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: PA200036 Atty Dkt. No.: THF-080PCT0SEPARATION OF VIRUS PARTICLES WITH LIGATING FILTER ELEMENTSNarendranath Bhokisham Ethan Laudermilch Semra Colak Atan Jerald K. Rasmussen Nisha Hollingsworth George W. Griesgraber Katie Fraass Wlaschin Logan Daniel Cruckson Bishop Patrick M. Crain Erick I. Soto Cantu Lindsay TraegerEmily Julik Qiuge Zhang Shri NiwasHector F. Espitia NavaarroCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application embodiments priority to U.S. Application No. 63 / 722,185, 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: PA200036 Atty Dkt. No.: THF-080PCT0DNA 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: PA200036 Atty Dkt. No.: THF-080PCT0

[0011] FIG. IB is an image from a computational model demonstrating a portion of a ligating polymer of the present disclosure docking to the 5-fold symmetry pore of an adeno-associated virus.

[0012] FIG. 1C is an image from a computational model demonstrating a portion of a Q chemistry polymer docking to the 5-fold symmetry pore of an adeno-associated virus.

[0013] 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.

[0014] 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.

[0015] FIG. 2C 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.

[0016] FIG. 2D-1, FIG. 2D-2 and FIG. 2D-3 are each 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.

[0017] FIG. 2E 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.

[0018] FIG. 2F 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.

[0019] FIG. 2G 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.

[0020] FIG. 2H 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.

[0021] FIG. 3 is a chromatogram illustrating the elution profiles for empty / full AAV5 separation processes with a Q chemistry filter element, each across a gradient of salt concentrations / conductivities.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0022] FIG. 4 is a chromatogram illustrating the elution profiles for empty / full AAV5 separation processes across a gradient of salt concentrations / conductivities with three filter elements of the present disclosure.

[0023] FIG. 5 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.

[0024] FIG. 6 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.

[0025] FIG. 7 is a chromatogram illustrating the elution profiles for an empty / full AAV2 separation process across a gradient of salt concentrations / conductivities with a filter element of the present disclosure.

[0026] FIG. 8 is a chromatogram illustrating the elution profiles for an empty / full AAV6 separation process across a gradient of salt concentrations / conductivities with a filter element of the present disclosure.

[0027] DESCRIPTION

[0028] Summary

[0029] In one embodiment, a ligating monomer of Formula I is described.CH2=C(R1)-C(O)-W-R2-X-C(O)-Y-R3-HA+-Q Z (I), wherein:R1is -H or a straight or branched Ci-4 alkyl,W is -O- or -N(R5)-,R2is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene,X and Y are independently selected from a bond, -O-, or -N(R5)-, provided that at least one of X and Y is -O-, or -N(R5)-,R3is a straight or branched C2-C6 alkylene, or a straight or branched C4-C12 alkoxyalkylene,HA+is a charged heteroaryl having 5-9 atoms and 1-3 heteroatoms selected from N, O, and S, provided that at least one heteroatom is a quadrivalent nitrogen,Q is a straight or branched C1-C12 alkyl or -R4-Ar,R4is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene,Ar is aryl,Z is a counterion, andPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 each R5is independently selected from -H and a straight or branched C1-4 alkyl.

[0030] 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.

[0031] In one embodiment, a ligating polymer is described. The ligating polymer is derived from one or more ligating monomer of the present disclosure.

[0032] 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 the present disclosure, wherein the ligating polymer is grafted to at least one of the one or more porous substrate.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] Detailed DescriptionPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0039] The present disclosure is directed toward chromatographic separations of full vs empty AAVs with polymeric filter media prepared from ligating monomers that have a charged heteroaryl 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 conductivity for separation.

[0040] Molecular docking computations illustrated 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 acids of the virus. Conversely, Q chemistries were observed to only interact with exterior regions of the 5f pore site. Without wishing to be bound by theory, the increased salt tolerance of the present chemistries compared to Q chemistries may be explained by these differences in binding activity. In other words, the ligating chemistries of the present disclosure 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.

[0041] 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 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 Q chemistries.

[0042] Separation of full AAVs from empty AAVs occurs over a greater window of conductivities with the chemistries of the present disclosure compared to 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 ~5-7 mS / cm compared to ~l-2 mS / cm for Q chemistries.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0043] Additionally, it has been demonstrated that the present chemistries are capable of separating AAVs from other viral contaminations, such as bacteriophages, e.g., Phi6.

[0044] The filter elements of the present disclosure, having novel ligating polymeric filtering media, provide a promising avenue toward the realization of many therapeutics.

[0045] Definitions

[0046] 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 the 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.

[0047] 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.

[0048] 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).

[0049] The use of “or” means “and / or” unless stated otherwise.

[0050] The use of “a” or “an” herein means “one or more” unless stated otherwise or where the use of “one or more” is clearly inappropriate.

[0051] The use of “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of’ and / or “consisting of.”

[0052] As used herein, the term “about” refers to a ±10% variation from the nominal value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0053] 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.

[0054] 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., -CH2PI1.

[0055] 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.

[0056] 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.

[0057] 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 as 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.

[0058] 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.

[0059] 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 C3-6PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 alkyl includes C3 alkyl (i.e., isopropyl -CH(CH3)2), C4 alkyl (e.g., ec-butyl - CH(CH3)CH2CH3), C5 alkyl (e.g., neopentyl -CPfc CHs^). 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-.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] As used herein, “exclude” means that the referenced substance is not present, i.e., 0 wt.%.

[0065] 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.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0066] 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.

[0067] 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.

[0068] 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.

[0069] As used herein, “A-vinyl lactam monomer” refers to a lactam moiety having a vinyl group directly attached to a nitrogen (i.e., -N-CILCH2) within a lactam ring system. A lactam is a cycloaliphatic system having an amide (i.e., -NR-C(O)-) within the ring atoms.

[0070] 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 at 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.

[0071] 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.

[0072] 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.

[0073] As used herein, “organic” defines a compound having carbon, whereas “inorganic” defines a compound without carbon.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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 would be / / -butyl (i.e., -CH2CH2CH2CH3), and a “branched” C4 alkyl group could be isobutyl, secbutyl, or tert-butyl.

[0078] As used herein, “ligating polymer” refers to a polymer derived from a ligating monomer.

[0079] 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).

[0080] As used herein, “full AAVs” refers to AAV capsids encapsulating a complete recombinant genome at or near the designed length, capable of transduction.

[0081] As used herein, “empty AAVs” refers to AAV capsids without encapsulated nucleic acid; structurally intact but non-functional for gene delivery.

[0082] 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.

[0083] 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.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0084] 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.

[0085] Ligands of Ligating Polymers for Filter Elements

[0086] 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.

[0087] In various embodiments, a ligating monomer represented by Formula I is described: CH2=C(R1)-C(O)-W-R2-X-C(O)-Y-R3-HA+-Q Z (I), wherein:R1is -H or a straight or branched Ci-4 alkyl,W is -O- or -N(R5)-,R2is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene,X and Y are independently selected from a bond, -O-, or -N(R5)-, provided that at least one of X and Y is -O-, or -N(R5)-,R3is a straight or branched C2-C6 alkylene, or a straight or branched C4-C12 alkoxyalkylene, HA+is a charged heteroaryl having 5-9 atoms and 1-3 heteroatoms selected from N, O, and S, provided that at least one heteroatom is a quadrivalent nitrogen,Q is a straight or branched C1-C12 alkyl or -R4-Ar,R4is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene, Ar is aryl,Z' is a counterion, and each R5is independently selected from -H and a straight or branched C1-4 alkyl.

[0088] In some embodiments, Q may be methyl or n-butyl.

[0089] In many embodiments, Q may be -R4-Ar, the ligating monomer represented by Formula la:CH2=C(R1)-C(O)-W-R2-X-C(O)-Y-R3-HA+-R4-Ar Z (la), wherein:R1is -H or a C1-4 alkyl,W is -O- or -N(R5)-,PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0R2is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene,X and Y are independently selected from a bond, -O-, or -N(R5)-, provided that at least one of X and Y is -O-, or -N(R5)-,R3is a straight or branched C2-C6 alkylene, or a straight or branched C4-C12 alkoxyalkylene,HA+is a charged heteroaryl having 5-9 atoms and 1-3 heteroatoms selected from N, O, andS, provided that at least one heteroatom is a quadrivalent nitrogen,R4is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene,Ar is aryl,Z' is a counterion, and each R5is independently selected from -H and a straight or branched C1-4 alkyl.

[0090] In some embodiments, W may be -O-.

[0091] In some embodiments, W may be -N(R5)-.

[0092] In some embodiments, X and Y may each be -N(R5)-.

[0093] In some embodiments, X may be a bond and Y may be -O-.

[0094] In some embodiments, X may be a bond and Y may be -N(R5)-.

[0095] In some embodiments, X may be -O- and Y may be a bond.

[0096] In some embodiments, X may be -N(R5)- and Y may be a bond.

[0097] In some embodiments, X may be -N(R5)- and Y may be -O-.

[0098] In some embodiments, Xmay be -N(R5)- and Y may be -O-.

[0099] In some embodiments, X may be -O- and Y may be -N(R5)-.

[0100] In some embodiments, HA+may be an imidazoyl ring, an oxazolyl ring, a pyrazolyl ring, a pyrrolyl ring, an indolyl ring, a pyridyl ring, or a benzimidazoyl ring.

[0101] In some embodiments, HA+may be an imidazolyl ring, an oxazolyl ring, a pyrazolyl ring, a pyrrolyl ring, an indolyl ring, or a benzimidazoyl ring.

[0102] In some embodiments, HA+may be benzimidazoyl ring.

[0103] In some embodiments, HA+may be an imidazolyl ring.

[0104] In some embodiments, HA+may be -N(R6)-R7-N+(R8)-, wherein R6and R8are taken together to form a heteroaryl ring, and R7is -C(H)=.

[0105] In some embodiments, HA+may be a benzimidazolyl ring.

[0106] In some embodiments, Ar may be a Ce, C9, or C10 aryl.

[0107] In some embodiments, Ar may be phenyl.

[0108] In some embodiments, Ar may be indenyl.

[0109] In some embodiments, Ar may be naphthyl.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0110] 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.

[0111] In some embodiments, Z' may be selected from chloride, bromide, methyl sulfate, butyl sulfate, tosylate, and tritiate.

[0112] In some embodiments, Z' may be selected from chloride, bromide, methyl sulfate, butyl sulfate, and tosylate.

[0113] In some embodiments, Z' may be selected from chloride, bromide, tosylate, and tritiate.

[0114] In some embodiments, Z' may be selected from chloride, bromide, and tosylate.

[0115] In some embodiments, Z' may be selected from chloride and bromide.

[0116] In some embodiments, Z' may be bromide.

[0117] In some embodiments, Z' is not a sulfate (e.g., methyl sulfate, butyl sulfate, or the like).

[0118] In some embodiments, R1is -H or -CH3.

[0119] In some embodiments, R2may be a straight or branched Ci-Ce alkylene.

[0120] In some embodiments, R2may be a straight or branched C2-C6 alkylene.

[0121] In some embodiments, R2may be a straight or branched C1-C4 alkylene

[0122] In some embodiments, R2may be a straight or branched C2-C4 alkylene.

[0123] In some embodiments, R2may be -CH2, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, or -CH2CH2CH2CH2-.

