination thereof.32. The ligating polymer of any one of embodiments 26-31, further being derived from one or more co-monomer, the one or more co-monomer being a vinyl co-monomer having at least one hydrogen-bond accepting group.33. The ligating polymer of any one of embodiments 26-32, further being derived from at least one (meth)acrylate monomer having one or more hydroxyl groups.34. The ligating polymer of any one of embodiments 26-33, further being derived from at least one (meth)acrylate monomer having one or more glycerol groups.PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT035. The ligating polymer of any one of embodiments 26-34, further being derived from a comonomer selected from A-vinyl pyrrolidone, hydroxyethyl methacrylate, di(ethylene glycol) methyl ether methacrylate, and a combination thereof.36. The ligating polymer of any one of embodiments 26-35, further being derived from a comonomer selected from glycerol (meth)acrylate, glycerol di(meth)acrylate, and a combination thereof.37. The ligating polymer of any one of embodiments 26-36, further being derived from one or more co-monomer, the one or more co-monomer is present within the ligating polymer in an amount of no greater than 95 %.38. The ligating polymer of any one of embodiments 26-37, excluding co-monomers characterized by water solubility of less than 5 g per L at 25 °C, an octanol-water partition coefficient of greater than 4, or a combination thereof.39. The ligating polymer of any one of embodiments 26-38, excluding co-monomers comprising a quaternary ammonium trialkyl groups of formula: -N+(Rq)3, wherein each Rqis independently a Ci-Cs alkyl group.40. The ligating polymer of any one of embodiments 26-39, excluding co-monomers comprising a guanidine group.41. The ligating polymer of any one of embodiments 26-40, wherein the one or more ligating monomer or Formula I is present within the ligating polymer in an amount of at least 5 mol%.42. The ligating polymer of any one of embodiments 26-41, wherein the ligating monomer of Formula I accounts for about 5 mol% to about 100 mol% of the ligating polymer.43. The ligating polymer of any one of embodiments 26-41, wherein the ligating monomer of Formula I accounts for about 5 mol% to about 95 mol% of the ligating polymer, and hydroxyethyl acrylate, A-vinyl pyrrolidone, di(ethylene glycol) methyl ether methacrylate, glycerol (meth)acrylate, glycerol di(meth)acrylate, or a combination thereof accounts for 5 mol% to 95 mol% of the ligating polymer.44. The ligating polymer of any one of embodiments 26-41, wherein the ligating monomer of Formula la accounts for about 5 mol% to about 100 mol% of the ligating polymer.45. The ligating polymer of any one of embodiments 26-41, wherein the ligating monomer of Formula la accounts for about 5 mol% to about 95 mol% of the ligating polymer, and hydroxyethyl acrylate, A-vinyl pyrrolidone, di(ethylene glycol) methyl ether methacrylate, glycerol (meth)acrylate, glycerol di(meth)acrylate, or a combination thereof accounts for 5 mol% to 95 mol% of the ligating polymer.PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT046. The ligating polymer of any one of embodiments 26-41, wherein the ligating monomer of Formula lb accounts for about 5 mol% to about 100 mol% of the ligating polymer.47. The ligating polymer of any one of embodiments 26-41, wherein the ligating monomer of Formula lb accounts for about 5 mol% to about 95 mol% of the ligating polymer, and hydroxyethyl acrylate, A-vinyl pyrrolidone, di(ethylene glycol) methyl ether methacrylate, glycerol (meth)acrylate, glycerol di(meth)acrylate, or a combination thereof accounts for 5 mol% to 95 mol% of the ligating polymer.48. A filter element comprising: one or more porous substrate; and a filter media, the filter media comprising: a ligating polymer derived at least from one or more ligating monomer of any one of embodiments 13-24, wherein the ligating polymer is grafted to each of the one or more porous substrate.49. A filter element comprising: one or more porous substrate; and a filter media, the filter media comprising: a ligating polymer of any one of embodiments 25-47, wherein the ligating polymer is grafted to each of the one or more porous substrate.50. The filter element of any one of embodiments 48-49, the one or more porous substrate characterized by an average pore size of about 0.1 pm - 10 pm.51. The filter element of any one of embodiments 48-50, the one or more porous substrate having fibers characterized by a fiber diameter of about 0.5 micrometers to about 15 micrometers.52. The filter element of any one of embodiments 48-51, the one or more porous substrate comprised of material selected from polyolefins, polyisoprenes, polybutadienes, fluorinated polymers, chlorinated polymers, polyamides, polyimides, polyethers, polyether sulfones, polysulfones, polyvinyl acetates, polyesters, copolymers of vinyl acetate, polyphosphazenes, polyvinyl esters, polyvinyl ethers, polyvinyl alcohols, polycarbonates, and a combination thereof.53. The filter element of any one of embodiments 48-52, the one or more porous substrate comprised of material selected from nylon and polypropylene.54. The filter element of any one of embodiments 48-53, the one or more porous substrate in the form of a membrane or a nonwoven.PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT055. The filter element of any one of embodiments 48-54, the one or more ligating monomer of Formula I is present on the porous substrate at a ligand density of about 0.05 mmol to about 2.0 mmol per gram of porous substrate.56. The filter element of any one of embodiments 48-54, the ligating monomer of Formula la is present on the porous substrate at a ligand density of about 0.05 mmol to about 2.0 mmol per gram of porous substrate.57. The filter element of any one of embodiments 48-54, the ligating monomer of Formula lb is