Multivalent cation-binding ligands and compositions comprising the same for ionic chelate affinity separations

Abstract

Disclosed herein are compositions comprising a solid substrate that is bound to a multivalent cation-binding ligand. The composition can be used to associate positively-charged ion species with the solid substrate so as to facilitate separation methods in the solid phase. Also disclosed are methods of making and using the composition.

Description

MULTIVALENT CATION-BINDING LIGANDS AND COMPOSITIONS COMPRISING THE SAME FOR IONIC CHELATE AFFINITY SEPARATIONSCROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of and priority to the earlier filing date of U.S. Provisional Patent Application No. 63 / 556,293, filed on February 21 , 2024, the entirety of which is incorporated herein by reference.FIELD

[0002] The present disclosure is directed to multivalent cation-binding ligands that can be bound to substrates to provide the ability to associate positively-charged species, along with methods of making and using the same.BACKGROUND

[0003] In the field of solid phase / separations chemistry, there is a lack of commercial compositions that can be used to provide multivalent cations having proper orbital orientation of the electron shell configuration in solid form. Further, conventional materials used in separations chemistry that utilize calcium as a multivalent cation are traditionally limited to the solution phase, which does not lend to use of such materials in solid phase separations; or, they are comprised of inorganic materials and thus is limited to separations based on ionic interactions, which introduces limitations, such as operating ranges of pH, temperature, and solvent. There is a need in the art for materials comprising a non-soluble surface with the ability to retain multivalent cations that further is stable under a variety of pH, temperature, and / or solvent parameters.SUMMARY

[0004] Disclosed herein is a composition, comprising: a substrate; and a multivalent cation-binding ligand covalently bound to a surface of the substrate, wherein the multivalent cation-binding ligand has a structure according to one of Formulas I or II— -Y— Z1Formula IFormula II wherein each Y, independently for each occurrence, is a heteroaliphatic group, a bond, or an aliphatic group; each Z1is selected from -O[X(A)s(A,)mA”]q-Hr or -[X(A)s(A’)mA”]q-Hr, wherein each X, independently for each occurrence, is P, S, B, N, or C; each A, independently for each occurrence, is selected from O or S; each A', independently for each occurrence, is selected from -OH, -SH, -O’, or -S'; each A”, independentlyfor each occurrence, is selected from O, S, O', or S'; each s, independently for each occurrence, is an integer selected from 0 or 1 ; each m, independently for each occurrence, is an integer selected from 0 or 1 ; q is an integer selected from 1 to 10; and r is an integer selected from 0 or 1 ; each Z2, independently for each occurrence, is selected from (i) -O[X(A)s(A’)mA”]q-Hr or -[X(A)s(A’)mA”]q-Hr, wherein each X, independently for each occurrence, is P, S, N, B, or C; each A, independently for each occurrence, is selected from O or S; each A', independently for each occurrence, is selected from -OH, -SH, -O , or -S ; each A”, independently for each occurrence, is selected from O, S, O', or S'; each s, independently for each occurrence, is an integer selected from 0 or 1 ; each m, independently for each occurrence, is an integer selected from 0 or 1 ; q is an integer selected from 1 to 10; and r is an integer selected from 0 or 1 ; or (II) an R3group, wherein R3is hydrogen, an aliphatic group, a heteroaliphatic group, or an aromatic group, provided that if an R3group is present, then at least one Z2is -O[X(A)s(A,)mA,’]q-Hr or -[X(A)s(A’)mA”]q-Hr; each of R1and R2, independently for each occurrence, is selected from hydrogen, aliphatic, heteroaliphatic, or aromatic; TG is a terminating group selected from hydrogen, hydroxyl, or alkoxy; n is an integer selected from 1 to 10,000 or higher; and u is an integer selected from 0 or 1 .

[0005] Also disclosed herein is a kit according to aspects of the present disclosure, comprising: the composition as described herein; and a container configured to house the composition during use of the composition.

[0006] Also disclosed herein is a separation method, comprising: exposing a sample to a composition according to the present disclosure to form a mixture; and exposing the mixture to a solution to facilitate separating a target from the sample.

[0007] Also disclosed is a composition according to aspects of the present disclosure, comprising: a solid substrate; and an associating means for associating a positively-charged ion species with the solid substate.

[0008] The foregoing and other objects, features, and advantages of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIGS. 1 A and 1 B are schematic illustrations showing how solid substrates bound to a multivalent cation-binding ligand according to the present disclosure can be used to bind multivalent ions.

[0010] FIG. 2 is a chromatogram showing protein separation and selectivity observed using a composition according to the present disclosure.

[0011] FIG. 3 is a chromatogram showing protein separation and selectivity observed using a composition according to the present disclosure.

[0012] FIG. 4 is a chromatogram showing protein separation and selectivity observed using a composition according to the present disclosure.

[0013] FIG. 5 is a chromatogram showing protein separation and selectivity observed using a composition according to the present disclosure.

[0014] FIG. 6 is a chromatogram showing protein separation and selectivity observed using a composition according to the present disclosure.

[0015] FIG. 7 is a bar graph showing binding results for compositions according to the present disclosure for mRNA as compared with a commercial hydroxyapatite material.DETAILED DESCRIPTION

[0016] Overview of Terms

[0017] The following explanations of terms are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, "comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements unless the context clearly indicates otherwise.

[0018] Although the steps of some of the disclosed methods are described in a particular, sequential order for convenient presentation, it should be understood that this manner of description encompasses rearrangement, unless a particular ordering is required by specific language set forth below. For example, steps described sequentially may in some cases be rearranged or performed concurrently. Additionally, the description sometimes uses terms like “produce” or “provide” to describe the disclosed methods. These terms are high-level abstractions of the actual steps that are performed. The actual steps that correspond to these terms will vary depending on the particular implementation and are readily discernible by one of ordinary skill in the art.

[0019] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting, unless otherwise indicated. Other features of the disclosure are apparent from the following detailed description and the claims.

[0020] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that can depend on the desired properties sought and / or limits of detection under standard test conditions / methods and in some aspects encompasses a range up to ± 15% of that numerical value, unless the context clearly dictates otherwise. When directly and explicitly distinguishing aspects of the disclosure from discussed prior art, the numbers used for aspects of the present disclosure are not approximates unless the word “about” is recited. Furthermore, not all alternatives recited herein are equivalents.

[0021] Certain functional group terms used herein include a symbol which is used to show how the defined functional group attaches to, or within, the compound to which it is bound. Also, a dashed bond (i.e. , “ — ”) as used in certain formulas described herein indicates an “optional” bond to a substituent or atom of the formula other than hydrogen in the sense that the bond (and in some aspects, the substituent) may or may not be present. In any formulas comprising a dashed bond, if the optional bond and / or any corresponding substituent is not present, then the valency requirements of any atom(s) bound thereto is completed by a bond to a hydrogen atom.

[0022] The symbol “ is used to indicate a bond disconnection in abbreviated structures / formulas provided herein. A person of ordinary skill in the art recognizes that the definitions provided below and the compounds and formulas included herein are not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 different groups, and the like). Such impermissible substitution patterns are easily recognized by a person of ordinary skill in the art. In formulas and compounds disclosed herein, a hydrogen atom is present and completes any formal valency requirements (but may not necessarily be illustrated) wherever a functional group or other atom is not illustrated. For example, a phenyl ring that is drawn as a comprises a hydrogen atom attached to each carbon atom of the phenyl ring other than the “a” carbon, even though such hydrogen atoms are not illustrated. Any functional group disclosed herein and / or defined above can be substituted or unsubstituted, unless otherwise indicated herein.

[0023] To facilitate review of the various aspects of the disclosure, the following explanations of specific terms are provided.