[0124] In some embodiments, R2may be a straight or branched C4-C12 alkoxyalkylene.

[0125] In some embodiments, R2may be a straight or branched C4-C12 alkoxyethylene.

[0126] In some embodiments, R2may be a straight or branched C4-C6 alkoxy ethylene.

[0127] In some embodiments, R3may be a straight or branched C2-C6 alkylene.

[0128] In some embodiments, R3may be a straight or branched C2-C4 alkylene.

[0129] In some embodiments, R3may be -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, or -CH2CH2CH2CH2-.

[0130] 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 a C1-C2 alkylene.

[0131] In some embodiments, R4may be -CH2-.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0132] In some embodiments, R4may be methylene, ethylene, propylene, isopropylene, butylene, isobutylene, or sec-butylene.

[0133] In some embodiments, each R5may be -H.

[0134] In some embodiments, each R5may be -CH3.

[0135] In some embodiments, each R5may be independently selected from -H and -CH3.

[0136] In many embodiments, the ligating monomer may be represented by the formula lb:

[0137] In some embodiments, the ligating monomer may be represented by formula lb and Z' may be bromide.

[0138] Polymerizable Compositions with Ligating Monomers

[0139] In various embodiments, a polymerizable composition is described. The polymerizable composition may include one or more ligating monomer of Formula I above. In some embodiments, the polymerizable composition may include a ligating monomer of Formula la.

[0140] In some embodiments, one or more ligating monomer of Formula I (e.g., Formula la, Formula lb) may be present within the polymerizable composition in an 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, 100, or a value within a range between any of the preceding values, e.g., between about 20 mol% and about 80 mol%, between about 45 mol% and about 95 mol%, or the like.

[0141] In some embodiments, one or more ligating monomer of Formula I (e.g., Formula la, Formula lb) may be present within the polymerizable composition in an amount of about 1 wt% to about 100 wt% with respect to the total weight of polymerizable components (i.e., not including any non-polymerizable components such as solvent) within the polymerizable composition. 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, 100, or a value within a range between any of the preceding values, e.g., between about 20 wt% and about 80 wt%, between about 45 wt% and about 95 wt%, or the like.

[0142] 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 aPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 combination thereof. In some embodiments, any of the polymerizable compositions may also further include a solvent, e.g., de-ionized water.

[0143] 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 amino groups. Thus, in some embodiments, any co-monomer may include one or more of the described hydrophilic groups.

[0144] 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.

[0145] 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-, -PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0C(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

[0146] 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.

[0147] 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.

[0148] In embodiments further including a co-monomer described herein, the one or more comonomer may be present within the polymerizable composition in an 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%.

[0149] In embodiments further including a co-monomer described herein, the one or more comonomer may be present within the polymerizable composition in an amount of about 5 wt% to about 95 wt% based on the total weight of 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%PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 and about 75 wt%, or the like. In other embodiments, the polymerizable composition may exclude co-monomers, i.e., 0 wt%.

[0150] 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.

[0151] In some embodiments, the polymerizable compositions may exclude co-monomers having a guanidine group.

[0152] 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. In other words, the polymerizable compositions may exclude “Q chemistry” ligand monomers.

[0153] 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.

[0154] Ligating Polymers

[0155] The ligating polymers of the present disclosure may be prepared from any of the polymerizable compositions described herein.

[0156] 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 10 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 10, 15, 20, 25, 30,PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT035, 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.

[0157] 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 molar amount of 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.

[0158] In some embodiments, the ligating polymer may be derived from one or more ligating monomer of Formula la (e.g., Formula lb) and HEMA. The ligating monomer(s) of Formula la and HEMA 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 la and HEMA 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 3:1 to about 1:3, or the like. In some embodiments, the ligating polymer may be derived solely from the ligating monomer(s) of Formula la and HEMA. In other embodiments, the ligating polymer may be derived from the ligating monomer(s) of Formula la, 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 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.

[0159] In some embodiments, the ligating polymer may be derived from one or more ligating monomer of Formula la (e.g., Formula lb) and NVP. The ligating monomer(s) of Formula la and NVP may be present within the ligating polymer at a molar ratio of about 20: 1 to 1 :4. For example, the ligating monomer(s) of Formula la and NVP may be present within the ligating polymer at a molar 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 la and NVP.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0In other embodiments, the ligating polymer may be derived from the ligating monomer(s) of Formula la, 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 total 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.

[0160] In some embodiments, the ligating polymer may be derived from one or more ligating monomer of Formula la (e.g., Formula lb) and DEGMEMA. The ligating monomer(s) of Formula la and DEGMEMA may be present within the ligating polymer at a molar ratio of about 20: 1 to 1 :4. For example, the ligating monomer(s) of Formula la and DEGMEMA may be present within the ligating polymer at a molar 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 la and DEGMEMA. In other embodiments, the ligating polymer may be derived from the ligating monomer(s) of Formula la, 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 total 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.

[0161] 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

[0162] Filter Elements with Ligating Polymers

[0163] 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.

[0164] In many embodiments, the filter element may be characterized by a 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 a binding to full adeno-associated viruses of up toPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 about 25 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, 22, 23, 24, or 25 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 25 mS / cm, full AAV (e.g., full AAV5) may be bound to the filter element allowing for flow- through (bind-elute) separation of full AAVs from 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 5-7 mS / cm less than the binding for full AAVs. For comparison, filter elements based on Q chemistry (i.e., ligating monomers having a trialkyl quaternary ammonium group) typically bind to full AAVs up to only 10 mS / cm, whereas binding for empty AAVs are only 1-2 mS / cm less than that.

[0165] Further details regarding features of the filter element are provided below.

[0166] Porous Substrate

[0167] 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.

[0168] 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.

[0169] 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,PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT01952. 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.

[0170] In some embodiments, the filter element may include a combination of membrane substrate(s) and nonwoven substrate(s). Membranes can have smaller and more narrowly distributed pore sizes than nonwovens, whereas nonwovens can have greater mechanical strength compared to membranes.

[0171] 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.

[0172] 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.

[0173] In some embodiments, the one or more porous substrate may be a polypropylene non woven.

[0174] 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,PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 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. For example, the one or more porous substrates may be characterized by an overall thickness (mm) 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, 10, or a value within a range between any of the preceding values, e.g., between about 0.2 mm and about 0.4 mm, or the like. 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. 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.

[0175] Filter Media

[0176] 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.

[0177] In many embodiments, the filter media may be characterized by an amount of the one or more ligating monomer of Formula I (e.g., Formulae 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 monomerPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 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.

[0178] In some embodiments, the filter media may exclude polymers other than the ligating polymers described herein.

[0179] 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.

[0180] In some embodiments, the filter media may exclude polymers derived from monomers having a guanidine group.

[0181] Separation Processes with Filter Elements having Ligating Polymers

[0182] 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.

[0183] 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 5 mS / cm to about 7 mS / cm on filter elements of the present disclosure. In other words, full AAV may elute at conductivities that are 5 mS / cm to about 7 mS / cm greater than the conductivities at which empty AAV elutes. In some embodiments, at least 75% of the adeno-associated virus containing the DNA payload (i.e., full AAV) from the loading solution may be recovered, e.g., 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 85% and 92%, 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 virusPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 containing a DNA payload may be recovered in an amount of at least 75% at an enrichment of at least 75%.

[0184] AAVs

[0185] 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.

[0186] 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.

[0187] In some embodiments, the DNA payload may be selected from any protein coding gene fragment or a regulatory gene fragment.

[0188] Loading Solutions

[0189] In many embodiments, the loading solution may further include one or more inorganic 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 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.

[0190] 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 200 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, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200, 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.

[0191] 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,PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 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.

[0192] 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.

[0193] 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.

[0194] In some embodiments, the loading solution may further include viruses other than the AAV(s) (e.g., enveloped viruses (e.g., Phi6) or non-enveloped viruses (e.g., SV40 virus)), viral fragments, bacteria, DNA or fragments thereof, host cell proteins, lipids, salts, or a combination thereof. It is not uncommon for prepared samples to be contaminated with other viruses and the like. Obviously, such contaminations will necessitate removal prior to any therapeutic use of full AAVs. It has been demonstrated herein that enveloped viruses, such as Pseudomonas virus “Phi6,” may be effectively separated from full AAVs with the filter elements of the present disclosure. Specifically, it has been shown that Phi6 remains bound to the filter elements of the present disclosure at conductivities of at least up to 40 mS / cm. Any such contaminants that may be present within the loading solution can be effectively removed via the filter elements of the present disclosure.

[0195] 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 in total or a value within a range between any of the preceding values, e.g., between about 1014and about 2*1014AAV capsids, or the like.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0196] 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*1014capsids per ml of filter media.

[0197] 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.

[0198] Conditions for Separation

[0199] 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

[0200] 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 said conductivity. Subsequently, contacted an eluting solution to the filter element such that the full AAV elutes from the filter media.

[0201] 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.

[0202] 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 byPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] Eluting solutions are described in greater detail below.

[0209] Eluting SolutionsPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0210] 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 21 mS / cm and 23 mS / cm, or the like.

[0211] In many embodiments, the eluting solution may include the same or different conductive components as the loading solutions described above. For example, eluting solutions may include salt(s), such as from 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.

[0212] In many embodiments, 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.

[0213] Processes for Preparing Filter Elements

[0214] In various embodiments, a process for preparing a filter element of the present disclosure is described. The process may include providing a polymerizable composition 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.

[0215] 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.

[0216] 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.

[0217] KitsPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0218] 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.

[0219] 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).