present on the porous substrate at a ligand density of about 0.05 mmol to about 2.0 mmol per gram of porous substrate.58. The filter element of any one of embodiments 48-57, characterized by a binding for the adeno-associated virus containing a DNA payload at a conductivity value of about 10 mS / cm to about 21 mS / cm.59. The filter element of any one of embodiments 48-58, characterized by a binding for an adeno-associated virus containing a DNA payload at a conductivity that is 2-4 mS / cm higher than a binding for an adeno-associated virus without a DNA payload.60. The filter element of any one of embodiments 48-59, the filter media excluding polymers other than a ligating polymer of any one of embodiments 14-45.61. The filter element of any one of embodiments 48-60, the filter media excluding polymers derived from monomers having quaternary ammonium trialkyl groups of formula -N+(Rq)3 wherein each Rqis independently a C1-C20 alkyl group.62. A process for separating an adeno-associated virus containing a DNA payload from an adeno-associated virus without a DNA payload, the process comprising: providing a filter element of any one of embodiments 48-61; providing a loading solution comprising the adeno-associated virus containing a DNA payload and the adeno-associated virus without a DNA payload; contacting the loading solution to the filter element; and separating the adeno-associated virus containing a DNA payload from the adeno-associated virus without a DNA payload.63. The process of any one of embodiments 62, wherein each of the adeno-associated virus containing a DNA payload and the adeno-associated virus without a DNA payload is an adeno- associated virus of serotype 2, serotype 5, or serotype 6.64. The process of any one of embodiments 62-63, wherein the DNA payload is a sequence effective to treat an inherited genetic disease.PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT065. The process of any one of embodiments 62-64, wherein the DNA payload is a sequence effective to treat hemophilia A, hemophilia B, muscular dystrophy, AADC deficiency, lipoprotein lipase deficiency, and retinal dystrophy.66. The process of any one of embodiments 62-65, the loading solution further comprising one or more water soluble inorganic salt.67. The process of any one of embodiments 62-66, the loading solution characterized by a conductivity of about 5 mS / cm to about 15 mS / cm.68. The process of any one of embodiments 62-67, the loading solution characterized by an inorganic salt concentration of about 50 mM to about 150 mM.69. The process of any one of embodiments 62-68, the loading solution characterized by a pH of about 8.5 to about 10.70. The process of any one of embodiments 62-69, the loading solution further comprising one or more virus other than an AAV, viral fragments, bacteria, DNA or fragments thereof, proteins, lipids, or a combination thereof.71. The process of any one of embodiments 62-70, the loading solution further comprising one or more enveloped virus.72. The process of any one of embodiments 62-71, the loading solution further comprising one or more non-enveloped virus.73. The process of any one of embodiments 62-72, the separating comprising centrifuging the filter element with the loading solution and collecting a solution enriched in the adeno- associated virus without a DNA payload.74. The process of any one of embodiments 60-71, the separating comprising contacting the filter element with an eluting solution, and collecting a solution enriched in the adeno- associated virus containing a DNA payload.75. The process of any one of embodiments 62-74, wherein the separating of the adeno- associated virus containing a DNA payload from the adeno-associated virus without a DNA payload occurs over an eluting conductivity window of about 2 mS / cm to about 4 mS / cm.76. The process of any one of embodiments 62-75, wherein the conditions are effective to recover the adeno-associated virus containing a DNA payload in an amount of at least 35 %.77. The process of any one of embodiments 62-76, wherein the conditions are effective to recover the adeno-associated virus containing a DNA payload at an enrichment of at least 75PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT078. The process of any one of embodiments 62-77, wherein the conditions are effective recover the adeno-associated virus containing a DNA payload in an amount of at least 35% at an enrichment of at least 75%.79. A process for preparing a filter element of any one of embodiments 48-62, the process comprising: providing a polymerizable composition of embodiment 25; providing a porous substrate; contacting the polymerizable composition to the porous substrate under conditions effective to polymerize the polymerizable composition onto the porous substrate.80. A process for preparing a filter element of any one of embodiments 38-62, the process comprising: providing a ligating polymer of any one of embodiments 28-39; providing a porous substrate; and contacting the ligating polymer to the porous substrate under conditions effective to graft the ligating polymer to the porous substrate.81. The process of any one of embodiments 79-80, the conditions comprising ionizing irradiation.82. The process of embodiment 81, wherein said irradiation is chosen from e-beam irradiation, X-ray irradiation, or gamma irradiation.83. The process of embodiment 82, wherein said irradiation is e-beam irradiation.84. The process of any one of embodiments 79-83, the conditions comprising e-beam irradiation at a dose of about 6 MRad to about 10 MRad.85. The process of any one of embodiments 79-82, the conditions comprising UV irradiation.86. A kit comprising: a filter element of any one of embodiments 48-61; and a set of instructions for separating adeno-associated virus containing a DNA payload from adeno-associated virus without a DNA payload.