[0024] Aliphatic: A hydrocarbon group having at least one carbon atom to 50 carbon atoms (C1-50), such as one to 25 carbon atoms (C1-25), or one to ten carbon atoms (C1-10), and which includes alkanes (or alkyl), alkenes (or alkenyl), alkynes (or alkynyl), including cyclic versions thereof, and further including straight- and branched-chain arrangements, and all stereo and position isomers as well. Aliphatic groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0025] Alkoxy: -O-aliphatic, such as -O-alkyl, -O-alkenyl, -O-alkynyl; with exemplary examples including, but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy (wherein any of the aliphatic components of such groups can comprise no double or triple bonds, or can comprise one or more double and / or triple bonds). Alkoxy groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0026] Amino: -NRbRc, wherein each of Rband Rcindependently is selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group, and can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0027] Antibody: The term "antibody" is used in the broadest sense to cover monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, immunoadhesins and antibody-immunoadhesin chimerias. An antibody may include, for example, an antibody of any molecule class, e.g., IgGi, lgG2, etc. that is to be purified from a mixture containing contaminants. An "antibody fragment" includes at least a portion of a full-length antibody and typically, an antigen binding or variable region thereof; for e.g., they include Fab, Fab', F(ab')2, and Fv fragments; single-chain antibody molecules like camelid antibodies; diabodies; linear antibodies; and multispecific antibodies formed from engineered antibody fragments. The term "monoclonal" antibodies, indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies described herein include "chimeric" and "humanized" antibodies, and "human" antibodies, which can be isolated from various sources, including, e.g., from the blood of a human patient or recombinantly prepared using transgenic animals.

[0028] Aromatic: A cyclic, conjugated group or moiety of, unless specified otherwise, from 5 to 15 ring atoms having a single ring (e.g., phenyl) or multiple condensed rings in which at least one ring is aromatic (e.g., naphthyl, indolyl, or pyrazolopyridinyl); that is, at least one ring, and optionally multiple condensed rings, have a continuous, delocalized Ti-electron system. Typically, the number of out of plane n-electrons corresponds to the Huckel rule (4n + 2). The point of attachment to the parent structure typically is through an aromatic portion of the condensed ring system. For example,zcou. However, in certain examples, context or express disclosure may indicate that the point of attachment is through a non-aromatic portion of the condensed ring system. For example,. An aromatic group or moiety may comprise only carbon atoms in the ring, such as in an aryl group or moiety, or it may comprise one or more ring carbon atoms and one or more ring heteroatoms comprising a lone pair of electrons (e.g. S, O, N, P, or Si), such as in a heteroaryl group or moiety. Aromatic groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0029] Carboxyl: -C(O)-.

[0030] Carboxylate: -C(O)O‘ or salts thereof.

[0031] Ester: -C(O)ORaor -OC(O)Ra, wherein Rais selected from aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Ester groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0032] Halo (or halide or halogen): Fluoro, chloro, bromo, or iodo. In some aspects, halo can also include astatine.

[0033] Heteroatom: An atom other than carbon or hydrogen, such as (but not limited to) oxygen, nitrogen, sulfur, silicon, boron, selenium, phosphorous, tellurium, polonium, and the like. In particular disclosed aspects of the disclosure, such as when valency constraints do not permit, a heteroatom does not include a halogen atom.

[0034] Heteroaliphatic: An aliphatic group comprising at least one heteroatom to 20 heteroatoms, such as one to 15 heteroatoms, or one to 5 heteroatoms, which can be selected from, but not limited to oxygen, nitrogen, sulfur, silicon, boron, selenium, phosphorous, and oxidized or protonated forms thereof within the group. Alkoxy, ether, amino, disulfide, peroxy, and thioether groups are exemplary (but non-limiting) examples of heteroaliphatic. Heteroaliphatic groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0035] Multivalent Cation-Binding Ligand: A ligand group that facilitates indirect binding of a multivalent cationic species to a solid substrate, wherein the ligand group is bound to a surface of the solid substrate. Multivalent cation-binding ligands include phosphorus-, sulfur-, boron-, nitrogen, and carboxyl-containing ligands disclosed herein.

[0036] Multivalent Cationic Species: A representative a positively-charged species that includes a 2+ or higher positive charge. Exemplary multivalent cationic species can include ions having a 2+, 3+, 4+ or higher positive charge.

[0037] Organic Functional Group: A functional group that may be provided by any combination of aliphatic, heteroaliphatic, aromatic, haloaliphatic, and / or haloheteroaliphatic groups, or that may be selected from, but not limited to, aldehyde; aroxy; acyl halide; halogen; nitro; cyano; azide; carboxyl (or carboxylate); amide; ketone; carbonate; imine; azo; carbamate; hydroxyl; thiol; sulfonyl (or sulfonate); oxime; ester; thiocyanate; thioketone; thiocarboxylic acid; thioester; dithiocarboxylic; phosphonate; phosphate; silyl ether; sulfinyl; sulfonamide; thial; or combinations thereof. Organic functional groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0038] Phosphate: -O-P(O)(ORa)2, wherein each Raindependently is hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group; or wherein one or more Ragroups are not present and the phosphate group therefore has at least one negative charge. The Ragroups of the phosphate can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0039] Phosphonate: -P(O)(ORa)2, wherein each Raindependently is hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group; or wherein one or more Ragroups are not present and the phosphate group therefore has at least one negative charge. The Ragroups of the phosphonate group can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0040] Positively-Charged Species: A chemical species (e.g., atom or molecule) that possesses a net positive charge. In particular aspects of the disclosure, the positively-charged species is a positively- charged ion.

[0041] Solid: A physical form that is not a liquid or a fluid. Solids can include rigid solids as well as gels, hydrogels, and the like.

[0042] Substrate: A solid component having a surface that is bound to a multivalent cation-binding, typically through one or more chemical bonds, such as covalent bonds.

[0043] Sulfonate: -SOa'.

[0044] Target (or Target Molecule): Any molecule of interest that is to be purified, concentrated, separated, isolated, enriched, or the like. Exemplary targets / target molecules are described herein.

[0045] Terminating Group: A functional group that is used to terminate polymerization of a multivalent cation-binding ligand group and that becomes covalently bound to the multivalent cation-binding ligand group at an end of the polymer chain, as illustrated in Formula II herein.

[0046] Introduction

[0047] In the field of solid phase / separations chemistry, there is a lack of commercial compositions that can be used to provide multivalent cations having proper orbital orientation of the electron shell configuration in solid form. Providing a non-soluble surface with the ability to retain multivalent cations would enable a product comprising a surface functionalized with multivalent cationic species that can be used to retain anionic species in an affinity mechanism not available with traditional cationic exchange media.

[0048] Phosphate-Cation-Phosphate-based interactions are used in many biological systems, particularly Phosphorus-Calcium-Phosphorus interactions. Conventional materials used in separations chemistry that utilize calcium as a multivalent cation are traditionally limited to the solution phase, which does not lend to their use in solid phase separations. While a solid-phase calcium-containing media exists (namely, hydroxyapatite), this material is inorganic and thus is limited to separations based on ionic interactions. As such, materials like hydroxyapatite (as well as ceramic variants, including ceramic hydroxyapatite and ceramic fluoroapatite) are limited to specific operating ranges of pH, temperature, and solvent. Such materials are therefore susceptible to degradation under various parameters.

[0049] Disclosed herein is a multivalent cation-binding ligand group that addresses drawbacks associated with current multivalent separation media (including those discussed above). The disclosed multivalent cation-binding ligand is able to covalently bind with a solid substrate and thus can link positively-charged species (e.g., multivalent cations) with the solid substrate. This capability to associate the positively- charged species with a solid substrate avoids the constraints of pH, temperature, and solvent that are associated with non-covalently bound inorganic media, such as the hydroxyapatite compounds discussed above. In addition, covalent linkages allow for multi elution mechanisms (e.g., competitive displacement or displacement and regeneration). The multivalent cation-binding ligand is able to associate with positively-charged species (e.g., multivalent cationic species) and can thus be used to retain a target while avoiding the limitations associated with non-covalently bound inorganic media.