[0220] LIST OF EMBODIMENTS1. A compound represented by Formula I:CH2=C(R1)-C(O)-W-R2-X-C(O)-Y-R3-HA+-Q Z (I), wherein:R1is -H or a straight or branched Ci-4 alkyl,W is -O- or -N(R5)-,R2is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene,X and Y are independently selected from a bond, -O-, or -N(R5)-, provided that at least one of X and Y is -O-, or -N(R5)-,R3is a straight or branched C2-C6 alkylene, or a straight or branched C4-C12 alkoxyalkylene,HA+is a charged heteroaryl having 5-9 atoms and 1-3 heteroatoms selected from N, O, and S, provided that at least one heteroatom is a quadrivalent nitrogen,Q is a straight or branched C1-C12 alkyl or -R4-Ar,R4is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene,Ar is aryl,Z is a counterion, and each R5is independently selected from -H and a C1-4 alkyl.2. The compound of embodiment 1, represented by Formula la:CH2=C(R1)-C(O)-W-R2-X-C(O)-Y-R3-HA+-R4-Ar Z (la).3. The compound of any one of the preceding embodiments, wherein W is -O-.4. The compound of any one of the preceding embodiments, wherein W is -N(R5)-.5. The compound of any one of the preceding embodiments, wherein X and Y are each -N(R5)-.6. The compound of any one of embodiments 1-4, wherein X is a bond and Y is -O-.7. The compound of any one of embodiments 1-4, wherein X is a bond and Y is -N(R5a)-.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT08. The compound of any one of embodiments 1-4, wherein X is -O- and Y is a bond.9. The ligating monomer of any one of embodiments 1-3, wherein X is -N(R5)- and Y is -O-.10. The compound of any one of embodiments 1-4, wherein X is -N(R5)- and Yais -O-.11. The compound of any one of embodiments 1-4, wherein X is -O- and Yais -N(R5)-12. The compound of any one of the preceding embodiments, wherein HA+is -N(R6)-R7-N+(R8)-, wherein R6and R8are taken together to form a heteroaryl ring, and R7ais -C(H)=.13. The compound of any one of the preceding embodiments, wherein HA+is an imidazolyl ring.14. The compound of any one of embodiments 1-12, wherein HA+is a benzimidazolyl ring.15. The compound of any one of the preceding embodiments, wherein R4is -CH2-.16. The compound of any one the preceding embodiments, wherein Ar is a Ce, C9, or C10 aryl.17. The compound of any one of the preceding embodiments, wherein Ar is phenyl.18. The compound of embodiment 1, represented by the formula lb:19. The compound of any one of the preceding embodiments, wherein Z is selected from chloride, bromide, tosylate, and tritiate.20. A ligating monomer represented by Formula I: CH2=C(R1)-C(O)-W-R2-X-C(O)-Y-R3-HA+-Q Z (I), wherein:R1is -H or a straight or branched C1-4 alkyl,W is -O- or -N(R5)-,R2is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene,X and Y are independently selected from a bond, -O-, or -N(R5)-, provided that at least one of X and Y is -O-, or -N(R5)-,R3is a straight or branched C2-C6 alkylene, or a straight or branched C4-C12 alkoxyalkylene,PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0HA+is a charged heteroaryl having 5-9 atoms and 1-3 heteroatoms selected from N, O, and S, provided that at least one heteroatom is a quadrivalent nitrogen,Q is a straight or branched C1-C12 alkyl or -R4-Ar,R4is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene,Ar is aryl,Z is a counterion, and each R5is independently selected from -H and a C1-4 alkyl.21. The ligating monomer of embodiment 20, represented by Formula la:CH2=C(R1)-C(O)-W-R2-X-C(O)-Y-R3-HA+-R4-Ar Z (la).22. The ligating monomer of any one of the preceding embodiments, wherein W is -O-.23. The ligating monomer of any one of the preceding embodiments, wherein W is -N(R5)-.24. The ligating monomer of any one of the preceding embodiments, wherein X and Y are each -N(R5)-.25. The ligating monomer of any one of embodiments 20-24, wherein X is a bond and Y is -O-.26. The ligating monomer of any one of embodiments 20-24, wherein X is a bond and Y is -N(R5a)-.27. The ligating monomer of any one of embodiments 20-24, wherein X is -O- and Y is a bond.28. The ligating monomer of any one of embodiments 20-23, wherein X is -N(R5)- and Y is -O-.29. The ligating monomer of any one of embodiments 20-24, wherein X is -N(R5)- and Yais -O-.30. The ligating monomer of any one of embodiments 20-24, wherein X is -O- and Yais - N(R5)-.31. The ligating monomer of any one of the preceding embodiments, wherein HA+is -N(R6)-R7-N+(R8)-, wherein R6and R8are taken together to form a heteroaryl ring, and R7ais -C(H)=.32. The ligating monomer of any one of the preceding embodiments, wherein HA+is an imidazolyl ring.33. The ligating monomer of any one of embodiments 20-32, wherein HA+is a benzimidazolyl ring.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT034. The ligating monomer of any one of the preceding embodiments, wherein R4is -CH2-.35. The ligating monomer of any one the preceding embodiments, wherein Ar is a Ce, C9, or C10 aryl.36. The ligating monomer of any one of the preceding embodiments, wherein Ar is phenyl.37. The ligating monomer of embodiment 20, represented by the formula lb:38. The ligating monomer of any one of the preceding embodiments, wherein Z is selected from chloride, bromide, tosylate, and tritiate.39. A polymerizable composition comprising: one or more ligating monomer of any one of embodiments 20-38, and optionally one or more co-monomer.40. A ligating polymer derived from: one or more ligating monomer of any one of embodiments 20-38.41. A ligating polymer derived from a polymerizable composition of embodiment 39.42. The ligating polymer of any one of embodiments 40-41, further being derived from one or more co-monomer characterized by octanol-water partition coefficient of about -4 to about 4.43. The ligating polymer of any one of embodiments 40-42, further being derived from one or more co-monomer characterized by a water solubility of at least 5 g per L at 25 °C.44. The ligating polymer of any one of embodiments 40-43, 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.45. The ligating polymer of any one of embodiments 40-44, 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.46. The ligating polymer of any one of embodiments 40-45, 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.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT047. The ligating polymer of any one of embodiments 40-46, further being derived from at least one (meth)acrylate monomer having one or more hydroxyl groups.48. The ligating polymer of any one of embodiments 40-47, further being derived from at least one (meth)acrylate monomer having one or more glycerol groups.49. The ligating polymer of any one of embodiments 40-48, further being derived from a comonomer selected from A-vinyl pyrrolidone, hydroxyethyl methacrylate, di(ethylene glycol) methyl ether methacrylate, and a combination thereof.50. The ligating polymer of any one of embodiments 40-49, further being derived from a comonomer selected from glycerol (meth)acrylate, glycerol di(meth)acrylate, and a combination thereof.51. The ligating polymer of any one of embodiments 40-50, 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 mol%.52. The ligating polymer of any one of embodiments 40-51, excluding co-monomers characterized by water solubility of less than 100 g per L at 25 °C, an octanol -water partition coefficient of greater than 4, or a combination thereof.53. The ligating polymer of any one of embodiments 40-52, excluding co-monomers comprising a quaternary ammonium trialkyl groups of formula: -N+(Rq)3, wherein each Rqis independently a C1-C20 alkyl group.54. The ligating polymer of any one of embodiments 40-53, excluding co-monomers comprising a guanidine group.55. The ligating polymer of any one of embodiments 40-54, wherein the one or more ligating monomer is present within the ligating polymer in an amount of at least 10 mol%.56. The ligating polymer of any one of embodiments 40-55, wherein the ligating monomer of Formula I accounts for about 10 mol% to about 100 mol% of the ligating polymer.57. The ligating polymer of any one of embodiments 40-55, wherein the ligating monomer of Formula I accounts for about 10 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 90 mol% of the ligating polymer58. The ligating polymer of any one of embodiments 40-55, wherein the ligating monomer of Formula lb accounts for about 10 mol% to about 100 mol% of the ligating polymer.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT059. The ligating polymer of any one of embodiments 40-55, wherein the ligating monomer of Formula lb accounts for about 10 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 90 mol% of the ligating polymer.60. 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 20-38, wherein the ligating polymer is grafted to each of the one or more porous substrate.61. 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 40-59, wherein the ligating polymer is grafted to each of the one or more porous substrate.62. The filter element of any one of embodiments 60-61, the one or more porous substrate characterized by an average pore size of about 0.1 pm - 10 pm.63. The filter element of any one of embodiments 60-62, the one or more porous substrate having fibers characterized by a fiber diameter of about 0.5 micrometers to about 15 micrometers.64. The filter element of any one of embodiments 60-63, 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.65. The filter element of any one of embodiments 60-64, the one or more porous substrate comprised of material selected from nylon and polypropylene.66. The filter element of any one of embodiments 60-65, the one or more porous substrate in the form of a membrane or a nonwoven.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT067. The filter element of any one of embodiments 60-66, 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.68. The filter element of any one of embodiments 60-67, 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.69. The filter element of any one of embodiments 60-68, characterized by a binding for the adeno-associated virus containing a DNA payload at a conductivity value of about 10 mS / cm to about 25 mS / cm.70. The filter element of any one of embodiments 60-69, characterized by a binding for the adeno-associated virus containing a DNA payload at a conductivity that is 5-7 mS / cm higher than binding for an adeno-associated virus without a DNA payload.71. The filter element of any one of embodiments 60-70, the filter media excluding polymers other than a ligating polymer of any one of embodiments 21-40.72. The filter element of any one of embodiments 41-52, 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.73. 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 41-53; 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.74. The process of any one of embodiments 54, 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 6.75. The process of any one of embodiments 54-55, wherein the DNA payload is a sequence effective to treat an inherited genetic disease.76. The process of any one of embodiments 54-56, wherein the DNA payload is a sequence effective to treat hemophilia A, hemophilia B, muscular dystrophy, AADC deficiency, lipoprotein lipase deficiency, and retinal dystrophy.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT077. The process of any one of embodiments 54-57, the loading solution further comprising one or more water soluble inorganic salt.78. The process of any one of embodiments 54-58, the loading solution characterized by a conductivity of about 5 mS / cm to about 15 mS / cm.79. The process of any one of embodiments 54-59, the loading solution characterized by an inorganic salt concentration of about 50 mM to about 200 mM.80. The process of any one of embodiments 54-60, the loading solution characterized by a pH of about 8.5 to about 10.81. The process of any one of embodiments 54-61, 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.82. The process of any one of embodiments 54-62, the loading solution further comprising one or more enveloped virus.83. The process of any one of embodiments 54-63, the loading solution further comprising one or more non-enveloped virus.84. The process of any one of embodiments 54-64, 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.85. The process of any one of embodiments 54-65, 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.86. The process of any one of embodiments 54-66, 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 5 mS / cm to about 7 mS / cm.87. The process of any one of embodiments 54-67, wherein the conditions are effective to recover the adeno-associated virus containing a DNA payload in an amount of at least 75 %.88. The process of any one of embodiments 54-68, wherein the conditions are effective to recover the adeno-associated virus containing a DNA payload at an enrichment of at least 75 %.89. The process of any one of embodiments 54-69, wherein the conditions are effective recover the adeno-associated virus containing a DNA payload in an amount of at least 75% at an enrichment of at least 75%.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT090. A process for preparing a filter element of any one of embodiments 60-72, the process comprising: providing a polymerizable composition of embodiment 39; providing a porous substrate; contacting the polymerizable composition to the porous substrate under conditions effective to polymerize the polymerizable composition onto the porous substrate91. A process for preparing a filter element of any one of embodiments 60-72, the process comprising: providing a ligating polymer of any one of embodiments 40-59; 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.92. The process of any one of embodiments 90-91, the conditions comprising ionizing irradiation.93. The process of embodiment 92, wherein said irradiation is chosen from e-beam irradiation, X-ray irradiation, or gamma irradiation.94. The process of embodiment 93, wherein said irradiation is e-beam irradiation.95. The process of any one of embodiments 90-94, the conditions comprising e-beam irradiation at a dose of about 6 MRad to about 10 MRad.96. The process of any one of embodiments 90-95, the conditions comprising UV irradiation.97. A kit comprising: a filter element of any one of embodiments 60-72; and a set of instructions for separating adeno-associated virus containing a DNA payload from adeno-associated virus without a DNA payload.

[0221] EXAMPLES

[0222] The following examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, described herein.

[0223] Example 1. Sample functionalization procedure for nonwoven and / or membrane layers (e-beam)

[0224] 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 withPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 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.

[0225] 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.

[0226] Washing and Drying: Grafted substrates are transferred to a stainless-steel pot of boiling deionized water (only for e-beam grafted samples) 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.

[0227] 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

[0228] Example 2. Sample functionalization procedure for nonwoven and / or membrane layers (UV)

[0229] 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 microlitersPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 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.