[0177] EXAMPLES

[0178] The following examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, described herein.

[0179] Example 1. Sample functionalization procedure for nonwoven and / or membrane layers (e-beam)PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0

[0180] Preparation: Substrates are cut to size and weighed. Substrates are subsequently inserted into an appropriately sized zip-top bag and transferred to a nitrogen-inerted glove box. Sample bags are left open while the glove box chamber, bags, and contents are purged with nitrogen to less than 20 ppm oxygen as measured by a trace oxygen analyzer. While the substrates are purging, monomer solutions are weighed at desired compositions in jars, capped, and shaken by hand to mix the contents. Jars are subsequently opened and the solutions are sparged with nitrogen for 2 min or longer to remove any dissolved oxygen from the solution. Sparging is accomplished by fully immersing a nitrogen line in the solution and bubbling nitrogen through the sample. To cap, the nitrogen line is slowly drawn out of the solution with the jar lid partially covering the jar so that as much nitrogen as possible remains in the headspace. Jars are then transferred into the glove box via double-door (“airlock”) chamber. Jars lids are removed to flush any residual air out of the jar headspace. The glove box remains purged at less than 20 ppm oxygen.

[0181] Functionalization (e-beam): Before functionalization, sample zip-top bags are sealed and samples are removed from the glove box. Samples inside their purged bags are taped to a PET carrier web being conveyed at 35 fpm through an Energy Sciences, Inc (Wilmington, MA) ElectroCure electron beam. Samples are irradiated at 300 kV to a specified dose of 6 to 10 Mrad. Sealed sample bags are immediately returned to the glove box and saturated with sparged monomer solution in the <20 ppm oxygen environment: bags are opened and solution is poured in. Monomer solution is distributed evenly throughout the substrate with a 6” hand roller. Bags are then sealed and the saturated, irradiated substrate is left to react for a specified amount of time. After reaction, sample bags are removed from the glove box and opened in a fume hood to allow atmospheric oxygen to quench the grafting reactions.

[0182] Washing and Drying: Grafted substrates are transferred to a stainless-steel pot of boiling deionized water and extracted for 60 min to remove excess monomer solution and any free (non-grafted) polymer in solution. Substrates are then dried at ambient conditions in a polyethylene-lined aluminum tray.

[0183] Grafting Characterization: Weight gain of the grafted substrate is defined as % mass added to the sample:Weight gain (%) = (Final mass of dry grafted substrate - Initial mass of the substrate) / (Initial mass of the substrate)xl00

[0184] Example 2. Sample functionalization procedure for nonwoven and / or membrane layers (UV)PCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT0

[0185] Grafting solutions (5 grams each) of monomers were prepared at various monomer concentrations in deionized water, based on the measured % solids of the monomer solution. Each monomer solution also contained 3 -carboxybenzophenone, sodium salt (62.5 microliters of a 0.033 g / mL aqueous solution). For each grafting solution, a nylon membrane substrate (#080ZN, reinforced nylon 6,6 membrane, 0.8 micrometer nominal pore size, obtained from the 3M Company, St. Paul, MN) was placed on a sheet of polyester film, and sufficient grafting solution was pipetted onto the top surface of the substrate to completely wet the substrate. The coating solution was allowed to soak into the substrate for about 1 minute, and then a second sheet of polyester film was placed on top of the substrate. A 2.28 kg cylindrical weight was rolled over the top of the resulting three-layer sandwich to squeeze out excess coating solution. Ultraviolet (UV)-initiated grafting was conducted by irradiating the sandwich using a UV stand (Classic Manufacturing, Inc., Oakdale, MN) equipped with 18 bulbs (Sylvania RG2 40W F40 / 350BL / ECO, 10 above and 8 below the substrate, 1.17 meters (46 inches) long, spaced 5.1 cm (2 inches) on center), with an irradiation time of 15 minutes. The polyester sheets were removed and the resulting grafted membrane was placed in a polyethylene bottle. The bottle was filled with 0.9% saline solution, sealed and placed on a laboratory bottle roller for 30 minutes to wash off any residual monomer or ungrafted polymer. The saline solution was poured off and replaced with deionized water for an additional 30 minutes of washing. Washing was repeated with fresh 0.9% saline solution for 30 minutes, followed by 30 minutes of washing with deionized water (2 times). Following the wash steps, the polymer grafted membrane was air dried. Ligand density of the polymer grafted membrane was estimated based on mass gain.

[0186] Example 3. Production and purification of rAAV using the TESSA System

[0187] HEK 293 cells and TESSA vectors were obtained from OXGENE. Cells were grown in BalanCD HEK293 media (91128, Fujifilm) supplemented with 4 mM GlutaMax (35050061, Thermo Fisher Scientific) at 37 °C in 8% CO2 in Erlenmeyer shake flasks in a shaking incubator. Cells were passaged every 2-3 days to maintain a cell density between 0.5-4 million cells / mL. To produce AAV using TESSA vectors, two separate Ad5 TESSA vectors (Oxgene) were used: TESSA-AAV-EGFP, containing the AAV genome, and TESSA-RepCapX, where X is the sequence for AAV2 or AAV5. In the morning before infecting cells with the TESSA vectors, cells were split to obtain a density of 1.5 million cells / mL in the desired volume. Two to four hours after splitting the cells, TESSA-AAV-EGFP and either TESSA-RepCap2 or TESSA-RepCap5 were mixed at an infectious unit (IU) ratio of 1 : 1 or 3 : 1 and 1 IU or 3 IU ofPCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 each TESSA vector per cell. The TESSA vectors were mixed and diluted in 500 pL of media and added dropwise to the cells while stirring. Cells were returned to the shaking incubator. After 48 h, the cells were lysed with 0.1% Triton-X 100, 2 mM MgCh. After 2 h, the conductivity was adjusted to 25 mS / cm using 5 M NaCl and the cell lysate was clarified with a 3M Harvest RC synthetic depth filter. The conductivity was then further adjusted to 50 mS / cm with 5 M NaCl and the pH was adjusted to 8.5 with 1 MNaOH. Then, 25 U / mL of SAN HQ nuclease (70920, Arctic Zymes) was added, and the solution was incubated at 37 °C with mixing for 2-3 h. After nuclease digestion, the sample was applied to a POROS AAVX affinity column and washed with three different buffers of 40 mS / cm conductivity with pH descending from 7.4 to 6.5 to 5.5. Bound AAV was then eluted from the AAVX column with a 50 mM citric acid, pH 2.5, 40 mS / cm buffer and immediately neutralized with Tris-Cl buffer. The buffer was then exchanged into lx PBS with 5% glycerol using dialysis cassettes.