[0050] Composition and Kit

[0051] Disclosed herein is a composition for use in various chemical applications, such as solid phase chemistry, separations chemistry, and / or catalysis. The composition comprises a multivalent cation-binding ligand and a substrate. The multivalent cation-binding ligand is bound to a surface of the substrate, as described herein. In some aspects of the disclosure, the composition further comprises a positively-charged species, a target, or any combination thereof. In yet additional aspects of the disclosure, the composition further comprises a positively-charged species, a contaminant, or any combination thereof. In some aspects of the disclosure, the composition can comprise a combination of a positively-charged species, a target, and a contaminant. Components of the composition are discussed in more detail below.

[0052] The multivalent cation-binding ligand of the composition can have a structure according to Formula I or Formula II:- -Y-Z1Formula IFormula II

[0053] With reference to Formulas I and II, the wavy line indicates the disconnection between the multivalent cation-binding ligand and the substrate; each Y, independently for each occurrence, is a bond, an aliphatic group, an aromatic group, a heteroaliphatic group, or any combination thereof (e.g., aromatic- heteroaliphatic-aromatic or aromatic-aliphatic). In aspects of the disclosure where Y is a bond, the Z1or Z2groups are directly bound to the carbon to which Y is attached in Formulas I and II.

[0054] With reference to Formula I, Z1is selected from -O[X(A)s(A’)mA’']q-Hr or -[X(A)s(A’)mA,’]q-Hr, wherein each X, independently for each occurrence, is P, S, C, N, or B; each A, independently for each occurrence, is selected from O or S and is bound to X via a double bond; each A', independently for each occurrence, is selected from -OH, -SH, -O‘, or -S‘; each A", independently for each occurrence, is selected from O, S, O‘, or S'; each s, independently for each occurrence, is an integer selected from 0 or 1 ; each m, independently for each occurrence, is an integer selected from 0 or 1 ; q is an integer selected from 1 to 10, such as 1 to 8, or 1 to 6, or 1 to 4, including 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10; and r is an integer selected from 0 or 1 (and when r is 0, the terminating A” group is O' or S ). In particular aspects, m is 1 when X is C, B, N, or S and m is 2 when X is P. In some aspects, s is 1 when X is P, S, C, or N and s is 0 when X is B. In particular aspects of the disclosure, when r is 0, A” is O' or S'; and when r is 1 , A” is O or S.

[0055] With reference to Formula II, u is 1 or 0; each Z2, independently for each occurrence, is selected from (I) -O[X(A)s(A’)mA”]q-Hr or -[X(A)s(A’)mA”]q-Hr, wherein each X, independently for each occurrence, is P, S, N, B, or C; each A, independently for each occurrence, is selected from O or S; each A’, independently for each occurrence, is selected from -OH, -SH, -O', or -S'; each A”, independently for each occurrence, is selected from O, S, O', or S'; each s, independently for each occurrence, is an integer selected from 0 or 1 ; each m, independently for each occurrence, is an integer selected from 0 or 1 ; q is an integer selected from 1 to 10, such as 1 to 8, or 1 to 6, or 1 to 4, including 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10; and r is an integer selected from 0 or 1 ; or (II) an R3group, wherein R3is hydrogen, an aliphatic group, a heteroaliphatic group, or an aromatic group, provided that if an R3group is present, then at least one Z2is -O[X(A)s(A’)mA"]q-Hr or - [X(A)s(A’)mA”]q-Hr; each of R1and R2, independently for each occurrence, is selected from hydrogen, aliphatic, heteroaliphatic, or aromatic; TG is a terminating group selected from hydrogen, hydroxyl, or alkoxy; and n is an integer selected from 1 to 10,000 or higher.

[0056] In particular aspects of the disclosure, each Y, independently for each occurrence in Formula I and / or Formula II, is a bond, an alkyl group, an alkenyl group, an alkynyl group, a heteroalkyl group, a heteroalkenyl group, a heteroalkynyl group, an aryl group, a heteroaryl group, or any combination thereof (e.g., aryl-heteroalkyl-aryl or ary l-alkyl). In representative aspects, Y is a bond, -Ph-CH2-N(H)-Ph, -Ph-CH2-, -[NH2]+[CH2CH2]t-, or -[NH2]+[CH2CH2Y’]t-, wherein Y’ is O, S, or NH, and t is an integer ranging from 1 to 8, such as 1 to 6, or 1 to 4, or 1 , 2, 3, 4, 5, 6, 7 or 8. In some aspects of the disclosure, Z1and / or each Z2(independently, for each occurrence) is selected from -0[P(0)(OH)0]i-ioH, -[P(0)(OH)0]i-ioH, -O[C(O)O]i- 10H, -[C(0)0]i-ioH, -0[S(0)20]I-IOH, -[S(0)20]I-IOH, -O[P(S)(OH)O]I-I0H, -[P(S)(OH)0]I-IOH, -0[C(S)0]I-IOH, - [C(S)0]i-ioH, -0[S(S)20]I-IOH, -[S(S)2O]I-I0H, -0[P(S)(SH)S]I-IOH, -[P(S)(SH)S]I-IOH, -0[C(S)S]I-IOH, - [C(S)S]i-ioH, -0[S(S)2S]I-IOH, -[S(S)2S]I-IOH, -O[P(S)(OH)S]I-I0H, -[P(S)(OH)S]I-IOH, -0[P(S)(SH)0]I-IOH, - [P(S)(SH)O]i-i0H, -0[P(0)(0 )0]i-ioH, -[P(0)(0 )0]i-ioH, -0[P(S)(0 )0]i-ioH, -[P(S)(O )O]i-i0H, -O[P(S)(S )S]i- 10H, -[P(S)(S-)S]i-ioH, -0[P(S)(0 )S]i-ioH, or -[P(S)(0 )S]i-ioH, -O[P(S)(S )O]i-i0H, -[P(S)(S )0]i-ioH, - OB(OH)2, -B(OH)2, -OB(O )2, -B(O )2, -ON(O)O', or -N(O)O'. In exemplary aspects of the disclosure, Z1and / or each Z2(independently, for each occurrence) is selected from -OP(O)(OH)2, -P(O)(OH)2, -O-C(O)OH,

[0057] In some aspects of the disclosure, at least one Z2group of a repeating “n” unit is selected from any such groups disclosed above and the other Z2is an R3group that is hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a heteroalkyl group, a heteroalkenyl group, a heteroalkynyl group, an aryl group, or a heteroaryl group. In some aspects of the disclosure, each of R1and R2independently, for each occurrence, is selected from hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a heteroalkyl group, a heteroalkenyl group, a heteroalkynyl group, an aryl group, or a heteroaryl group. In some aspects of the disclosure, the TG group is hydrogen or hydroxyl. In some particular aspects, one Z2is an R3group and R3is styrene group and the styrene group can be further coupled (e.g., via radical couplings / polymerization) to another styrene group, another Z2group that is selected from the groups listedabove in the definition for Z1and / or Z2, or a combination thereof. In some aspects, the external aliphatic portion of the R3styrene group is positioned para to the bond connecting the Z2 / R3group to the rest of Formula II.

[0058] In representative aspects of Formula I, Y is a bond and Z1is -P(O)(OH)2; -P(O)(O’)2; or - [P(O)(OH)O]2-4H or -[P(O)(O )O]2-4 (e.g., -P(O)(OH)O-P(O)(OH)OH, -P(O)(OH)O-P(O)(OH)O-P(O)(OH)OH, - P(O)(OH)O-P(O)(OH)O-P(O)(OH)O-P(O)(OH)OH, -P(O)(OjO-P(O)(Oj2, -P(O)(OH)O-P(O)(OH)O-P(O)(Oj2, or -P(O)(OH)O-P(O)(OH)O-P(O)(OH)O-P(O)(Oj2). In representative aspects of Formula II, and for each “n” repeat, the following options can apply: (i) one Y is a bond, alkyl (e.g., lower alkyl, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl), or -[NH2]+[CH2CH2]t-, or -[NH2]+[CH2CH2O]t-, wherein t is an integer ranging from 1 to 8, such as 1 to 6, or 1 to 4, or 1 , 2, 3, 4, 5, 6, 7 or 8, and the Z2attached to such Y is selected from -OP(O)(OH)2; -P(O)(OH)2; -OP(O)(O )2; -P(O)(O )2; or -O[P(O)(OH)O]2-P(O)(O )2); (II) the other Y is a bond and the Z2bound to such Y is R3, which in turn is hydrogen; and (iii) each of R1and R2is H.