[0230] Example 3. Production and purification of AAV using transient transfection process

[0231] HEK293-F cells (R79007, Thermo Fisher Scientific) suspended in LVmax cell culture / growth media (A3583401, Thermo Fisher Scientific) were grown in an incubator using 2.8 L shaker flasks. The incubator was maintained at 37 °C with 8% CO2. When the cell density reached approximately 2 x 106cells / mL, a transfection cocktail was prepared and administered to the shaker flask. The transfection cocktail consisted of three plasmids namely pAAV- ZsGreenl (6231, Takara Bio), pALD-X80 (5017-10, Aldeveron), pALD-AAVx (Aldeveron) and the transfection reagent FectoVIR-AAV (101000022, Polyplus Transfection). X in pALD- AAVx is the sequence for AAV2 (5057-10, Aldeveron) or AAV5 (5058-10, Aldeveron) serotypes. Transfection cocktail is prepared by adding equimolar amounts of all three plasmids and the total plasmid amount was adjusted to a pg of plasmid mixture per million cells used for transfection. Later, the 5% v / v of DMEM (10569010, Thermo Fisher Scientific) is added to the plasmid mixture (v / v calculations are based on total cell culture volume) and mixed well.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0After addition of DMEM, for every pg of plasmid used in transfection cocktail, 1 pl of FectoVIR-AAV transfection reagent is added to the cocktail and mixed gently by pipetting and the cocktail is incubated at room temperature for 45 min. After incubation, the transfection cocktail is once again mixed gently by pipetting and added dropwise to the cell culture. After addition of the transfection cocktail, cells are grown in the 37 °C incubator for 72 h. After 72 h, the cells were lysed with 0.1% Triton-X 100 (X100, Millipore Sigma) for 2 h. 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 (EMP301HRC2FA, 3M). After clarification, the sample was applied to a POROS AAVX affinity column (A36651, Thermo Fisher Scientific) and washed with three different buffers of 25 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, 25 mS / cm buffer and immediately neutralized with Tris-Cl buffer. The buffer was then exchanged into lx PBS (10010001, Thermo Fisher Scientific) using dialysis cassettes (66003, Thermo Fisher Scientific).

[0232] To produce empty AAV viruses, a similar strategy of transfection and purification was performed. One exception was that instead of a three plasmids transfection cocktail, 2 plasmids were used. The plasmid encoding the viral genome, pAAV-ZsGreenl was removed from the transfection cocktail.

[0233] Example 4. Production and purification of rAAV using the TESSA system

[0234] 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 usingPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT05 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.

[0235] Example 5. Total AAV capsid estimation using AAV ELISA

[0236] AAV2 and AAV5 capsid estimations were performed using the AAV2 (PRAAV2XP, Progen) and AAV5 Xpress ELISA kits (PRAAV5XP, Progen) respectively as per manufacturers instruction.

[0237] Example 6. Genome copy estimation using qPCR

[0238] 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 finally held at 4 °C until qPCR was performed.

[0239] 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 pipetted 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.

[0240] 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 vivoPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 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 AriaMx 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).

[0241] Example 7. Calculation of full percentages using ELISA and qPCR

[0242] 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.

[0243] Example 8. Stunner based measurements of AAV full percentages

[0244] Empty / full percentages of AAV viruses were measured using the Stunner Instrument (Unchained Labs, CA) according to the protocol provided by the instrument manufacturer.

[0245] Example 9. Size Exclusion Chromatography-Multi-angle Light scattering (“SEC- MALS”)

[0246] Two important attributes in evaluating the quality of an adeno-associated virus (AAV) sample are the total AAV particle concentration (Cp) and the empty / full ratio (Vg / Cp). These attributes can be efficiently determined using methodology developed around SEC- MALS analytical measurements as described in Michelle Chen, Ph.D. and Anatolii Purchel; AN 1617: “Quantifying quality attributes of AAV gene therapy vectors by SEC-UV-MALS- dRI”, Wyatt Technology Application Note and Nicole L. McIntosh, et.al.; “Comprehensive characterization and quantification of adeno associated vectors by size exclusion chromatography and multi angle light scattering” ; Scientific Reports (2021), 11:3012, each of which are incorporated herein by reference. The SEC column separates the monomeric AAV capsids from the larger capsid agglomerates as well as the smaller nucleotide impurities and buffer components. With the use of two concentration detectors (dRI and / or UV_280nm, and / or UV_260nm), particle concentration and relative capsid content can be determined along with other quality characteristics of the AAV sample.

[0247] SEC-MALS Chromatography: In this work, the AAV particle concentration (Cp) and the empty / full ratio (Vg / Cp) measurements were conducted using an Agilent Technologies 1260 Infinity II liquid chromatography instrument with the data processed using Wyatt Technologies Astra 8 software. The LC system used lx phosphate buffered saline (PBS)PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 aqueous mobile phase at 0.3 ml\min, and a Sepax Technology Inc. SRT SEC-1000 (300 mm x 4.6 mm i.d.). The detectors attached to the system consisted of an Agilent 1260 Infinity II DAD WR, Wyatt DAWN MALS-QELS, and Wyatt Optilab dRI / aRI. The document “SOP Guidance Manual; Critical Quality Attributes of AAV by SEC-MALS” MG1000 Rev. B; Wyatt Technology Corporation; Copyright 2021, which is incorporated herein by reference, was followed in determining and defining the extinction coefficients and serotype specific material parameters. Empty and partially full AAV5 samples obtained using “Production and purification of rAAV using the TESSA system” mentioned above were used to determine the protein and DNA extinction coefficients that were then used in the Astra 8 software to determine Cpand Vg / Cp. It should be noted that for different serotypes and mutated capsid proteins, the extinction coefficients of the corresponding capsid may vary slightly and need to be determined for that specific serotype. To determine total AAV (Cp) concentration with this system, two concentration sources are needed. Generally, the UV signals at 260 nm and 280 nm are used; alternatively, the dRI signal could be used in place of either UV signal. The capsid concentration is calculated via ASTRA 8 software using the two concentration signals and expected molar masses of the protein capsid and nucleic acid payload. The following equations further describe the calculations.CAAV = mpx NA / (MCapsid x v) where:CAAV = concentration of total AAV capsidsNA = Avogadro’s number mp= total protein eluted Mcapsid = molar mass of viral capsid v = injected volume andCfuii = mDNA x NA / (Mfuii x v) where:Cfuii = concentration of full AAV capsidsNA = Avogadro’s number mDNA = total DNA elutedMfuii = molar mass of full-length DNA v = injected volume such that:PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0Cempty = CAAV—Cfull where:Cempty = concentration of empty AAV capsidsCfuii = concentration of full AAV capsidsCAAV = concentration of total AAV capsid

[0248] Example 10. Plaque assay to quantitate Phi6 phage

[0249] Preparation of Pseudomonas syringe soft agar overlay plates: A soft agar overlay containing Pseudomonas syringae (DSMZ 21482) host bacteria are used for propagation and harvest of Phi6 virus, as well as for quantification of virus by plaque assay. Soft agar is prepared by autoclaving IX Tryptic Soy Broth (TSB) with 1.5% agar and autoclaving to sterilize. The sterilized molten soft agar is then cooled to 38 °C - 40 °C for use. To prepare the overlay, overnight culture of Pseudomonas syringae is added to soft agar along with MgSCU at the following ratio:5 mL soft agar / 50 pL overnight culture / 25uL IM MgSC . The molten mixture of bacteria is then poured over a standard TSA agar plate (TSB Broth + 3% agar plate).

[0250] Preparation of Phi6 virus stock: Phi6 bacteriophage (DSMZ 21518) was obtained from the DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. A volume of 50-100 pL of Phi6 virus containing stock of spread over a Pseudomonas syringae soft agar overlay plate. The plate is then incubated overnight at room temperature. The next day, the plate should be clear in appearance (indicating lysis of the host bacteria). The produced virus is harvested from the agar by scraping the layer of top ager from the plates into a 50 mL conical tube, adding 20 mL of phage storage buffer (50 mM NaHPCU, 22 mM KH2PO4, 85.5 mM NaCl, 1 mM MgSCU, 1 mM CaCh) and vortexing for 15-30 min to release the virus into the buffer. The mixture is then centrifuged at 3000 x g for 15 min and filtered through a 0.2 pm PES membrane.

[0251] Determination of Phi6 virus concentration by surface spotting method: To enumerate Phi6 in a sample, each sample was serially diluted (10-fold) in a 96 well plate down to a 100,000X dilution in PBS. 3 pL of each dilution was then spotted onto the surface of a pseudomonas syringe soft agar overlay plate (described above) in an array. The spotted plates were incubated overnight at 25 °C. Following incubation, the virus was quantified by counting particle forming units in the 3 pL spot at the highest dilution where plaques were countable. The virus particle concentration (particles / mL) was calculated from the pfu count adjusted for dilution.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0252] Example 11. AAV Infectious titer assay

[0253] HT1080 cells (# CCL-121, ATCC) were maintained in a maintenance medium consisting of Eagle’s Minimum Essential Medium (# 10009CV, Coming) supplemented with 10% heat-inactivated fetal bovine serum (# 16140089, Gibco) and IX penicillin-streptomycin (# 15140122, Gibco) in a humidified incubator at 37 °C and 8% CO2. To determine AAV infectious titer, HT1080 cells were seeded in 96-well, CellBIND-treated, flat-bottom microplates with black walls and clear bottoms (# 10-009-CV, Coming) at a density of 1 x 104viable cells in 100 pL medium per well and incubated 2-4 h to allow cells to attach. Once cells were attached, 10-fold serial dilutions of vims samples were prepared in triplicate in maintenance medium. AAV2-GFP (# 7004, Vector Biolabs) served as a positive control for viral infection, while the diluent alone served as a negative control for viral infection. The culture medium was removed from the wells of the 96-well plates seeded with HT1080 cells and replaced with 100 pL of vims sample dilutions. The plates were then incubated as described above for three days. Brightfield and GFP fluorescence images of the assay plate wells were acquired with the BiokTek Cytation5 Multimode Imaging Reader (Agilent) using a 4X objective. The percentage of GFP+ cells, as determined by dividing the GFP+ cell count by the cell count in the image, was then used to calculate the infectious titer of the vims sample in transducing units per milliliter (TU / mL) by the following equation:Titer = (F*C / V)*D where:F is the frequency of GFP+ cells (percentage GFP+ cells / 100)]C is the cell number at the time of transduction (1 x 104cells)V is the volume of the inoculum in mL (0.1 mL)D is the viral dilution factor

[0254] Only wells with 1-20% GFP+ cells were used in viral titer calculation. If multiple dilutions from the same dilution series resulted in 1-20% GFP+ cells, the titers determined from those wells were averaged and reported as the titer from that dilution series. The final vims sample titer was then calculated as the average of the three titers determined for the replicate dilution series prepared for that sample.