[0188] Example 4. Production and purification of rAAV using the TESSA system

[0189] HEK 293 cells and TESSA vectors were obtained from OXGENE. Cells were grown in BalanCD HEK293 media (91128, Fujifilm) supplemented with 4 mM GlutaMax (35050061, Thermo Fisher Scientific) at 37 °C in 8% CO2 in Erlenmeyer shake flasks in a shaking incubator. Cells were passaged every 2-3 days to maintain a cell density between 0.5-4 million cells / mL. To produce AAV using TESSA vectors, two separate Ad5 TESSA vectors (Oxgene) were used: TESSA-AAV-EGFP, containing the AAV genome, and TESSA-RepCapX, where X is the sequence for AAV2 or AAV5. In the morning before infecting cells with the TESSA vectors, cells were split to obtain a density of 1.5 million cells / mL in the desired volume. Two to four hours after splitting the cells, TESSA-AAV-EGFP and either TESSA-RepCap2 or TESSA-RepCap5 were mixed at an infectious unit (IU) ratio of 1 : 1 or 3 : 1 and 1 IU or 3 IU of each TESSA vector per cell. The TESSA vectors were mixed and diluted in 500 pL of media and added dropwise to the cells while stirring. Cells were returned to the shaking incubator. After 48 h, the cells were lysed with 0.1% Triton-X 100, 2 mM MgCh. After 2 h, the conductivity was adjusted to 25 mS / cm using 5 M NaCl and the cell lysate was clarified with a 3M Harvest RC synthetic depth filter. The conductivity was then further adjusted to 50 mS / cm with 5 M NaCl and the pH was adjusted to 8.5 with 1 MNaOH. Then, 25 U / mL of SAN HQ nuclease (70920, Arctic Zymes) was added, and the solution was incubated at 37 °C with mixing for 2-3 h. After nuclease digestion, the sample was applied to a POROS AAVX affinity column and washed with three differentPCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 buffers of 40 mS / cm conductivity with pH descending from 7.4 to 6.5 to 5.5. Bound AAV was then eluted from the AAVX column with a 50 mM citric acid, pH 2.5, 40 mS / cm buffer and immediately neutralized with Tris-Cl buffer. The buffer was then exchanged into lx PBS with 5% glycerol using dialysis cassettes.

[0190] Example 5. Total AAV capsid estimation using AAV ELISA

[0191] AAV2 and AAV5 capsid estimations were performed using the AAV2 (PRAAV2XP, Progen) and AAV5 Xpress ELISA kits (PRAAV5XP, Progen) respectively as per manufacturers instruction.

[0192] Example 6. Genome copy estimation using qPCR

[0193] Preparation of samples for qPCR analysis: Prior to setting up qPCR reactions, extracapsular DNA was digested with DNase as described here. Samples were serially diluted 1000X in PBS (10010-023, ThermoFisher Scientific), and lOuL of each sample reacted in 50 pL final volume with 1 pL DNaseXT (M0570S, New England Biolabs, Ipswitch, MA) 5 pL DNase buffer, and 34 pL molecular biology-grade water (10977015, Invitrogen). Samples were incubated at 37 °C for 16 h, followed by 20 min at 75 °C enzyme inactivation and final held at 4 °C until qPCR was performed.

[0194] qPCR standard: A well-characterized reference AAV2 strain was used as the standard in qPCR (VR-1616, ATCC). Upon receipt, the vial was thawed, and samples were pipette into low bind tubes in single use aliquots and were stored at -80 °C until use. To create a standard curve, the AAV2 standard was serial diluted in molecular biology-grade water final concentrations ranging from 102- 108copies / ml in 10X increments.

[0195] qPCR reactions: DNase-treated samples were diluted 10X in water and 5 pL was subsequently analyzed with qPCR with the following protocol: The qPCR mix contained the following in 50 pL: IX PrimeTime Mastermix (IDT, Coralville, IA, USA), 0.5 pM forward primer (5'-GTCAATGGGTGGAGTATTTACGG-3'), reverse primer (5'-AGGTCATGTACTGGGCATAATGC-3') and CMV Probe (5’- / 56-FAM / AA GTG TAT C / ZEN / A TAT GCC AAG TAC GCC CCC / 3IABkFQ / -3’). Primers were synthesized by Integrated DNA Technologies (Coralville, IA) and described in De, Bishnu P., et al. "In vivo potency assay for adeno-associated virus-based gene therapy vectors using AAVrh. 10 as an example." Human gene therapy methods 29.3 (2018): 146-155, incorporated herein by reference. The qPCR was run in a skirted PCR plate (Agilent 401490) sealed with optically clear strip caps (401425, Agilent Technologies, Santa Clara, CA) using an Agilent AriaMxPCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 instrument with the following parameters: 10 min at 95 °C, 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Data was then viewed and exported using AriaMX software (Agilent).

[0196] Example 7. Calculation of full percentages using ELISA and qPCR

[0197] The full percentage of AAV particles was determined by dividing the total genome copies / mL (as determined by qPCR) by the total capsids / mL (as determined by ELISA) and multiplying by 100.

[0198] Example 8. Stunner based measurements of AAV full percentages

[0199] Empty / full percentages of AAV viruses were measured using the Stunner Instrument (Unchained Labs, CA) according to the protocol provided by the instrument manufacturer.