[0059] Compounds of Formula I can also have structures according to Formula IA, wherein each of Z1and t can be as recited above for Formula I.Formula IA

[0060] Compounds of Formula II can also have structures according to any of Formulas IIA-IIF. With reference to Formulas I IB and / or I IC, each Y independently can be aliphatic or heteroaliphatic. With reference to Formulas IIA-IIF, each of Y, Z2, R1, R2, R3, TG, and n can be as recited above for Formulas I and / or II.Formula IICFormula HF

[0061] In representative aspects, the multivalent cation-binding ligand can be selected from:

[0062] The substrate of the disclosed composition is a solid substrate. The substrate comprises a functional group that extends from a surface of the substrate to which the multivalent cation-binding ligand is bound. In some aspects, the functional group is an aliphatic group, and aromatic group, or a heteroatomcontaining functional group. In some aspects of the disclosure, the functional group can be part of a coatingthat is formed on a base substrate, wherein the coating and the base substrate together provide the substrate to which the multivalent cation-binding ligand is bound; or it can be a functional group inherent to the substrate or that has been formed on the substrate. In some such aspects, the coating can be a polyhydroxyl surface coating and the base substrate can comprise cross-linked polystyrene] divinylbenzene. In exemplary aspects of the disclosure, the substrate comprising a base substrate and a coating can be a POROS® substrate sold by Applied Biosystems / Thermo Fisher Scientific.

[0063] Any substrate suitable for applications disclosed herein can be used. In some aspects, the substrate can be in the form of a resin, a bead, a sphere, a particle, a microcarrier, a membrane, a web, a bag, a bioreactor, a tube, a plate, an array, a filter, a fiber, a fabric, or any combination thereof. In some aspects of the disclosure, the substrate comprises a ceramic material, a glass material, a metal material, a silica material, a synthetic polymeric material (e.g., polystyrene-containing polymers), a natural polymeric material (e.g., carbohydrate-based materials, such as polysaccharides, including cellulose materials and the like), or any combination thereof. In some aspects, the substrate is a chromatographic resin, a membrane, a porous bead, a porous monolith, a winged fiber, a hollow fiber, a woven fabric, a non-woven fabric, silica, a Sepharose™ material, a porous polyvinylether polymeric bead, a non-porous bead (and derivatives thereof), a styrenic bead (and derivatives thereof), an acrylate bead (and derivatives thereof), an acrylamide containing one or more polymerizable vinyl groups, or any combination thereof. In exemplary aspects, the substrate can be selected from a substrate described by any of the following U.S. patents / publications: US Patent No. 5,334310, US Patent No. 5,453185, US Patent No. 5,593,729, US Patent No. 5,728,457, US Patent No. 5,929214, US Patent No. 6,238,565, US Patent No. 6,616,825, US Patent No. 5,833,861 , US Patent No. 5,605,623, US Patent No. 5,552, 041 , US Patent No. 8,940,172, US Patent No. 3,997,482, US Patent No. 8,356,717, US Patent No. 9,028683, US Patent Application Publication No. 20100160605 Al, US Patent Application Publication No. 20140073769 Al, US Patent Application Publication No. 20130245139 Al, US Patent Application Publication No. 20020043499 Al, and US Patent Application Publication No.20140316017 Al; the disclosures of each of which are incorporated herein by reference with respect to their relevant disclosure related to solid supports and methods of making solid supports, to the extent that such teachings are not in conflict with the present disclosure.

[0064] In aspects of the disclosure wherein the surface functional group is a heteroatom-containing functional group, the heteroatom-containing functional group of the substrate can comprise a heteroatom selected from an element belonging to Group 16 of the periodic table (e.g., oxygen, sulfur, selenium, tellurium, and polonium); nitrogen; boron; and the like. In particular aspects of the disclosure, the heteroatom is selected from (I) oxygen and the heteroatom-containing functional group is a hydroxyl group; or (II) sulfur and the heteroatom-containing functional group is a thiol group. A plurality of heteroatomcontaining functional groups can be present on the surface of the substrate and thus one or more of the heteroatom-containing functional groups independently can be bound to a multivalent cation-binding ligands to provide a substrate comprising a plurality of multivalent cation-binding ligands. In certain aspects of the disclosure, the heteroatom of the heteroatom-containing functional group is bound directly to a Z1group of the multivalent cation-binding ligand, wherein Z1is -O[X(A)s(A’)mA”]q-Hr or -[X(A)s(A')mA”]q-Hr, as defined for Formula I. In such aspects, a covalent bond typically is formed between the X atom of the Z1group and the heteroatom. In yet other aspects of the disclosure, the heteroatom is bound to a carbon atom of themultivalent cation-binding ligand, which in turn is directly or indirectly bound to a Z2group of the multivalent cation-binding ligand, wherein Z2is -O[X(A)s(A’)mA”]q-Hr or -[X(A)s(A’)mA”]q-Hr, as defined for Formula II. In such aspects, a covalent bond is formed between the carbon atom and the heteroatom.

[0065] In aspects of the disclosure wherein the surface functional group is an aliphatic or aromatic group, the aliphatic group can be selected from an alkyl-, alkenyl-, or alkynyl-containing group and the aromatic group can be selected from an aryl or heteroaryl group. In such aspects, the multivalent cation-binding ligand can be bound to a carbon atom of the aliphatic or aromatic group via a covalent bond. In exemplary aspects, the aliphatic group is a lower alkyl group (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, septyl, octyl, nonyl, or decyl) and the aromatic group is phenyl. In some aspects wherein the multivalent cationbinding ligand comprises a structure according to Formula IA, the amine group of the multivalent cationbinding ligand can be bound to an aliphatic group (e.g., a CH2 group) that is part of an epoxide ring. In such aspects, the amine group becomes bound to the substrate by attacking a methylene group of the epoxide and ring-opening the epoxide ring.

[0066] Representative formulas (Formulas III and IV) showing binding options for attaching the multivalent cation-binding ligand to the substrate via the functional group are provided below, with Formulas IVA and IVB showing further representative formulas for Formula IV. As illustrated with the wavy bond in Formulas I, IA, II, and 11 A- HF, the multivalent cation-binding ligand can be bound to the substrate at the positions indicated. With reference to Formulas III and IV (and IVA and IVB), X' represents a heteroatom provided by the heteroatom-containing functional group of the substrate that becomes bound to the multivalent cationbinding ligand; an aliphatic group; or an aromatic group. In particular aspects of the disclosure, X’ is O, S, NR’ (wherein R’ is hydrogen, aliphatic, or aromatic), Se, Te, Po, methyl, or phenyl; and the remaining variables of the Formula are as recited herein for Formulas I, IA, II, or IIA-IIF.Substrate— X'—Y—Z1Formula IIIFormula IVAFormula IVB

[0067] The composition can further comprise a positively-charged species, such as a multivalent cation species or a combination of a multivalent cation species and monovalent cation species. In some aspects of the disclosure, the positively-charged species can be associated with the multivalent cation-binding ligand. For example, these two groups can be associated by chemical interactions, such as covalent bonds, ionicbonds, and / or electrostatic interactions. In particular aspects, the positively-charged species associates with the multivalent cation-binding ligand through chemical interactions between the positively-charged species and oxygen atoms of the multivalent cation-binding ligand, such as through negatively-charged oxygen atoms, lone pair electrons of the oxygen atoms, or combinations thereof. FIGS. 1 A and 1 B provide exemplary illustrations of such interactions. In such aspects, the positively-charged species is a multivalent cationic species. In particular aspects, the multivalent cationic species has a 2+ oxidation state; however, other higher oxidations states, including 3+, 4+, or higher, are contemplated. In exemplary aspects, the multivalent cationic species can be selected from Ca2+, Mg2+, Fe2+, Zn2+, Cu2+, Cr2+, Ag2+, Co2+, Ni2+, Mn2+, Be2+, Sr2+, Ba2+, V2+, or any combination thereof. The multivalent cation-binding ligand can be associated with a plurality of positively-charged species, wherein each positively-charged species is the same or different. In exemplary aspects of the disclosure, the positively-charged species is Ca2+, Ni2+, Co2+, or any combination thereof.