[0255] Example 12. Computational Modeling

[0256] Molecular docking was performed using the InducedFit Docking Module licensed by Schrodinger LLC (2023-2 release). Ligating monomer of Formula I and Q-Chemistry pentamers were docked to a receptor grid centered in the 5-fold symmetry pore region of AAV5PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0(PDB: 7KP3). Select poses were first docked using the Glide Standard Precision (SP) module using the OPLS2005 forcefield modulated with a receptor van der Waals scaling of 0.5 and a ligand van der Waals scaling of 0.5. Protein residues within 5 ang of the ligands were then optimized by the Prime module (Schrodinger, 2023-2 release) before being redocked with Glide extra precision (XP). The lowest energy poses were then reported using the Induced FitDocking score and visualized using Maestro (Schrodinger LLC, 2023-2).Table 1. Materials Table.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0257] Example 12. Ligand Synthesis

[0258] Preparatory Example 1 : 2-[3-(3-benzylimidazol-l-yl)propylcarbamoylamino]ethyl 2- methylprop-2-enoate bromide (“IEM-BIB”)

[0259] A-(3-aminopropyl)imidazole (25.0 g, 0.2 mol) was dissolved in 100 mL dichloromethane and cooled in an ice-water bath with magnetic stirring and a slow nitrogen sweep. Isocyanatoethyl methacrylate (31.0 g, 0.2 mol) dissolved in dichloromethane (50 mL) and then added dropwise to the reaction flask over a 30 min period. The reaction was stirred for 30 min, at which time NMR analysis indicated that the reaction was complete. Benzyl bromide (34.21 g, 0.2 mol) was then added dropwise to the reaction flask over a 10 min period and the reaction was allowed to come to room temperature and then refluxed overnight. The mixture was concentrated on a rotary evaporator to give a viscous syrup. Deionized water (100 mL) and 200 pL of a 10,000 ppm stock solution of 4-hydroxy TEMPO in deionized water was added to the mixture, and the mixture was further concentrated on the rotary evaporator until the solution became homogeneous. Percent solids (32.3% solids) was determined using an Ohaus moisture balance (Model Number MB35, obtained from the Ohaus Corporation, Parsippany, NJ).

[0260] 'H NMR (500 MHz, D2O) 8 1.67 (s, 3H), 1.85 (p, 2H), 2.95 (t, 2H), 3.20 (t, 2H), 3.97 (t, 2H), 4.03 (t, 2H), 5.19 (s, 2H), 5.47 (s, 1H) , 5.90 (s, 1H), 7.25 (m, 5H), 7.32 (s, 1H), 7.34 (s, 1H), 8.68 (s, 1H).PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0261] Preparatory Example 2: 2-Isocyanatoethyl Methacryl ate-Methyl Imidazolium Methylsulfate (lEM-Methyl Imidazolium Methyl sulfate, ZEM-MelMS)

[0262] Prepared according to Preparatory Example 1, replacing benzyl bromide with dimethyl sulfate.

[0263] Preparatory Example 3: 2-Isocyanatoethyl Methacryl ate-Butyl Imidazolium Butylsulfate (lEM-Butyl Imidazolium Butylsulfate, IEM-BUIBS)

[0264] Prepared according to Preparatory Example 1, replacing benzyl bromide with dibutyl sulfate.

[0265] Preparatory Example 4: A-[3-(3-benzylimidazol-l-yl)propyl]-2-methyl-2-(prop-2- enoylamino)propanamide;bromide (“VDM-BIB”)

[0266] A-f3-aminopropyl)imidazole (6.26 g, 0.05 mol) was dissolved in 40 mL dichloromethane and cooled in an ice-water bath with magnetic stirring and a slow nitrogen sweep. Vinyldimethylazlactone (6.95 g, 0.05 mol) was dissolved in di chloromethane (10 mL) and then added to the reaction flask. The reaction was stirred for 4 hours, at which time NMR analysis indicated that the reaction was complete. Benzyl bromide (8.55 g, 0.05 mol) was then added to the reaction flask and the reaction was allowed to come to room temperature and then stirred overnight. The mixture was concentrated on a rotary evaporator to give a viscous syrup. Deionized water (40 mL) and 100 pL of a 10,000 ppm stock solution of 4-hydroxy TEMPO in deionized water was added to the mixture, and the mixture was further concentrated on the rotary evaporator until the solution became homogeneous. Percent solids (35.9% solids) wasPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 determined using an Ohaus moisture balance (Model Number MB35, obtained from the Ohaus Corporation, Parsippany, NJ).

[0267] JHNMR (500 MHz, D2O) 8 1.66 (s, 6H), 2.27 (m, 2H), 3.41 (m, 2H), 4.40 (m, 2H), 5.58 (s, 2H), 5.95 (br. d, 1H), 6.35 (br. d, 1H), 6.50 (m, 1H), 7.66 (m, 6H), 7.73 (br. s, 1H), 9.00 (br. s, 1H).

[0268] Preparatory Example 5: 2-[(l-benzyl-3-pyridyl)methylcarbamoylamino]ethyl 2- methylprop-2-enoate;bromide (“IEM-BPyB”)

[0269] 3 -Aminomethylpyridine (5.41 g, 0.05 mol) was dissolved in 50 mL dichloromethane and cooled in an ice-water bath with magnetic stirring and a slow nitrogen sweep. IEM (7.75 g, 0.05 mol) was then added to the reaction flask. The reaction was stirred for 4 hours, at which time NMR analysis indicated that the reaction was complete. Benzyl bromide (8.55 g, 0.05 mol) was then added to the reaction flask and the reaction was allowed to come to room temperature and then stirred overnight. The mixture was concentrated on a rotary evaporator to give a viscous syrup. Deionized water (40 mL) and 100 pL of a 10,000 ppm stock solution of 4-hydroxy TEMPO in deionized water was added to the mixture, and the mixture was further concentrated on the rotary evaporator until the solution became homogeneous. Percent solids (34.9% solids) was determined using an Ohaus moisture balance (Model Number MB35, obtained from the Ohaus Corporation, Parsippany, NJ).

[0270] 'H NMR (500 MHz, D2O) 6 1.63 (s, 3H), 2.08 (t, 2H), 3.97 (t, 2H), 4.30 (s, 2H), 5.43 (s, 1H), 5.61 (s, 2H), 5.84 (s, 1H), 7.28 (m, 5H), 7.83 (m, 1H), 8.25 (d, 1H), 8.62 (s, 1H), 8.66 (d, 1H).

[0271] Example 13. IEM-BIB FILTER MEDIA SALT TOLERANCE WITH AAV5

[0272] Filter Media FM1 and comparative filter media CFM1-CFM3 were grafted onto a 0.8-micron nylon membrane by either UV or e-beam. The resulting filter elements are represented in Table 2.

[0273] Table 2. Filter Media “FM” for Filter element 1 and Comparative Filter Elements 1-3PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0274] Small circular discs of 5 mm diameter were punched out of each material listed in Table 2 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).

[0275] 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.

[0276] Table 2A. Filter Elements illustrating that IEM Benzyl Imidazolium Bromide (FM1) filter media is more salt tolerant than IEM alkyl imidazolium sulfates (CFM2 andCFM3) and MAPTAC Q Chemistry (CFM1).PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0277] The percentage of virus (i.e., total AAV) in the flow through compared to the Feed AAV is listed in Table 2 and graphically shown in FIG. 1 A. The results indicate that IEM-BIB binds to AAV5 and there is no AAV5 in flow through at any of the tested conductivities, up to and including the 200 mM NaCl condition. Conversely, lEM-MelMS, IEM-BUIBS, andMAPTAC display complete binding to AAV5 up until 100 mM NaCl and lets AAV5 flow through the material at 150 mM NaCl and 200 mM NaCl. This result indicates that the methyl and butyl version of imidazolium bromide ligands perform similarly to MAPTAC (Q chemistry), and it is the benzyl version of imidazolium bromide that displays salt tolerant binding to AAV5 at least up to 200 mM NaCl (22 mS / cM). In other words, IEM-BIB is a more salt tolerant ligand for binding to AAV at pH 9.0 than the lEM-MelMS, IEM-BuIBS, and MAPTAC (standard quaternary ammonium chemistry).

[0278] FIG. IB is a computational model illustrating an IEM-BIB pentamer docking to the 5-fold symmetry pore of AAV5. FIG. 1C is a computational model illustrating a MAPTAC pentamer docking to the 5-fold symmetry pore of AAV5. Details of the computational modeling method are provided above. The IEM-BIB pentamer was shown to have a stronger binding energy (Induced Fit Docking Score: -5477.54) with the 5-fold symmetry region of thePCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0AAV5 capsid than did MAPTAC (Induced Fit Docking Score: -5461.72). As can be seen in FIG. IB, IEM-BIB accesses a deeper region of the 5-fold symmetry pore and can mediate hydrophobic interactions within the capsid structure. Comparatively, interactions between the MAPTAC (Q-chemistry) and AAV5 are shallow, as seen in FIG. 1C. The resulting structural arrangements of ligand and receptor provide a rationalization for IEM-BIB’ s enhanced binding to AAV (both empty and full) at higher salt concentrations than the Q chemistry. Additionally, is well known that DNA, when present, is inserted into the full capsid via the 5-fold symmetry pore. Thus, it is further speculated that IEM-BIB interacts with the DNA in the depths of the 5-fold symmetry pore, which may explain the greater salt tolerance of full AAV with IEM-BIB compared to empty AAV, as demonstrated below.

[0279] Example 14. EMPTY / FULL AAV5 SEPARATIONS WITH IEM-BIB FILTER MEDIA

[0280] Filter Media FM2 (25 mol% IEM-BIB, 75 mol% HEMA) was grafted via e-beam onto a BA080 membrane and the resulting filter element is described in Table 3. Filter element in Table 3 is employed in the examples below.

[0281] Table 3. Filter Element 2

[0282] Example 15. Evaluation of Virus Binding and Elution Conductivity

[0283] The filter element in Table 3 was attached to an AKTA Avant 150 purification system and equilibrated with 20 column volumes (CV) of buffer E (50 mM Tris- Acetate, pH 9.0, 2 mM MgCh, 100 mM sodium chloride). An AAV5 virus sample, comprising a mix of empty and full viruses, was loaded onto the filter element using buffer E (conductivity of about 11.35 mS / cm) at a flow rate of 3 CV / min. After loading, a wash step was performed with buffer E to wash off any unbound viruses. Elution was performed via step gradients by mixing varying percentages of buffer E with buffer F (50 mM Tris- Acetate, pH 9.0, 2 mM MgCh, 400 mMPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 sodium chloride) at a constant flow rate of 10 CV / min. Elution of AAV5s began at a conductivity of 17 mS / cm and continued until 26 mS / cm, with AAV5 appearing across 4 fractions (E2, E3, E4 and E5).

[0284] FIG. 2A is a separation chromatogram illustrating the elution profile of AAV5 (indicated by OD 280 and 260 on the left y axis) across various salt concentrations (indicated by conductivity on the right y axis). Total AAV5 in each fraction is measured by ELISA (see methods) and the full percentages for each fraction were measured via SEC-MALS (see methods). The elution data is tabulated in Table 2A.

[0285] Table 3A. Empty full AAV separations on filter element described in Table 3

[0286] The data demonstrates that AAV5 largely remains bound to the filter element until a conductivity of about 17 mS / cm and past 17 mS / cm, empty AAV preferentially elutes off the filter media in the E2 fraction with a full percentage of just 2.3%. In the E3 fraction, the full percentage was measured to be 64.8% and in the E4 fraction, full percentage was 104%. While E2 is comprised majorly of empty AAV, and E4 fraction with full AAV5, the E3 fraction was found to be a mix of empty and full, with a full percentage of 64.8. We hypothesized that by altering the conductivities, we could aim to collect empty and full AAVs in two separate fractions. This was the premise for Example 2B below.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0287] Example 16. Simple two-step separation of Empty and Full AAV5

[0288] AAV5 virus was produced and purified with the TESSA system was estimated to be about 37% full by SEC-MALS (see methods). A single layer of filter element of Table 2 was loaded onto a column holder and attached to the AKTA Avant 150 purification system. A total of 3.8E+13 AAV5 was diluted into loading buffer E and used as feed. The filter element was first equilibrated with 20 CV of loading buffer E at 10 CV / min and then the AAV5 feed was applied to the column at a flow rate of 3 CV / min with the aim of binding all viruses. After sample application, the membrane was washed with 30 CV of loading buffer E at 10 CV / min and viruses in El fraction were eluted by buffer G which comprised of 69% of buffer E and 31% of buffer F. The conductivity of the buffer G was ~21 mS / cm. Later, full viruses were preferentially eluted in E2 fraction with buffer H comprising of 50% of buffer E and 50% of buffer F with a conductivity of ~26 mS / cm. A strip step of buffer F was used to collect bound AAVs (E3 fraction) at the end.