[0200] Table 1. Materials Table.PCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT0PCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT0

[0201] Example 9. Ligand Synthesis

[0202] Preparatory Example 1 : Acrylamidopropyl dimethylphenacylammonium chloride (“APDMPAC”).

[0203] A-[3-(Dimethylamino)propyl] acrylamide (“DMAPA”) (7.81 g, 0.05 mol) was dissolved in 25 mL of dichloromethane and cooled in an ice-water bath with magnetic stirring and a slow nitrogen sweep. Phenacyl chloride (7.73 g, 0.05 mol) dissolved in dichloromethane (20 mL) was added rapidly to the reaction flask and the reaction was allowed to slowly warm to room temperature and then stirred overnight. The mixture was extracted into about 50 mL of deionized water and the dichloromethane was removed using a rotary evaporator (% solidsPCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT0 of 23.9%). Proton NMR analysis confirmed that the desired product dissolved in the remaining water fraction. About 50 pL of a 10,000 ppm stock solution of 4-hydroxy TEMPO in deionized water was added to the mixture. Percent solids (% solids) was determined using an Ohaus moisture balance (Model Number MB35, obtained from the Ohaus Corporation, Parsippany, NJ).

[0204] ‘HNMR (500 MHz, D2O) 6 1.87 (m, 2 H), 3.17 and 3.19 (m and s, 8 H), 3.57 (m, 2 H), 5.58 (d, 1 H), 5.97 (d, 1 H), 6.06 (dd, 1 H), 7.40 (t, 2 H), 7.57 (t, 1 H), 7.76 (d, 2 H).

[0205] Preparatory Example 2: Methacryl amidopropyl dimethylbenzylammonium bromide (“MAPDMB”).

[0206] A-[3-(Dimethylamino) propyl]methacrylamide (“DMAPMA”) (42.56 g, 0.25 mol) was dissolved in 250 mL dichloromethane and cooled in an ice-water bath with magnetic stirring and a slow nitrogen sweep. Benzyl bromide (42.76 g, 0.25 mol) dissolved in dichloromethane (50 mL) and then added dropwise to the reaction flask over a 35 min period and the resulting mixture was allowed to slowly warm to room temperature and then stirred overnight. The mixture was extracted into about 150 mL of deionized water (% solids of 36.1%). Proton NMR analysis confirmed that the desired product was dissolved in the water fraction. About 250 microliters of a 10,000 ppm stock solution of 4-hydroxy TEMPO in deionized water was added to the water fraction. Percent solids (% solids) was determined using an Ohaus moisture balance (Model Number MB35, obtained from the Ohaus Corporation, Parsippany, NJ). The monomer was designated as MAPDMB.

[0207] 'H-NMR (500 MHz, D2O): 1.73 (s, 3H), 1.97 (m, 2H), 2.88 (s, 6H), 3.09 (m, 2H), 3.20 (t, 2H), 4.32 (s, 2H), 5.31 (s, 1H), 5.54 (s, 1H), 7.35 (m, 5H).

[0208] Preparatory Example 3: Aery 1 amidopropyl dimethylbenzylammonium bromide(“APDMB”).

[0209] This monomer was prepared by a procedure similar to that of Preparatory Example 2, substituting 3-(dimethylaminopropyl)acrylamide (“DMAPA”, 39.06 g, 0.25 m) for the DMAPMA. The measured % solids was 29.05%. The monomer was designated APDMB.PCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT0

[0210] Preparatory Example 4: Methacrylamidopropyl dimethylphenoxy ethyl ammonium bromide (“MAPDMP”)

[0211] A-[3-(Dimethylamino) propyl]methacrylamide (“DMAPMA”) (8.51 g, 0.05 mol) was dissolved in 50 mL acetonitrile and cooled in an ice-water bath with magnetic stirring and a slow nitrogen sweep. Phenoxyethyl bromide (10.01 g, 0.05 mol) added to the reaction flask and the resulting mixture was allowed to warm to room temperature and then stirred for seven days. 50 microliters of a 10,000 ppm stock solution of 4-hydroxy TEMPO in deionized water was added to the mixture. The mixture was stripped on a rotary evaporator to give a viscous syrup. Deionized water (55 mL) was added and the mixture was further stripped to remove residual acetonitrile. Percent solids (41.25% solids) was determined using an Ohaus moisture balance (Model Number MB35, obtained from the Ohaus Corporation, Parsippany, NJ). The monomer was designated as MAPDMP.

[0212] 1H-NMR (500 MHz, D2O): 8 1.65 (s, 3H), 1.84 (m, 2H), 2.94, (s, 6H), 3.12 (t, 2H), 3.17 (m, 2H), 3.49 (m, 2H), 4.15 (m, 2H), 5.21 (s, 1H), 5.48 (s, 1H), 6.78 (d, 2H), 6.85 (t, 1H), 7.16 (t, 2H).

[0213] Table 2. Phenalkyl and Phenacyl Quat. Filter Media (“FM”) of the present disclosure: Polymerizable Compositions for Preparing Ligating Polymers*values in mol%

[0214] Table 3. Comparative Filter MediaPCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT0*values in mol%

[0216] Example 10. Phenacyl and Phenalkyl Quat Filter Media Salt Tolerance with AAV5

[0217] Filter Media FM1, filter media FM2, and comparative filter medias CFM1 were grafted onto a 0.8 micron pore size nylon membrane by UV. The resulting filter elements are represented in Table 4.