[0068] In some aspects of the disclosure, the composition can further comprise a target (also referred to as a target molecule). In such aspects, the target can become part of the composition upon exposure of the multivalent cation-binding ligand-bound substrate to the target, which can be present in a separate solution, solid mixture, or some combination thereof. When the composition comprises the target, the target can either be associated or not associated with the multivalent cation-binding ligand. In aspects wherein the target is associated with the multivalent cation-binding ligand, the association of the target can occur through chemical interactions between functional groups of the target and (i) a positively-charged species that in turn is associated with the multivalent cation-binding ligand; and / or (ii) one or more A, A', and / or A” atoms of the multivalent cation-binding ligand (as defined for Formulas I and / or II). In aspects wherein the target is not associated with the multivalent cation-binding ligand, the target can be in a free state such that it exists with the multivalent cation-binding ligand and the substrate but is not bound thereto by any chemical interactions.

[0069] In some aspects of the disclosure, the composition can further comprise contaminants that are desired to be separated from a target or other media. In such aspects, the contaminants can become part of the composition upon exposure of the multivalent cation-binding ligand-bound substrate to the contaminants, which can be present in a separate solution, solid mixture, or some combination thereof. When the composition comprises the contaminants, the contaminants can either be associated or not associated with the multivalent cation-binding ligand. In aspects wherein the contaminants are associated with the multivalent cation-binding ligand, the association of a contaminant can occur through chemical interactions between functional groups of a contaminant and (i) a positively-charged species that in turn is associated with the multivalent cation-binding ligand; and / or (ii) one or more A, A’, and / or A” atoms of the multivalent cation-binding ligand (as defined for Formulas I and / or II). In aspects wherein the contaminants are not associated with the multivalent cation-binding ligand, the contaminants can be in a free state such that they exist with the multivalent cation-binding ligand and the substrate but are not bound thereto by any chemical interactions. In some aspects of the disclosure, the composition can comprise both contaminants and one or more targets in addition to the multivalent cation-binding ligand-bound substrate. In such aspects, the multivalent cation-binding ligand can be used to isolate the target from the contaminants either by associating with the target, in which instance contaminants can be removed; or by associating with the contaminants, in which instance the one or more targets can be removed.

[0070] In some aspects, the composition can comprise the multivalent cation-binding ligand as bound to the substrate and a mixture of contaminants and targets. In such aspects, the contaminants and targets can be separated from one another to isolate contaminants from any targets and / or to further isolate different targets from one another. In such aspects, the contaminants and / or targets can be separated / isolated as described above. In additional aspects of the disclosure, the composition can comprise solvents, buffers, and / or any other components that might be needed to adjust parameters of the composition during use (e.g., pH, temperature, viscosity, polarity, etc.).

[0071] A kit comprising the composition according to aspects disclosed herein also are described. The kit can comprise the multivalent cation-binding ligand and the substrate, with particular aspects having these two components coupled together as described herein. In some aspects, the substrate comprising the bound multivalent cation-binding ligand can be included in a container (e.g., pouch, wrapper, bottle, or other suitable container) when in the kit. In some aspects, the kit can further comprise a container that is configured to house the composition during use. For example, in some aspects of the disclosure, the container can be a column (e.g., a separatory column, a chromatographic column, or the like), a reactor, a well-plate, a cartridge, or other such container. The kit can further comprise instructions for use, buffers, or other reagents for use in combination with the composition.

[0072] Methods

[0073] Methods for making and using the composition disclosed here are described.

[0074] The method for making the composition can comprise combining a substrate and a multivalent cation-binding ligand as described herein. In some aspects, the method can comprise exposing a substrate as described herein to a multivalent cation-binding ligand precursor in the presence of suitable coupling reagents. In some aspects, the multivalent cation-binding ligand precursor can be selected from a halogenated phosphorus-containing reagent (such as a phosphate, phosphonate, or phosphate anhydride), a halogenated sulfur-containing reagent (such as a sulfate), a carboxyl-containing reagent (such as an anhydride, ester, or the like), a vinyl phosphorus-containing reagent, a vinyl sulfur-containing reagent, a vinyl carboxyl-containing reagent, or the like. In some aspects of the disclosure, the multivalent cation-binding ligand precursor is selected from phosphorous oxychloride, chlorosulfonic acid, succinic anhydride, vinyl phosphoric acid, vinylsulfonic acid, acrylic acid, bis(pinacolato)diboron, 2-allyl-4,4,5,5-tetramethyl-1 ,3,2- dioxaborolane, 4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)aniline, 4-(aminomethyl)benzeneboronic acid pinacol ester hydrochloride, or trimethyl borate. Coupling reagents can include reagents known to those in the art, with the benefit of the present disclosure, for forming covalent bonds between a functional group of the substrate surface (e.g., hydroxyl, thiol, amine, aromatic, or aliphatic groups) and the multivalent cationbinding ligand. Exemplary coupling reagents can include, but are not limited to, bases (e.g., triethylamine, diisopropylethylamine, KOt-Bu, and the like), Lewis acids (e.g., AICIs, AIF3, BF3-Et2O, etc.), redox reagents (ceric ammonium nitrate or “CAN,” ceric sulfate, iron persulfate, and the like), and suitable solvents.Schematic illustrations of different methods for making a composition according to the present disclosure are provided below in Scheme 1 and specific examples are also described herein.U Coupling Reagents oCl — P— Cl11Substrate I Substrate — rFGCl 'OHScheme 1

[0075] The method of using compositions according to aspects of the present disclosure can comprise exposing a sample to a composition disclosed herein. In some aspects, the method comprises exposing the sample to a composition comprising the substrate and the multivalent cation-binding ligand wherein the composition has first been exposed to the positively-charged species. In yet other aspects, the sample can be exposed to the composition before or concurrently with the positively-charged species. The sample canbe exposed to the composition by flowing the sample through a column comprising the composition. In yet other aspects of the disclosure, the method can comprise mixing the sample with the composition using suitable techniques (e.g., shaking, agitating, or the like). The sample can be in the form of a solid or a solution. In some aspects, the sample is in the form of a solution that can further comprise contaminants or other reagents (e.g., buffers, etc.). The method can be performed such that target species contained within the sample are extracted by the composition. In such aspects of the disclosure, the target species become associated with the multivalent cation-binding ligand and can thus be indirectly bound to the substrate. The target species can thus be separated from extraneous contaminants and sample solution. In yet other aspects of the disclosure, the method can be performed such that contaminants contained within the sample are extracted and removed from a target species also contained within the sample. In such aspects of the disclosure, the contaminants become associated with the multivalent cation-binding ligand and can thus be indirectly bound to the substrate. The contaminants can thus be separated from any target species that might be present in the sample.