[0289] FIG. 2B is a separation chromatogram illustrating the elution profile of AAV5 (indicated by OD 280 and 260 on the left y axis) across various salt concentrations (indicated by conductivity on the right y axis). Total AAV5 in each fraction is measured by ELISA (see methods) and the full percentages for each fraction were measured via SEC-MALS (see methods). The elution data is tabulated in Table 3B.

[0290] Table 3B. Simple two-step separation empty and full AAV5 using filter element 2

[0291] Example 17. Flowing though the empty AAV and separating full AAVPCT Application Applicant Ref PA200036 Atty Dkt. No.: THF-080PCT0

[0292] AAV5 virus was produced and purified with the TESSA system was estimated to be about 40% full by SEC-MALS (see methods). A single layer of filter element of Table 3 was loaded onto a column holder and attached to the AKTA Avant 150 purification system. A total of 4E+13 AAV5 was diluted into loading buffer G with a conductivity of ~21 mS / cm and used as feed. The membrane was first equilibrated with 20 CV of loading buffer at 10 CV / min and then the AAV5 feed was applied to the column at 3 CV / min with the aim of binding full viruses and allowing empty viruses to flow through the membrane. After sample application, the membrane was washed with 30 CV of loading buffer G at 10 CV / min and then bound virus was eluted with buffer H. A strip step with buffer F was used to collect bound AAVs at the end.

[0293] FIG. 2C is a separation chromatogram illustrating the elution profile of AAV5 (indicated by OD 280 and 260 on the left y axis) across various salt concentrations (indicated by conductivity on the right y axis). Total AAV5 in each fraction is measured by ELISA (see methods) and the full percentages for each fraction were measured via SEC-MALS (see methods). The elution data is tabulated in Table 3C.

[0294] Table 3C. Flowing though the empty AAV and separating full AAV using filter element2PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0295] The results show that the full percentage of flow through was lower than the feed full percentage and the elution fraction showed an enrichment in the percentage of full virus from 40% to 103%.

[0296] Example 18. Effect of magnesium chloride concentration on Empty Full Separations

[0297] Data in the examples was performed with 2mM MgCh. There is literature evidence that with increase in MgCh concentrations, there is an increase in AAV full percentages when separations are performed with quaternary ammonium chemistry. Here, we evaluated the effect of MgCh concentrations on the AAV full percentages obtained with IEM-BIB chemistry.

[0298] A single layer of filter element in Table 3 was attached to an AKTA Avant 150 purification system and equilibrated at 10 CV / min with 20 CV of buffer I (50 mM Tris-Acetate, pH 9.0, 5 mM MgCh, 100 mM sodium chloride) in Run 1, buffer J (50 mM Tris-Acetate, pH 9.0, 10 mM MgCh, 100 mM sodium chloride) in Run 2, or buffer K (50 mM Tris-Acetate, pH 9.0, 15 mM MgCh, 100 mM sodium chloride) in Run 3. An AAV5 virus sample comprising a mix of empty and full viruses was loaded onto the filter element at 3 CV / min using buffer I (loading conductivity 13 mS / cm) in Run 1, using buffer J (loading conductivity 13.47 mS / cm) in Run 2, and using buffer K (loading conductivity 14.75 mS / cm) in Run 3. After collecting the flow through fraction, a wash step was performed with the respective loading buffers at a flow rate of 10 CV / min to wash off any unbound viruses. Elution was then performed via step gradients at a flow rate of 3 CV / min by mixing varying percentages of buffer I with buffer L (50 mM Tris-Acetate, pH 9.0, 10 mM MgCh, 400 mM sodium chloride) in Run 1; buffer J with buffer M (50 mM Tris-Acetate, pH 9.0, 15 mM MgCh, 400 mM sodium chloride) in Run 2; and buffer K with buffer N ( 50 mM Tris-Acetate, pH 9.0, 10 mM MgCh, 400 mM sodium chloride) in Run 3. The elution fractions were measured via SEC-MALS to determine the percentage of full virus in each fraction. In addition, the total AAV5 recovery across the entire process was measured via ELISA. The results are represented in Tables 3D-1, 3D-2 and 3D-3 and the chromatogram is represented in FIG. 2D-1, FIG. 2D-2, and FIG. 2D-3, respectively.

[0299] Table 3D-1. Empty full separation of AAV5 using filter element 2 described in Table 3 with 5 mM MgCh concentrations.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0300] Table 3D-2. Empty full separation of AAV5 using filter element 2 described in Table3 with 10 mM MgCh concentrations.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0301] Table 3D-3. Empty full separation of AAV5 using filter element described 2 in Table3 with 15 mM MgCh concentrations.

[0302] The results show that there was no breakthrough of full viruses in the flow through fractions up until when MgCh concentration was increased to 15 mM in Run 3. With increasingMgCh concentrations, earlier onset of elution was observed, and this was consistent with the increased conductivity of the solution. Further, a trend of reduced total recovery was observed with 15mM MgCh concentrations, which may indicate enhanced hydrophobic interactions between the filter media and the virus that could be potentially mediated by Mg2+ions. Nevertheless, no significant trend of increasing AAV full % with increase in Mg2+ion concentration was observed with filter element in Table 3 indicating that a 2 mM Mg2+is sufficient to obtain high AAV full percentages.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0303] Example 19. Estimation of total capacity of filter media

[0304] Examples 15-18 had total AAV5 recoveries from 36% to 68%. To determine ways to improve the total recovery to >80%, firstly we estimated the total binding capacity of the filter element in Table 2. One layer of filter element in Table 3 was attached to an AKTA Avant 150 purification system and equilibrated with 20 CV of buffer E (50 mM Tris-Acetate, pH 9.0,2 mM MgCh, 100 mM sodium chloride) at a flow rate of 10 CV / min. Excess amounts of AAV5 virus sample, comprising of only empty viruses produced via plasmid transfection (see methods) was loaded onto the filter element using buffer E (conductivity of about 12 mS / cm) at a flow rate of 3 CV / min. Viruses that were not bound and flowing through the filter were collected in flow throughs (FT). After loading, a wash step was performed with buffer E to wash off any unbound viruses. Elution was performed via step gradients by mixing varying percentages of buffer E with buffer F at a constant flow rate of 10 CV / min.

[0305] FIG. 2E is a separation chromatogram illustrating the elution profile of AAV5 across various salt concentrations. Total AAV5 in each fraction is measured by ELISA and the elution data is tabulated in Table 3E.

[0306] Table 3E. Estimation of AAV capacity of filter element 2 described in Table 3.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0307] Table 3E indicates that the total capacity of the filter element detailed in Table 2 was 4.7E+14 AAV5. Further increases in conductivity resulted in elution of 67.7 percent of bound AAV from filter element. So even while fully loading the filter to its maximum capacity, the recovery was low with close to -32% of AAV5 still bound to the filter element.

[0308] Example 20. Improving the total recovery from Empty Full separation

[0309] To determine ways to improve the total AAV5 recovery to >80%, we performed experiments where the filter element was equilibrated with two different conditions: High conductivity and a low conductivity equilibration condition. High equilibration condition involved using buffer F with a conductivity adjusted to 40 mS using sodium chloride. Buffer F was used equilibrate the filter element in Table 3 to up to 100 CV. In a few runs, buffer F’s conductivity was also adjusted to 80 mS using sodium chloride and used for experiments as well. After high salt equilibration, filters were equilibrated with 20 CV of low salt buffer E to remove excess salt in the lines in the AKTA system. A low equilibration condition indicated equilibrating with buffer E with a conductivity of 12 mS adjusted using sodium chloride. Buffer E was used equilibrate the filter element in Table 3 to up to 20 CV at 10 CV / min. In addition to the equilibration conditions, we simultaneously tested AAV5 loading at two different levels: high and low AAV load. High AAV loading indicated loading to greater than 10% of capacity of filter (> 4* E13 total AAV5) and low AAV loading indicated loading lower than 10% of capacity of filter (< 4* E13 total AAV5). Post loading, conductivity of solutions was increased in a step-by-step manner to elute AAVs in various fractions, as done in previous examples. The AAVs eluted in each fraction was measured by ELISA and recovery against feed from all fractions is calculated and listed as total recovery against feed in Table 3F along with various loading and equilibration conditions employed.

[0310] Table 3F. Total AAV5 recoveries from various loading and equilibration conditionsPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0312] Data in Table 3F indicated that when AAV empty full experiments were performed with high equilibration condition and a high AAV load condition, the total AAV recovery was -90% (average across 6 different runs). Alternatively, in runs where either the high equilibration and / or high AAV load condition was not met, the average recovery was -66% (average across 8 different runs). This illustrates that filter element listed in Table 2 needed to be equilibrated to at least 100CV with a buffer of conductivity > 40 mS and loaded to at least 10% of capacity of filter to have total recoveries of > 80% of AAV.

[0313] Example 21. Simultaneous high enrichment and high recovery of full AAV

[0314] Learnings from Example 20 on high recovery by using high salt equilibration coupled with loading to at least 10% of filter capacity was employed to perform AAV empty full separation to achieve simultaneous high enrichment and high recovery of full AAV.

[0315] A two-step method to separate empty and full AAV displayed in Example 2B was employed here. AAV5 virus was produced and purified with the TESSA system was estimated to be about 15% full by SEC-MALS (see methods). A single layer of filter element of Table 3 was loaded onto a column holder and attached to the AKTA Avant 150 purification system. A total of 5.2E+13 AAV5 was diluted into loading buffer E and used as feed. The filter element was first equilibrated with 100 CV of loading buffer F at 10 CV / min and followed it up with low salt equilibration comprising of 20 CV of low salt buffer E to remove excess salt in thePCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 lines in the AKTA system. Then the AAV5 feed was applied to the column at a flow rate of 3 CV / min with the aim of binding all viruses. After sample application, the membrane was washed with 30 CV of loading buffer E at 10 CV / min and viruses in El fraction were eluted by buffer G which comprised of 69% of buffer E and 31% of buffer F. The conductivity of the buffer G was ~21 mS / cm. Later, full viruses were preferentially eluted in E2 fraction with buffer H comprising of 50% of buffer E and 50% of buffer F with a conductivity of -26 mS / cm. A strip step of buffer F was used to collect bound AAVs (E3 fraction) at the end.

[0316] FIG. 2F is a separation chromatogram illustrating the elution profile of AAV5 (indicated by OD 280 and 260 on the left y axis) across various salt concentrations (indicated by conductivity on the right y axis). Total AAV5 in each fraction is measured by ELISA (see methods) and the full percentages for each fraction were measured via SEC-MALS (see methods). The elution data is tabulated in Table 3G.