[0218] Small circular discs of 5 mm diameter were punched out of each material listed in Table 4 and secured inside the wells of a 3M Empore plate with an O-ring. A 1 mL 96-deep well collection plate was placed beneath the Empore plate and each of the discs were flushed with buffer A (50 mM Tris-Acetate, pH 9.0, 50 mM sodium chloride). Flushing was mediated by spinning the Empore plate together with the collection plate in a centrifuge at 500xg for 3 min. An AAV5 virus sample suspended in buffer A (i.e., loading conductivity 5.5 mS / cm) was loaded onto the filter element and centrifuged to collect the flow through from each well. Both the feed sample and the flow throughs from each well were collected and analyzed via AAV5 ELISA (see methods above).

[0219] The above experiment was repeated for each filter element with buffers B-D having sodium chloride concentrations of 100 mM, (11.3 mS / cm), 150 mM (16.5 mS / cm), and 200 mM (22 mS / cm), respectively.

[0220] Table 4. Filter Elements illustrating that Phenacyl Quat (FM1) and Phenalkyl Quat (FM2) filter media are more salt tolerant than MAPTAC Q ChemistryPCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT0

[0221] The percentage of virus (i.e., total AAV) in the flow through compared to the Feed AAV is listed in Table 4 and graphically shown in FIG. 1. The results indicate that the phenacyl quaternary ammonium ligands bind to AAV5 and there is no AAV5 in flow through at any of the tested conductivities, up to 150 mM NaCl. At 200 mM NaCl condition, there was flow through of the AAV viruses for phenacyl quat. For phenalkyl quat and MAPTAC, viruses start to flow through the material at 150 mM and 200 mM NaCl conditions. At 150 mM NaCl condition, flow through of viruses was more for MAPTAC than phenalkyl quat indicating phenalkyl quat to be a more salt tolerant than MAPTAC at 150 mM NaCl condition. This result indicates that the more hydrophobic regions afforded by the phenacyl and phenalkyl quaternary ammonium groups allow for greater salt tolerance compared to MAPTAC (current Q chemistry).

[0222] It is theorized that the phenacyl and phenalkyl quaternary ammonium groups may access a deeper region of the 5-fold symmetry pore of AAV5, which may offer stronger binding energies, and can mediate hydrophobic interactions within the capsid structure. Comparatively, interactions between the MAPTAC (Q-chemistry) and AAV5 are relatively shallow. The resulting structural arrangements of ligand and receptor provide a rationalization for the phenacyl and phenalkyl quaternary ammonium ligands enhanced binding to AAV (both empty and full) at higher salt concentrations than the current Q chemistry. Additionally, it is well known that DNA, when present, is contained within the 5-fold symmetry pore of a fullPCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 capsid. Thus, it is further speculated that these types of hydrophobic quaternary ammonium groups may interact with the DNA in the depths of the 5-fold symmetry pore, which may further explain the greater salt tolerance of full AAV with these novel ligands compared to empty AAV, as demonstrated below. It is also possible that the ligands may induce mixedmode interactions with AAV, i.e., both hydrophobic and ionic interactions.

[0223] Example 11. Empty / Full AAV5 Separations with Phenalkyl and Phenacyl Quaternary Ammonium Filter Media.

[0224] Phenacyl Quat Filter Media FM3 (74 mol% APDMPAC, 24 mol% HEMA, 2 mol% GDMA, 1 mM MSA) was grafted via e-beam onto a BA080 nylon membrane and the resulting filter element was then attached to an AKTA Avant 150 purification system.

[0225] Phenalkyl Quat Filter Media FM4 (75 mol% MAPDMB, 23 mol% HEMA, 2 mol% GDMA) was grafted via e-beam onto a B A080 nylon membrane and the resulting filter element was then attached to an AKTA Avant 150 purification system.

[0226] Table 5 describes the resulting filter elements 3 and 4.

[0227] Table 5. Filter elements 3-4.

[0228] The filter elements containing FM 3 and FM4 were each equilibrated with 20 column volumes of buffer E (50 mM Tris-Acetate, pH 9.0, 2mM MgCh, 100 mM sodium chloride, conductivity of 11.5 mS / cm). An AAV5 virus sample comprising a mix of empty and full viruses with a full percentage of 20% was diluted into buffer E and loaded onto the filter element (loading conductivity -14.7 mS / cm). After loading, a wash step is performed with buffer E to wash off any unbound viruses. Elution was performed via step gradient by mixingPCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT0 varying percentages of buffer E with buffer F (50 mM Tris-Acetate, pH 9.0, 2mM MgCh, 400 mM sodium chloride).

[0229] The elution data is tabulated in Table 6 and Table 7 and represented in the chromatogram of FIG. 2A and FIG. 2B, respectively. The fractions with the most virus were analyzed for empty-full percentages, via Stunner as well as ELISA and qPCR. In addition, the ratio of UV 260 and UV 280 signals of the peaks were recorded for each of the fractions, which gives an estimate of the empty-full ratios.

[0230] Table 6. Separation Data for Phenacyl Quat Filter Element 3

[0231] Results indicated that the phenacyl quat chemistries mediated enrichment in full percentages and a 20% full AAV5 sample was enriched to 82% for filter element 3.

[0232] Table 7. Separation Data for Phenalkyl Quat Filter Element 4.PCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT0

[0233] Results indicated that the phenalkyl quat chemistries mediated enrichment in full percentages and a 20% full AAV5 sample was enriched to 85% for filter element 4.