[0076] In some aspects, the method can comprise eluting, from the composition, any contaminants or target species that have been bound indirectly to the substrate. In such aspects, the composition comprising the bound contaminant or target is subjected to an elution solution capable of removing the contaminant or the target from the composition and thus isolating the target, either by affirmatively eluting the target with the elution solution or eluting the contaminants with the elution solution, leaving the target associated with the substrate through the multivalent cation-binding ligand. In such aspect, the target can be finally stripped from the composition using another suitable elution solution. In some aspects, the composition can further be stripped of any positively-charged species that is associated with the multivalent cation-binding ligand using a stripping solution. The stripping solution can comprise an acidic species that facilitates providing hydrogen ions that can displace positively-charged ion species that are associated with the multivalent cation-binding ligand. In other aspects, the stripping solution can comprise a basic species that facilitates either providing 1 + or 2+ charged metal ions that can displace positively-charged ion species that are associated with the multivalent cation-binding ligand. Exemplary stripping solutions can comprise an acid selected from hydrochloric acid, nitric acid, acetic acid, phosphoric acid, sulfuric acid, formic acid, boric acid, citric acid, and the like or a base selected from sodium hydroxide, lithium hydroxide, and the like.

[0077] In certain aspects of the disclosure, the target molecule is a biological molecule of interest, for e.g., antibodies, proteins (including processed and / or modified proteins, such as a glycosylated protein), peptides, glycoproteins, lipoproteins, enzymes, nucleic acids (e.g., RNA, DNA, and the like), nucleoproteins, viruses, viral fragments, viral capsids, viral antigens, antigenic proteins, cellular markers, cells or particular cell types (e.g., certain types of T cells), a cellular component or cell parts, organelles, receptor proteins, vaccines, or any combinations thereof. In certain aspects of the disclosure, a "target molecule" of interest may have pharmaceutical, diagnostic, agricultural, and / or any of a variety of other properties that are useful in commercial, experimental, or other applications. In yet additional aspects of the disclosure, a "target molecule" of interest can be an antibody or protein therapeutic. In exemplary aspects of the disclosure, the "target molecule" is an antibody. Exemplary target molecules of interest are described herein in Table 1.

[0078] In aspects where the method is used to separate one or more contaminants from a target, the contaminants can include unwanted species other than a target. In some aspects, the contaminant may be undesired biological components, such as host ceil proteins, host cell metabolites, antibody fragments, protein fragments, nucleic acids, endotoxins, viruses or viral particles or viral protein fragments, impurities from cell culture (e.g., cells and their fragments, ceil culture media components, media additives, media derivatives, lipids, etc.); product-related contaminants, such as charge variants of a target molecule, truncated forms of a target molecule, or aggregates of a target molecule: process-related contaminants, such as reagents and / or by-products that originate from components used in a separation method (e.g., unwanted charged species, etc.); or combinations thereof.

[0079] In some aspects, the disclosed composition can be used as an anion exchange resin. In other aspects, the disclosed composition can be used as a cation exchange resin. In yet additional aspects, the composition can be used as a zwitterionic resin. Table 1 , below, details various applications in which compositions of the present disclosure that include a multivalent cation-binding ligand can be used, along with targeted modalities, conditions to be used (binding and elution conditions), the type of ion exchange involved, and the expected mode of operation. Those in the art will recognize how to conduct methods utilizing the conditions described in Table 1 with the benefit of the present disclosure.

[0080] Overview of Several Aspects

[0081] Disclosed herein is a composition, comprising: a substrate; and a multivalent cation-binding ligand covalently bound to a surface of the substrate, wherein the multivalent cation-binding ligand has a structure according to one of Formulas I or II- -Y-Z1Formula IFormula II wherein each Y, independently for each occurrence, is a bond, an aliphatic group, or a heteroaliphatic group; each Z1is selected from -O[X(A)s(A')mA"]q-Hror -[X(A)s(A')mA"]q-Hr, wherein each X, independently for each occurrence, is P, S, B, N, or C; each A, independently for each occurrence, is selected from O or S; each A’, independently for each occurrence, is selected from -OH, -SH, -O', or -S'; each A”, independently for each occurrence, is selected from O, S, O', or S'; each s, independently for each occurrence, is an integer selected from 0 or 1 ; each m, independently for each occurrence, is an integer selected from 0 or 1 ; q is an integer selected from 1 to 10; and r is an integer selected from 0 or 1 ; each Z2, independently for each occurrence, is selected from (I) -O[X(A)s(A’)mA”]q-Hror -[X(A)s(A’)mA”]q-Hr, wherein each X, independently for each occurrence, is P, S, N, B, or C; each A, independently for each occurrence, is selected from O or S; each A', independently for each occurrence, is selected from -OH, -SH, -O , or -S ; each A”, independently for each occurrence, is selected from O, S, O', or S'; each s, independently for each occurrence, is an integer selected from 0 or 1 ; each m, independently for each occurrence, is an integer selected from 0 or 1 ; q is an integer selected from 1 to 10; and r is an integer selected from 0 or 1 ; or (II) an R3group, wherein R3is hydrogen, an aliphatic group, a heteroaliphatic group, or an aromatic group, provided that if an R3group is present, then at least one Z2is -O[X(A)s(A,)mA,’]q-Hr or -[X(A)s(A’)mA”]q-Hr; each of R1and R2, independently for each occurrence, is selected from hydrogen, aliphatic, heteroaliphatic, or aromatic; TG is a terminating group selected from hydrogen, hydroxyl, or alkoxy; n is an integer selected from 1 to 10,000 or higher; and u is an integer selected from 0 or 1 .

[0082] In any or all aspects, m is 1 when X is C, B, N, or S, and m is 2 when X is P.

[0083] In any or all of the above aspects, each Y independently for each occurrence is selected from a bond, an alkyl group, an alkenyl group, an alkynyl group, a heteroalkyl group, a heteroalkenyl group, or a heteroalkynyl group.

[0084] In any or all of the above aspects, each Y is a bond, -Ph-CH2-N(H)-Ph, -Ph-CHz-. or - [NH2]+[CHzCH2]t-, or -[NH2]+[CH2CH2Y’]t-, wherein Y’ is O, S, or NH, and t is an integer ranging from 1 to 8.

[0085] In any or all of the above aspects, wherein Z1is selected from -OP(O)(OH)2, -P(O)(OH)2, -O- C(O)OH, -C(O)(OH), -O-S(O)2(OH), -S(O)2(OH), -OP(S)(OH)2, -P(S)(OH)2, -O-C(S)OH, -C(S)(OH), -O- S(S)2(OH), -S(S)2(OH), -OP(S)(SH)2, -P(S)(SH)2, -O-C(S)SH, -C(S)(SH), -O-S(S)2(SH), -S(S)2(SH), - OP(S)(OH)(SH), -P(S)(OH)(SH), -OP(O)(O )2, -P(O)(O )2, -O-C(O)O', -0(0)0', -O-S(O)2(O ), -S(O)2(Oj, - OP(S)(O )2, -P(S)(O )2, -O-C(S)O , -C(S)O-, -O-S(S)2O-, -S(S)2O , -OP(S)(Sj2, -P(S)(S )2, -O-C(S)S-, -C(S)S-, -O-S(S)2S-, -S(S)2S-, -OP(S)(O )(S ), or -P(S)(O )(Sj.

[0086] In any or all of the above aspects, Z1is selected from -P(O)(OH)2, -C(O)(OH), -S(O)2(OH), - P(S)(OH)2, -C(S)(OH), -S(S)2(OH), -P(S)(SH)2, -C(S)(SH), -S(S)2(SH), -P(S)(OH)(SH), -P(O)(O2, -C(O)O-, - S(O)2(O-), -P(S)(O-)2, -C(S)O-, -S(S)2O-, -P(S)(S-)2, -C(S)S-, or -P(S)(O )(S ).

[0087] In any or all of the above aspects, Z1is selected from -P(O)(OH)O-P(O)(OH)OH, -P(O)(OH)O- P(O)(OH)O-P(O)(OH)OH, -P(O)(OH)O-P(O)(OH)O-P(O)(OH)O-P(O)(OH)OH, -P(O)(O O-P(O)(O )2, - P(O)(OH)O-P(O)(OH)O-P(O)(O')2, or -P(O)(OH)O-P(O)(OH)O-P(O)(OH)O-P(O)(O )2.