[0317] Table 3G. Simultaneous high enrichment and high recovery of full AAV

[0318] Data in Table 3G indicated that when AAV empty full experiments were performed with high equilibration condition and a high AAV load condition, the total AAV recovery was -90%. In addition, a 15% full AAV sample was enriched to 78% full with a 96% recovery of full AAVs in the E2 fraction.

[0319] Example 22. Using choline chloride to separate empty and full AAVPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0320] Apart of sodium chloride used to elute AAV viruses in all above examples, here we explored other salts including choline chloride that can be employed to separate empty and full AAVs using filter element listed in Table 3.

[0321] To test choline chloride, we prepared buffer O (50 mM Tris-Acetate, pH 9.0, 2 mM MgCh, 100 mM choline chloride) and buffer P (50 mM Tris-Acetate, pH 9.0, 2 mM MgCh,400 mM choline chloride). One layer of filter element in Table 3 was inserted into the filter holder and attached to an AKTA Avant 150 purification system, and equilibrated with 100 CV of buffer P at a flow rate of 10 CV / min. An AAV5 virus sample, comprising a mix of empty and full viruses, was loaded onto the filter element in Table 3 using buffer O (conductivity of about 10.64 mS / cm) at 3 CV / min. After loading, a wash step was performed with buffer O at3 CV / min to wash off any unbound viruses. Elution was performed via step gradients by mixing varying percentages of buffer P with buffer O at a constant flow rate of 10 CV / min. FIG. 2G is a separation chromatogram illustrating the elution profile of AAV5 across various choline chloride concentrations. Total AAVs in each fraction was measured by ELISA and AAV full percentages are calculated via SEC-MALS and the data is listed in Table 3H.

[0322] Table 3H. Using choline chloride to separate empty and full AAVPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0323] Data in Table 2H indicated that there was 100% total recovery of all AAV from the filter. Further, the AAV full percentage dropped from 36% in feed to 0% in El fraction indicating selective removal of empty AAVs in El fraction. In E2 fraction, AAV full percentage was increased to 76% displaying enrichment using choline chloride. In addition, all the full viruses in feed were eluted in E2 fraction indicating a -100% recovery of full viruses in the E2 fraction.

[0324] Example 23. Using tetraethylammonium chloride to separate empty and full AAV

[0325] Apart of sodium chloride and choline chloride used in prior examples, here we explored the use of tetraethylammonium chloride for separation of empty and full AAVs using filter element listed in Table 3.

[0326] To test of tetraethylammonium chloride, we prepared buffer Q (50 mM Tris- Acetate, pH 9.0, 2 mM MgCh, 100 mM tetraethylammonium chloride) and buffer R (50 mM Tris-Acetate, pH 9.0, 2 mM MgCh, 300 mM tetraethylammonium chloride). One layer of filter element in Table 3 was attached to an AKTA Avant 150 purification system and equilibrated with 100 CV of buffer R at a flow rate of 10 CV / min. An AAV5 virus sample, comprising a mix of empty and full viruses, was loaded onto the filter element in Table 3 using buffer Q (conductivity of about 10.54 mS / cm) at 3 CV / min. After loading, a wash step was performed with buffer Q at 10 CV / min to wash off any unbound viruses. Elution was performed via step gradients by mixing varying percentages of buffer Q with buffer R at a constant flow rate of 10 CV / min FIG. 3H is a separation chromatogram illustrating the elution profile of AAV5 across various tetraethylammonium chloride concentrations. Total AAVs and AAV full percentages are calculated via SEC-MALS and the data is listed in Table 31.

[0327] Table 31. Using tetraethylammonium chloride to separate empty and full AAVPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0328] Data in Table 21 indicated that there was total recovery of all AAV from the filter. Further, the AAV full percentage dropped from 36% in feed to 22% in El fraction indicating selective removal of empty AAVs in El fraction. In E2 fraction, AAV full percentage was increased to 115% displaying enrichment using tetraethylammonium chloride.

[0329] Example 24. Empty / Full aav5 separation using q chemistry and comparison of Q and iem-bib chemistry.

[0330] To display empty full separations using Q chemistry, the Emphaze™ filter was used. Emphaze™ filter element was attached to an AKTA Avant 150 purification system. The Emphaze™ filter was equilibrated as per manufacturer’s instruction prior to separation experiments. The filter was equilibrated with 20 CV of buffer S (50 mM Tris-Acetate, pH 9.0, 2mM MgCh, 50 mM sodium acetate) at 10 CV / min. An AAV5 virus sample comprising a mix of empty and full viruses was loaded onto each filter element using buffer S (loading conductivity 5.45 mS / cm) at 3 CV / min. After loading, a wash step was performed with buffer S at 10 CV / min to wash off any unbound viruses. Elution was performed at 10 CV / min by mixing varying percentages of buffer S with buffer T (50 mM Tris-Acetate, pH 9.0, 2 mM MgCh, 250 mM sodium acetate).

[0331] FIG. 3 is a separation chromatogram illustrating the elution profile of AAV5 (indicated by OD 280 and 260 on the left y axis) across various salt concentrations (indicated by conductivity on the right y axis). Table 4A indicates the various fractions, conductivities at which each fraction is collected, total AAV recovery and AAV full percentage in each fraction. Q chemistry in Emphaze™ filter elutes AAV5 viruses across a conductivity range of 2.6 mS / cm between 8.04 and 10.67 mS / cm, with an enrichment of AAV full percentage from 45% to 88%. The total recovery of AAV from the Emphaze™ filter was 78%.

[0332] Table 4A. Using Emphaze™ to separate empty and full AAVPCT Application Applicant Ref PA200036 Atty Dkt. No.: THF-080PCT0

[0333] In comparison to the Q chemistry in Emphaze™ filter, the IEM-BIB chemistry is more salt tolerant. Table 4B directly compares the conductivity ranges across which the Q chemistry in Emphaze™ filter and IEM-BIB chemistry in filter element described in Table 3 elutes the AAV virus.

[0334] Table 4B. Comparison of Emphaze™ and IEM-BIB to separate empty and full AAVPCT Application Applicant Ref PA200036 Atty Dkt. No.: THF-080PCT0

[0335] Q chemistry in Emphaze™ filter elutes AAV5 viruses across a narrow conductivity range of 2.6 mS / cm between 8.04 and 10.67 mS / cm, IEM-BIB chemistry elutes AAV5 viruses across a broader range of conductivity of 6 mS / cm between 17 and 23 mS / cm. With regards to enrichment, filter element 2 in Table 3 enriches a 15% full AAV sample to 78% full AAV, signifying a 5.2x enrichment in full percentage which was higher than a ~2x enrichment with Emphaze™. With regards to total AAV recovery, the filter element in Table 3 yields 90% recovery, higher than 78.3% with Emphaze™. These results demonstrate that IEM-BIB filter media listed in Table 3 is more salt tolerant than Q chemistry media enabling loading of the viruses to the IEM-BIB filters at a much higher salt concentration (up to 15 mS / cm conductivity), i.e., higher than the elution conductivity shown for Q chemistry (10 mS / cm) and Table 2 filter displays better enrichment and recovery.

[0336] Example 25. EMPTY / FULL AAV5 SEPARATIONS IEM-BIB + C0-M0N0MERS

[0337] Filter Medias FM3a (50 mol% IEM-BIB, 40 mol% NVP, 10 mol% GMA), FM3b (50 mol% IEM-BIB, 50 mol% HEMA) and FM3c (50 mol% IEM-BIB, 50 mol% DEGMEMA) were each grafted via e-beam onto a BA080 nylon membrane. Table 5 describes the filter elements.PCT Application Applicant Ref PA200036 Atty Dkt. No.: THF-080PCT0

[0338] Table 5. Filter elements 3a-3c

[0339] Filter elements 3a-3c were attached to an AKTA Avant 150 purification system and equilibrated with 20 CV of buffer E at 10 CV / min. An AAV5 virus sample comprising a mix of empty and full viruses was loaded onto the filters using buffer E at 3 CV / min. A wash step was performed with buffer E to wash off any unbound viruses and elution was performed via step gradients by mixing varying percentages of buffer E with buffer F. FIG. 4 is separation chromatogram illustrating the elution profile of AAV5 (indicated by OD 280 and 260 on the left y axis) from each filter element across various salt concentrations (indicated by conductivity on the right y axis). The percentages of AAV for each fraction were measured via ELISA. The elution data is tabulated in Table 5A.

[0340] Table 5a. Separation data for Filter Elements 3a-3cPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0341] The chromatogram in FIG. 4 indicates the windows of conductivity across which there is elution of AAV5. AAV5 elutes between 15-23 mS / cm with NVP / GMA as comonomer. AAV5 elutes between 15-23 mS / cm with DEGMEMA as co-monomer. AAV5 elutes between 25.7-30.8 mS / cm with HEMA as co-monomer. The results indicate that IEM- BIB with HEMA is the most salt tolerant formulation for AAV binding to the material.

[0342] Example 26. VIRAL CLEARANCE WITH EMPTY / FULL AAV5 SEPARATIONS

[0343] Filter Media FM4 (92 mol% IEM-BIB, 5 mol% HEMA, 3 mol% GDMA) was grafted via e-beam onto a BA080 nylon membrane. Table 6 describes the filter element.

[0344] Table 6. Filter Element 4

[0345] Filter element 4 was attached to an AKTA Avant 150 purification system and equilibrated with 20 CV of buffer E at 10 CV / min. A feed sample comprising of AAV5 and Phi6 virus was loaded onto the filters using buffer E (loading conductivity 11 mS / cm). A wash step was then performed with buffer E to wash off any unbound viruses. Elution performed via step gradient by mixing varying percentages of buffer E with buffer F. The chromatogram in FIG. 5 indicates the windows of conductivity across which there is elution of AAV5. AAV levels within the fractions were measured by ELISA and the Phi6 levels within the fractions was measured by the plaque assay.

[0346] Table 6a. Separation data for filter element 4PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0347] The results show that full AAV primarily eluted in fractions 5, 6 and 7 with a total recovery of 76%. No observable levels of Phi 6 virus appeared in the elution fractions (detection limit of the assay was 2.2 log). Thus, there was at least 5.5 log removal of Phi6 during the AAV5 empty / full separation experiment.

[0348] Example 27. EMPTY / FULL AAV5 SEPARATIONS IEM-BIB ON NON-WOVENS

[0349] Filter Media FM5 (24 mol% IEM-BIB, 59 mol% NVP, 17% GMA) was grafted via e-beam onto a 4-micron polypropylene nonwoven substrate and the resulting filter element was then attached to an AKTA Avant 150 purification system. Table 7 describes the resulting filter element.

[0350] Table 7. Filter element 5PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0351] The filter element containing FM5 attached to the AKTA Avant 150 purification system and was equilibrated with 20 CV of buffer E at 10 CV / min. An AAV5 virus sample comprising a mix of empty and full viruses was loaded onto the filter element using buffer E (loading conductivity 12.85 mS / cm) at 3 CV / min. After loading, a wash step was performed with buffer E to wash off any unbound viruses and elution was performed via step gradient by mixing varying percentages of buffer E with buffer F. FIG. 6 is separation chromatogram illustrating the elution profile of AAV5 (indicated by OD 280 and 260 on the left y axis) across various salt concentrations (indicated by conductivity on the right y axis). The full percentage for each fraction was measured via qPCR and ELISA. The elution data is tabulated in Table 7A.