[0234] Example 12. Equivalents

[0235] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

[0236] REFERENCES

[0237] A number of patents and publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

Claims

PCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT0CLAIMSWe claim:

1. A compound represented by Formula I:CH2=C(R1)-C(O)-W-R2-N+(R3)2-R4-Ar Z (I) wherein:R1is -H or a straight or branched C1-4 alkyl,W is -O- or -N(R5)-,R2is a straight or branched C2-Ce alkylene, or a straight or branched C4-Ci2alkoxyalkylene, or -R6-X-C(O)-Y-R7-, each R3is independently a straight or branched Ci-Ce alkyl,R4is a straight or branched Ci-Ce alkylene optionally substituted with oxo or oxa, or a straight or branched Ci-Ce alkylene,Ar is aryl,Z is a counterion,R5is -H or a straight or branched C1-4 alkyl,R6is a straight or branched Ci-Ce alkylene, or a straight or branched C4-Ci2alkoxyalkylene,R7is a straight or branched Ci-Ce alkylene, or a straight or branched C4-Ci2alkoxyalkylene, andX and Y are independently selected from -O-, or -N(R5)-, provided that at least one of X and Y is -O-, or -N(R5)-, and with the proviso that when R4is -CH2- and R3is methyl, Ar is not phenyl.

2. The compound of any one of the preceding claims, wherein each R3is methyl.

3. The compound of any one of the preceding claims, wherein Ar is phenyl.

4. The compound of claim 1, represented by the formula:PCT Application Applicant Ref: PA200156US01 Atty Dkt. No.: THF-084PCT05. The compound of claim 1, represented by the formula:

6. The compound of claim 1, wherein each of R3is methyl, and Ar is phenyl.

7. The compound of any one of the preceding claims, wherein Z' is selected from chloride, bromide, tritiate, and tosylate.

8. The compound of claim 1, wherein said compound is a monomer.

9. The compound of claim 8, wherein monomer is a ligating monomer.

10. A polymerizable composition comprising: one or more ligating monomer of any one of claims 1-9, and optionally one or more co-monomer.

11. A ligating polymer derived from: one or more ligating monomer of any one of claims 1-9.

12. A ligating polymer derived from a polymerizable composition of claim 11.

13. The ligating polymer of any one of claims 13-14, further being derived from one or more co-monomer characterized by octanol-water partition coefficient of about -4 to about 4.

14. The ligating polymer of any one of claims 13-15, further being derived from one or more co-monomer characterized by a water solubility of at least 5 g per L at 25 °C.

15. The ligating polymer of any one of claims 13-16, further being derived from one or more co-monomer, the one or more co-monomer selected from a (meth)acrylate co-monomer, a di(meth)acrylate co-monomer, an acrylamide co-monomer, or a combination thereof.PCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT016. The ligating polymer of any one of claims 13-17, further being derived from one or more co-monomer, the one or more co-monomer being a (meth)acrylate co-monomer or a di(meth)acrylate co-monomer, each having at least one hydrogen bond donating group, at least one hydrogen-bond accepting group, or a combination thereof.

17. The ligating polymer of any one of claims 13-18, further being derived from one or more co-monomer, the one or more co-monomer being a vinyl co-monomer having at least one hydrogen-bond accepting group.

18. The ligating polymer of any one of claims 13-19, further being derived from at least one (meth)acrylate monomer having one or more hydroxyl groups.

19. The ligating polymer of any one of claims 13-20, further being derived from at least one (meth)acrylate monomer having one or more glycerol groups.

20. The ligating polymer of any one of claims 13-21, further being derived from a co-monomer selected from A-vinyl pyrrolidone, hydroxyethyl methacrylate, di(ethylene glycol) methyl ether methacrylate, and a combination thereof.

21. The ligating polymer of any one of claims 13-22, further being derived from a co-monomer selected from glycerol (meth)acrylate, glycerol di(meth)acrylate, and a combination thereof.

22. The ligating polymer of any one of claims 13-23, further being derived from one or more co-monomer, the one or more co-monomer is present within the ligating polymer in an amount of no greater than 95 %.

23. The ligating polymer of any one of claims 13-24, excluding co-monomers characterized by water solubility of less than 5 g per L at 25 °C, an octanol -water partition coefficient of greater than 4, or a combination thereof.

24. The ligating polymer of any one of claims 13-25, excluding co-monomers comprising a quaternary ammonium trialkyl groups of formula: -N+(Rq)3, wherein each Rqis independently a Ci-Cs alkyl group.PCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT025. The ligating polymer of any one of claims 13-28, wherein the ligating monomer of Formula I accounts for about 5 mol% to about 100 mol% of the ligating polymer.

26. The ligating polymer of any one of claims 13-28, wherein the ligating monomer of Formula I accounts for about 5 mol% to about 95 mol% of the ligating polymer, and hydroxyethyl acrylate, A-vinyl pyrrolidone, di(ethylene glycol) methyl ether methacrylate, glycerol (meth)acrylate, glycerol di(meth)acrylate, or a combination thereof accounts for 5 mol% to 95 mol% of the ligating polymer.

27. The ligating polymer of any one of claims 13-28, wherein the ligating monomer of Formula la accounts for about 5 mol% to about 100 mol% of the ligating polymer.

28. The ligating polymer of any one of claims 13-28, wherein the ligating monomer of Formula la accounts for about 5 mol% to about 95 mol% of the ligating polymer, and hydroxyethyl acrylate, A-vinyl pyrrolidone, di(ethylene glycol) methyl ether methacrylate, glycerol (meth)acrylate, glycerol di(meth)acrylate, or a combination thereof accounts for 5 mol% to 95 mol% of the ligating polymer.

29. The ligating polymer of any one of claims 13-28, wherein the ligating monomer of Formula lb accounts for about 5 mol% to about 100 mol% of the ligating polymer.