[0088] In any or all of the above aspects, at least one Z2is selected from -OP(O)(OH)2, -P(O)(OH)2, -O- C(O)OH, -C(O)(OH), -O-S(O)2(OH), -S(O)2(OH), -OP(S)(OH)2J-P(S)(OH)2, -O-C(S)OH, -C(S)(OH), -O- S(S)2(OH), -S(S)2(OH), -OP(S)(SH)2, -P(S)(SH)2, -O-C(S)SH, -C(S)(SH), -O-S(S)2(SH), -S(S)2(SH), - OP(S)(OH)(SH), -P(S)(OH)(SH), -OP(O)(O )2, -P(O)(O )2, -O-C(O)O', -0(0)0', -O-S(O)2(O ), -S(O)2(Oj, - OP(S)(O-)2, -P(S)(O')2, -O-C(S)O-, -C(S)O-, -O-S(S)2O-, -S(S)2O', -OP(S)(S-)2, -P(S)(S')2, -O-C(S)S-, -C(S)S-, -O-S(S)2S , -S(S)2S , -OP(S)(O -)(S ), -P(S)(O )(S -), -OB(OH)2, -B(OH)2, -OB(O )2, -B(O )2, -ON(O)O , or - N(O)O-.

[0089] In any or all of the above aspects, at least one Z2is selected from -P(O)(OH)2, -C(O)(OH), - S(O)2(OH), -P(S)(OH)Z, -C(S)(OH), -S(S)2(OH), -P(S)(SH)Z, -C(S)(SH), -S(S)2(SH), -P(S)(OH)(SH), -P(O)(O- )2, -0(0)0-, -S(O)2(O ), -P(S)(O-)2, -C(S)O-, -S(S)2O-, -P(S)(S-)2, -C(S)S-, or -P(S)(O )(S ), -OB(OH)2, -B(OH)2, -OB(O')2, -B(O-)2, -ON(O)O-, or -N(O)O'.

[0090] In any or all of the above aspects, at least one Z2is selected from -P(O)(OH)O-P(O)(OH)OH, - P(O)(OH)O-P(O)(OH)O-P(O)(OH)OH, -P(O)(OH)O-P(O)(OH)O-P(O)(OH)O-P(O)(OH)OH, -P(O)(O )O- P(O)(O')2, -P(O)(OH)O-P(O)(OH)O-P(O)(O-)2, or -P(O)(OH)O-P(O)(OH)O-P(O)(OH)O-P(O)(O )2, - OP(O)(OH)O-P(O)(OH)OH, -OP(O)(OH)O-P(O)(OH)O-P(O)(OH)OH, -OP(O)(OH)O-P(O)(OH)O-P(O)(OH)O- P(O)(OH)OH, -OP(O)(O )O-P(O)(O')2, -OP(O)(OH)O-P(O)(OH)O-P(O)(O')2, or -OP(O)(OH)O-P(O)(OH)O- P(O)(OH)O-P(O)(O-)2.

[0091] In any or all of the above aspects, at least one Z2is R3, wherein R3is hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a heteroalkyl group, a heteroalkenyl group, a heteroalkynyl group, an aryl group, or a heteroaryl group.

[0092] In any or all of the above aspects, each of R1and R2independently, for each occurrence, is selected from hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a heteroalkyl group, a heteroalkenyl group, a heteroalkynyl group, an aryl group, or a heteroaryl group.

[0093] In any or all of the above aspects, the TG group is hydrogen or hydroxyl.

[0094] In any or all of the above aspects, the multivalent cation-binding ligand has a structure according toFormula IAFormula IA.

[0095] In any or all of the above aspects, the multivalent cation-binding ligand has a structure according to one of Formulas IIA-IIFFormula IICFormula HF.

[0096] In any or all of the above aspects, the multivalent cation-binding ligand is selected from any of the species disclosed herein.

[0097] In any or all of the above aspects, the substrate is a porous material, a non-porous material, a microporous material, a woven material, a non-woven material, a polymeric material, a non-polymeric material, or a fibrous material.

[0098] In any or all of the above aspects, the substrate is in the form of a resin, a bead, a sphere, a particle, a microcarrier, a membrane, a web, a bag, a bioreactor, a tube, a plate, an array, a filter, a fiber, a fabric, or any combination thereof.

[0099] In any or all of the above aspects, the substrate comprises a ceramic material, a glass material, a metal material, a silica material, a synthetic or natural polymeric material, or any combination thereof.

[0100] In any or all of the above aspects, the substrate comprises a functional group that extends from the surface of the substrate to which the multivalent cation-binding ligand according to Formula I is bound and wherein the multivalent cation-binding ligand is bound to the functional group via a direct covalent bond between Z1and the heteroatom.

[0101] In any or all of the above aspects, the multivalent cation-binding ligand and the substrate are bound as illustrated in Formula III, wherein X' is O; S; NR' wherein R’ is hydrogen, aliphatic, or aromatic; Se; Te;Po; methyl; or phenylSubstrate— X'—Y—Z1Formula III.

[0102] In any or all of the above aspects, the substrate comprises a functional group that extends from the surface of the substrate to which the multivalent cation-binding ligand according to Formula II is bound and wherein the multivalent cation-binding ligand is bound to the functional group via a covalent bond formed between the heteroatom and a carbon atom to which Z2is bound, directly or indirectly.

[0103] In any or all of the above aspects, the multivalent cation-binding ligand and the substrate are bound as illustrated in Formula IV wherein X’ is O; S; NR’ wherein R’ is hydrogen, aliphatic, or aromatic; Se; Te;Po; methyl; or phenylFormula IV.

[0104] In any or all of the above aspects, the functional group is a heteroatom-containing functional group that comprises a heteroatom selected from oxygen, sulfur, or nitrogen.

[0105] In any or all of the above aspects, the functional group is a heteroatom-containing functional group selected from a thiol group, a hydroxyl group, or an amine group.

[0106] In any or all of the above aspects, the composition further comprises a positively-charged species.

[0107] In any or all of the above aspects, the positively-charged species is associated with the multivalent cation-binding ligand.

[0108] In any or all of the above aspects, the positively-charged ion species is associated with the multivalent cation-binding ligand through coordination bonds formed between the positively-charged ion species and (i) negatively charged oxygen atoms of the multivalent cation-binding ligand, (ii) lone pair electrons of oxygen In any or all of the above aspects, the positively-charged ion species is a multivalent cationic species.

[0109] In any or all of the above aspects, the multivalent cationic species has a 2+, 3+, or 4+ oxidation state.

[0110] In any or all of the above aspects, the multivalent cationic species has a 2+ oxidation state.

[0111] In any or all of the above aspects, the positively-charged ion species is selected from Ca2+, Mg2+, Fe2+, Zn2+, Cu2+, Cr2+, Ag2+, Co2+, Ni2+, Mn2+, V2+, or any combination thereof.

[0112] In any or all of the above aspects, the composition comprises a plurality of positively-charged ion species, wherein each positively-charged ion species of the plurality is the same or different.

[0113] In any or all of the above aspects, the composition further comprises a target, a contaminant, or a combination thereof.

[0114] In any or all of the above aspects, the composition comprises the target and the target is associated with the substrate through interactions between the target and the positively-charged ion species.

[0115] In any or all of the above aspects, the target is a biological molecule of interest.

[0116] In any or all of the above aspects, the biological molecule of interest is selected from antibodies, proteins, peptides, glycoproteins, lipoproteins, enzymes, nucleic acids, nucleoproteins, viruses, virus-like particles, viral fragments, viral capsids, viral antigens, antigenic proteins, cellular markers, cells or particular cell types, a cellular component or cell parts, organelles, receptor proteins, vaccines, or any combinations thereof.

[0117] In any or all of the above aspects, the composition comprises the contaminant and the contaminant is associated with the substrate through interactions between the contaminant and the positively-charged ion species.