[0352] Table 7A. Separation Data for Filter Element 5

[0353] The results indicated that the BIB functionalized non-wovens effectively separated the empty and full AAV5 viruses. 4 elution peaks (E2, E3, E4 and E5) were observed and theOD 260 / 280 ratios of these peaks were 0.57, 0.80, 1.3 and >1.3, respectively. The E2 peak with OD 260 / 280 ratio of 0.57 is predominantly empty and the E4 and E5 peaks with OD 260 / 280 ratios of 1.3 and > 1.3 were predominantly full AAV. The same is reflected in the AAV fullPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 percentages calculated via the qPCR / ELISA as well with AAV5 full percentages being 58.6% in feed, 4.23% in El, 2.92% in E2, 54.7% in E3, 112.6% on E4 and 108.5% in E5.

[0354] Example 28. EMPTY / FULL AAV2 SEPARATIONS WITH IEM-BIB FILTERMEDIA

[0355] Filter Media FM6 (72 mol% IEM-BIB, 25 mol% HEMA, 3% GDMA) 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. Table 8 describes the resulting filter element.

[0356] Table 8. Filter element 6

[0357] The filter element containing FM6 was attached to an AKTA Avant 150 purification system and was equilibrated with 10 column volumes of buffer N (50 mM Tris-Acetate, pH 9.0, 2mM MgCh, 100 mM sodium chloride). An AAV2 virus sample comprising a mix of empty and full viruses was loaded onto the filter element using buffer N (loading conductivity 10.5 mS / cm). After loading, a wash step is performed with buffer N to wash off any unbound viruses. Next, elution was performed via step gradient by mixing varying percentages of buffer N with buffer O (50 mM Tris-Acetate, pH 9.0, 2mM MgCh, 400 mM sodium chloride). The elution fractions were taken and measured for AAV2 full percentages via qPCR and ELISA and further measured for AAV infectivity according to the AAV infectious titer assay. The data are presented in Table 8A and the chromatogram is represented in FIG. 7.

[0358] Table 8A. Separation Data for Filter Element 6PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0

[0359] The data shows that fractions E2 and E3 had the most AAV2 virus. A feed of -12% full was enriched to -53% full AAV2 using the IEM-BIB chemistry.

[0360] The data demonstrates that there was a 2.19x increase in infectivity per capsid of AAV2 and a 4.2x increase in full % AAV2 in fraction 3 over the feed.

[0361] Example 29. EMPTY / FULL AAV6 SEPARATIONS WITH IEM-BIB FILTER MEDIA

[0362] Filter Media FM2 (25 mol% IEM-BIB, 75 mol% HEMA) was grafted via e-beam onto a 0.8 micron double-sided nylon membrane and the resulting filter element was then attached to an AKTA Avant 150 purification system.

[0363] AAV6 virus was produced with the TESSA method and purified. The feed was estimated to be about 27% full by SEC / MALS. The AAV6 was diluted into loading buffer E to a concentration of -1E12 cp / mL in 40 mL of loading buffer (loading conductivity 12.7 mS / cm). The filter elements were equilibrated with buffer E for 100 CV at a flow rate of 10 CV / ml. Later, the AAV6 containing feed was applied to the column at a flow rate of 3 CV / ml.The filter elements were then washed with 30 CV of buffer E at 10 CV / min to wash off any unbound viruses. Next, elution was performed via step gradient by mixing varying percentages of buffer E with buffer U (50 mM Tris-Acetate, pH 9.0, 2mM MgCh, 350 mM sodiumPCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 chloride). Fractions from the flow through and elution were analyzed by ELISA and SEC- MALS to determine the recovery and percent of full virus in each sample. Data is indicated in Table 9 and the chromatogram is represented in FIG. 8.

[0364] Table 9. Separation Data with AAV6

[0365] EQUIVALENTS

[0366] 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.

[0367] REFERENCES

[0368] 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 Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0CLAIMSWe claim:

1. A compound represented by Formula I:CH2=C(R1)-C(O)-W-R2-X-C(O)-Y-R3-HA+-Q Z (I), wherein:R1is -H or a straight or branched C1-4 alkyl,W is -O- or -N(R5)-,R2is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene,X and Y are independently selected from a bond, -O-, or -N(R5)-, provided that at least one of X and Y is -O-, or -N(R5)-,R3is a straight or branched C2-C6 alkylene, or a straight or branched C4-C12 alkoxyalkylene,HA+is a charged heteroaryl having 5-9 atoms and 1-3 heteroatoms selected from N, O, and S, provided that at least one heteroatom is a quadrivalent nitrogen,Q is a straight or branched C1-C12 alkyl or -R4-Ar,R4is a straight or branched Ci-Ce alkylene, or a straight or branched C4-C12 alkoxyalkylene,Ar is aryl,Z is a counterion, and each R5is independently selected from -H and a C1-4 alkyl.

2. The compound of claim 1, represented by Formula la:CH2=C(R1)-C(O)-W-R2-X-C(O)-Y-R3-HA+-R4-Ar Z (la).

3. The compound of any one of the preceding claims, wherein HA+is -N(R6)-R7-N+(R8)- , wherein R6and R8are taken together to form a heteroaryl ring, and R7ais -C(H)=.

4. The compound of any one of the preceding claims, wherein HA+is an imidazolyl ring.

5. The compound of any one of claims 1-4, wherein HA+is a benzimidazolyl ring.

6. The compound of any one of the preceding claims, wherein R4is -CH2-.

7. The compound of any one the preceding claims, wherein Ar is a Ce, C9, or C10 aryl.

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

9. The compound of claim 1, represented by the formula lb:(lb).PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT010. The compound of any one of the preceding claims, wherein Z is selected from chloride, bromide, tosylate, and tritiate.

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

12. The compound of claim 11, wherein said monomer is a ligating monomer.

13. A polymerizable composition comprising: one or more ligating monomer of any one of claims 1-12, and optionally one or more co-monomer.

14. A ligating polymer derived from: one or more ligating monomer of any one of claims 1-12.

15. A ligating polymer derived from a polymerizable composition of claim 13.

16. The ligating polymer of any one of claims 14-15, further being derived from one or more co-monomer characterized by octanol-water partition coefficient of about -4 to about 4.

17. The ligating polymer of any one of claims 14-16, further being derived from one or more co-monomer characterized by a water solubility of at least 5 g per L at 25 °C.

18. The ligating polymer of any one of claims 14-17, 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.

19. The ligating polymer of any one of claims 14-18, 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.

20. The ligating polymer of any one of claims 14-19, 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.

21. The ligating polymer of any one of claims 14-20, further being derived from at least one (meth)acrylate monomer having one or more hydroxyl groups.

22. The ligating polymer of any one of claims 14-21, further being derived from at least one (meth)acrylate monomer having one or more glycerol groups.

23. The ligating polymer of any one of claims 14-22, further being derived from a comonomer selected from A-vinylpyrrolidone, hydroxyethyl methacrylate, di(ethylene glycol) methyl ether methacrylate, and a combination thereof.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT024. The ligating polymer of any one of claims 14-23, further being derived from a comonomer selected from glycerol (meth)acrylate, glycerol di(meth)acrylate, and a combination thereof.

25. The ligating polymer of any one of claims 14-24, 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 mol%.

26. The ligating polymer of any one of claims 14-25, excluding co-monomers characterized by water solubility of less than 100 g per L at 25 °C, an octanol -water partition coefficient of greater than 4, or a combination thereof.

27. The ligating polymer of any one of claims 14-26, excluding co-monomers comprising a quaternary ammonium trialkyl groups of formula: -N+(Rq)3, wherein each Rqis independently a C1-C20 alkyl group.

28. The ligating polymer of any one of claims 14-27, excluding co-monomers comprising a guanidine group.

29. The ligating polymer of any one of claims 14-28, wherein the ligating monomer of Formula I accounts for about 10 mol% to about 100 mol% of the ligating polymer.

30. The ligating polymer of any one of claims 14-29, wherein the ligating monomer of Formula I accounts for about 10 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 90 mol% of the ligating polymer31. The ligating polymer of any one of claims 14-29, wherein the ligating monomer of Formula lb accounts for about 10 mol% to about 100 mol% of the ligating polymer.

32. The ligating polymer of any one of claims 14-29, wherein the ligating monomer of Formula lb accounts for about 10 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 90 mol% of the ligating polymer.

33. 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 1-19,PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT0 wherein the ligating polymer is grafted to at least one or more porous substrate.

34. 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 23-42, wherein the ligating polymer is grafted to each of the one or more porous substrate.

35. The filter element of any one of claims 33-34, the one or more porous substrate characterized by an average pore size of about 0.1 pm - 10 pm.

36. The filter element of any one of claims 33-35, the one or more porous substrate having fibers characterized by a fiber diameter of about 0.5 micrometers to about 15 micrometers.

37. The filter element of any one of claims 33-36, 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.

38. The filter element of any one of claims 33-37, the one or more porous substrate comprised of material selected from nylon and polypropylene.

39. The filter element of any one of claims 33-38, the one or more porous substrate in the form of a membrane or a nonwoven.

40. The filter element of any one of claims 33-39, 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.

41. The filter element of any one of claims 33-40, 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.

42. The filter element of any one of claims 33-41, characterized by a binding for the adeno- associated virus containing a DNA payload at a conductivity value of about 10 mS / cm to about 25 mS / cm.

43. The filter element of any one of claims 33-42, characterized by a binding for the adeno- associated virus containing a DNA payload at a conductivity that is 5-7 mS / cm higher than binding for an adeno-associated virus without a DNA payload.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT044. The filter element of any one of claims 33-43, 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.

45. 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 43-55; 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.

46. The process of a claim 45, 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.

47. The process of any one of claims 45-46, wherein the DNA payload is a sequence effective to treat hemophilia A, hemophilia B, muscular dystrophy, AADC deficiency, lipoprotein lipase deficiency, and retinal dystrophy.

48. The process of any one of claims 45-47, the loading solution further comprising one or more water soluble inorganic salt.

49. The process of any one of claims 45-48, the loading solution characterized by a conductivity of about 5 mS / cm to about 15 mS / cm.

50. The process of any one of claims 45-49, the loading solution characterized by an inorganic salt concentration of about 50 mM to about 200 mM.

51. The process of any one of claims 45-50, the loading solution characterized by a pH of about 8.5 to about 10.

52. The process of any one of claims 45-51, 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.

53. The process of any one of claims 45-52, 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 5 mS / cm to about 7 mS / cm.PCT Application Applicant Ref: PA200036 Atty Dkt. No.: THF-080PCT054. The process of any one of claims 45-53, wherein the conditions are effective recover the adeno-associated virus containing a DNA payload in an amount of at least 75% at an enrichment of at least 75%.

55. A process for preparing a filter element of any one of claims 33-45, the process comprising: providing a polymerizable composition of claim 13; providing a porous substrate; contacting the polymerizable composition to the porous substrate under conditions effective to polymerize the polymerizable composition onto the porous substrate56. A process for preparing a filter element of any one of claims 33-45, 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.

57. The process of any one of claims 55-56, the conditions comprising ionizing irradiation.

58. The process of claim 57, wherein said irradiation is chosen from e-beam irradiation, X- ray irradiation, or gamma irradiation.

59. The process of claim 58, wherein said irradiation is e-beam irradiation.

60. The process of any one of claims 55-59, the conditions comprising e-beam irradiation at a dose of about 6 MRad to about 10 MRad.

61. The process of any one of claims 55-50, the conditions comprising UV irradiation.

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