30. The ligating polymer of any one of claims 13-28, wherein the ligating monomer of Formula lb accounts for about 5 mol% to about 95 mol% of the ligating polymer, and hydroxyethyl acrylate, A-vinyl pyrrolidone, di(ethylene glycol) methyl ether methacrylate, glycerol (meth)acrylate, glycerol di(meth)acrylate, or a combination thereof accounts for 5 mol% to 95 mol% of the ligating polymer.

31. A filter element comprising: one or more porous substrate; and a filter media, the filter media comprising: a ligating polymer derived at least from one or more ligating monomer of any one of claims 1- H,PCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT0 wherein the ligating polymer is grafted to each of the one or more porous substrate.

32. A filter element comprising: one or more porous substrate; and a filter media, the filter media comprising: a ligating polymer of any one of claims 12-34, wherein the ligating polymer is grafted to each of the one or more porous substrate.

33. The filter element of any one of claims 35-36, the one or more porous substrate characterized by an average pore size of about 0.1 pm - 10 pm.

34. The filter element of any one of claims 35-37, the one or more porous substrate having fibers characterized by a fiber diameter of about 0.5 micrometers to about 15 micrometers.

35. The filter element of any one of claims 35-38, the one or more porous substrate comprised of material selected from polyolefins, polyisoprenes, polybutadienes, fluorinated polymers, chlorinated polymers, polyamides, polyimides, polyethers, polyether sulfones, polysulfones, polyvinyl acetates, polyesters, copolymers of vinyl acetate, polyphosphazenes, polyvinyl esters, polyvinyl ethers, polyvinyl alcohols, polycarbonates, and a combination thereof.

36. The filter element of any one of claims 35-39, the one or more porous substrate comprised of material selected from nylon and polypropylene.

37. The filter element of any one of claims 35-40, the one or more porous substrate in the form of a membrane or a nonwoven.

38. The filter element of any one of claims 35-41, the one or more ligating monomer of Formula I is present on the porous substrate at a ligand density of about 0.05 mmol to about 2.0 mmol per gram of porous substrate.

39. The filter element of any one of claims 35-44, characterized by a binding for the adeno- associated virus containing a DNA payload at a conductivity value of about 10 mS / cm to about 21 mS / cm.PCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT040. The filter element of any one of claims 35-45, characterized by a binding for an adeno- associated virus containing a DNA payload at a conductivity that is 2-4 mS / cm higher than a binding for an adeno-associated virus without a DNA payload.

41. The filter element of any one of claims 35-47, the filter media excluding polymers derived from monomers having quaternary ammonium trialkyl groups of formula -N+(Rq)3 wherein each Rqis independently a C1-C20 alkyl group.

42. A process for separating an adeno-associated virus containing a DNA payload from an adeno-associated virus without a DNA payload, the process comprising: providing a filter element of any one of claims 35-48; providing a loading solution comprising the adeno-associated virus containing a DNA payload and the adeno-associated virus without a DNA payload; contacting the loading solution to the filter element; and separating the adeno-associated virus containing a DNA payload from the adeno-associated virus without a DNA payload.

43. The process of any one of claims 49, wherein each of the adeno-associated virus containing a DNA payload and the adeno-associated virus without a DNA payload is an adeno-associated virus of serotype 2, serotype 5, or serotype 6.

44. The process of any one of claims 49-50, wherein the DNA payload is a sequence effective to treat an inherited genetic disease.

45. The process of any one of claims 49-51, wherein the DNA payload is a sequence effective to treat hemophilia A, hemophilia B, muscular dystrophy, AADC deficiency, lipoprotein lipase deficiency, and retinal dystrophy.

46. The process of any one of claims 49-52, the loading solution further comprising one or more water soluble inorganic salt.PCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT047. The process of any one of claims 49-53, the loading solution characterized by a conductivity of about 5 mS / cm to about 15 mS / cm.

48. The process of any one of claims 49-54, the loading solution characterized by an inorganic salt concentration of about 50 mM to about 150 mM.

49. The process of any one of claims 49-55, the loading solution characterized by a pH of about 8.5 to about 10.

50. The process of any one of claims 49-56, the loading solution further comprising one or more virus other than an AAV, viral fragments, bacteria, DNA or fragments thereof, proteins, lipids, or a combination thereof.

51. The process of any one of claims 49-61, wherein the separating of the adeno-associated virus containing a DNA payload from the adeno-associated virus without a DNA payload occurs over an eluting conductivity window of about 2 mS / cm to about 4 mS / cm.

52. The process of any one of claims 49-64, wherein the conditions are effective recover the adeno-associated virus containing a DNA payload in an amount of at least 35% at an enrichment of at least 75%.

53. A process for preparing a filter element of any one of claims 35-49, the process comprising: providing a polymerizable composition of claim 12; providing a porous substrate; contacting the polymerizable composition to the porous substrate under conditions effective to polymerize the polymerizable composition onto the porous substrate.

54. A process for preparing a filter element of any one of claims 35-49, the process comprising: providing a ligating polymer of any one of claims 13-34; providing a porous substrate; and contacting the ligating polymer to the porous substrate under conditions effective to graft the ligating polymer to the porous substrate.PCT ApplicationApplicant Ref: PA200156US01Atty Dkt. No.: THF-084PCT055. The process of any one of claims 53-54, the conditions comprising ionizing irradiation.

56. The process of claim 55, wherein said irradiation is chosen from e-beam irradiation, X-ray irradiation, or gamma irradiation.

57. The process of claim 56, wherein said irradiation is e-beam irradiation.

58. The process of any one of claims 53-57, the conditions comprising e-beam irradiation at a dose of about 6 MRad to about 10 MRad.

59. The process of any one of claims 53-58, the conditions comprising UV irradiation.

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