[0118] Also disclosed is a kit, comprising: the composition according to any or all of the above composition aspects; and a container configured to house the composition during use of the composition.

[0119] In any or all of the above aspects, the container is a chromatography column, a reactor, a wellplate, or a cartridge.

[0120] In any or all of the above aspects, the kit further comprises instructions for use, buffers, or reagents for use with the composition.

[0121] Also disclosed herein is a separation method, comprising: exposing a sample to the composition according to any or all of the above compositions to form a mixture; and exposing the mixture to a solution to facilitate separating a target from the sample.

[0122] In any or all of the above aspects, the sample comprises the target and one or more contaminants.

[0123] In any or all of the above aspects, the target is a biological molecule.

[0124] In any or all of the above aspects, the biological molecule of interest is selected from antibodies, proteins, peptides, glycoproteins, lipoproteins, enzymes, nucleic acids, nucleoproteins, viruses, viral fragments, viral capsids, viral antigens, antigenic proteins, cellular markers, cells or particular cell types, a cellular component or cell parts, organelles, receptor proteins, vaccines, or any combinations thereof.

[0125] In any or all of the above aspects, the solution is an elution solution formulated to dissociate the target from the multivalent cation-binding ligand so as to isolate the target from the sample.

[0126] In any or all of the above aspects, the method further comprises exposing the composition to a stripping solution after separating the target from the sample.

[0127] In any or all of the above aspects, the stripping solution comprises an acidic species or a basic species.

[0128] In any or all of the above aspects, the method further comprises forming the composition prior to exposing the sample to the composition, wherein forming the composition comprises combining a composition according to any or all of the above composition aspects with a positively-charged ion species.

[0129] In any or all of the above aspects, the composition is used as an anion exchange resin, a cation exchange resin, or a zwitterionic resin.

[0130] In any or all of the above aspects, the composition according to any or all composition aspects described above is used for isolating an antibody and / or an antibody-derived product, wherein isolating includes removing aggregates, homodimers, fragments, DNA, host cell proteins, viral agents, or any combination thereof.

[0131] In any or all of the above aspects, the composition according to any or all composition aspects described above is used for capturing a recombinant protein, peptide, or enzyme; a virus or a virus-like particle; an adeno-associated virus; a nucleic acid; a plasma-derived protein; or any combination thereof.

[0132] Also disclosed is a composition comprising: a solid substrate; and an associating means for associating a positively-charged ion species with the solid substate.

[0133] In any or all of the above aspects, the associating means is a multivalent cation-binding ligand.

[0134] ExamplesExample 1

[0135] In this example, a multivalent cation-binding ligand was prepared wherein phosphate groups were attached directly to hydroxyl groups present on a surface of a substrate. The synthesis is summarized in Scheme 2 below.Scheme 2

[0136] Prior to reaction, all reaction components and glassware exposed to reagents were oven dried at 100°C for 24 hours. A three neck round bottom flask assembled with overhead stirring and addition funnel sealed with septum stoppers was flushed with dry nitrogen. To the reactor was added 10.0 g (4.2 mmol) of an oven dried polyhydroxylated resin (an exemplary substrate) followed by sealing the reaction vessel with septum stoppers and flushing the reactor with dry nitrogen for 5 minutes.

[0137] To the resin / reactor was added 10OmL of anhydrous toluene via cannula. The resin slurry was stirred (200rpm) until uniform. To the slurry was added triethyl amine 0.90 mL (6.3 mmol) in a single portion via syringe and stirred until uniform.

[0138] 20 mL of anhydrous toluene was added to the addition funnel followed by 9.8 mL (105 mmol) of phosphorous oxychloride. The phosphorous oxychloride solution was added in a single addition after which the reaction was allowed to stir at ambient temperature for 18 hours.

[0139] After 18 hours, the reaction solid was isolated by filtration and washed (30 minutes per wash, 100 mL per wash) with a solution comprising 3 x Acetone:D.L water 1 :1 , 2 x D.l. Water, EthanokD.L Water and Acetone. After final wash, the substrate-bound multivalent cation-binding ligand was isolated by filtration and dried under vacuum at 60°C until at constant weight.Example 2

[0140] In this example, a multivalent cation-binding ligand was prepared wherein phosphate groups were attached indirectly to hydroxyl groups present on a surface of a substrate. The synthesis is summarized in Scheme 3 below.Scheme 3

[0141] To a three neck round bottom flask assembled with overhead stirring, a reflux condenser, and heating mantle was added 10.0 g oven dried polyhydroxylated resin (an exemplary substrate) followed by 40 mL of D.L water. The mixture was stirred (200 rpm) and sparged with anhydrous nitrogen for 30 minutes then kept under nitrogen atmosphere.

[0142] To the resin / reactor was added 5.22 mL (34.0 mmol) vinyl phosphoric acid. The resin slurry was stirred (200rpm) until uniform and sparged with anhydrous nitrogen for an additional 30 minutes. The sparge line was removed and to the reactor was added 2.08 g (3.8 mmol) ammonium cerium (IV) nitrate in a single addition. To the reactor was added a reflux condenser and it was kept under positive nitrogen atmosphere.

[0143] To the reactor was added an internal feedback probe and it was heated to 70°C over 20 minutes, causing an internal exothermic reaction (100°C) before settling to 70°C. The reaction continued to stir for 2 hours under nitrogen atmosphere with heat. After 2 hours, heat was removed and the reaction continued to stir while cooling to ambient temperature. The reaction continued to stir at ambient temperature for 16 hours.

[0144] After 18 hours, the reaction solid was isolated by filtration and washed with using 30 minutes per wash, 200 mL per wash. Washes comprised 7 x D.L water and 1 x Acetone. After the final wash, the substrate-bound multivalent cation-binding ligand was isolated by filtration and dried under vacuum at 60°C until at constant weight.Example 3

[0145] In this example, a multivalent cation-binding ligand was prepared wherein sulfate groups were attached directly to hydroxyl groups present on a surface of a substrate. The synthesis is summarized in Scheme 4 below. j| 1 . anhyd Tol / NEt3 0\+Cl—s— OH- II _Substrate — f-OH ] II 2. H2O Substrate — FO 'u0H)V / m O V S 'mScheme 4

[0146] Prior to reaction, all reaction components and glassware exposed to reagents were oven dried at 100°C for 24 hours. A three neck round bottom flask assembled with overhead stirring and an addition funnel sealed with septum stoppers was flushed with dry nitrogen. To the reactor was added 10.0 g of oven dried polyhydroxylated resin (an exemplary substrate) followed by sealing the reaction vessel with septum stoppers and flushing the reactor with dry nitrogen for 5 minutes.

[0147] To the resin / reactor was added 100 mL anhydrous toluene (10mL per gram of resin) via cannula. The resin slurry was stirred (200rpm) until uniform. To the slurry was added triethyl amine in a single portion via syringe and stirred until uniform. The reaction vessel was then cooled with an external ice bath for 15 minutes until an internal temperature of 0°C was achieved.

[0148] 16 mL of anhydrous toluene was added to the addition funnel via syringe followed by 6.98 mL (12.23g) of chlorosulfonic acid via glass syringe. Chlorosulfonic acid solution was added drop-wise over 5 minutes after which the ice bath was removed and the reaction was allowed to stir while warming to ambient temperature for 18 hours.

[0149] After 18 hours, to the reaction was added 100mL of a 1 :1 (v:v) of D.L water:2-propanol solution dropwise over 20 minutes to quench the reaction. The solid was isolated by filtration and washed (30 mins per wash, 100 mL per wash) with a solution comprising 3 x Acetone:D.I. water 1 :1 , 2 x D.l. Water, Ethanol:D.L Water and Acetone. After final wash, substrate-bound multivalent cation-binding ligand was dried under vacuum at 60°C until at constant weight.Example 4

[0150] In this example, a multivalent cation-binding ligand was prepared wherein carboxylate groups were attached directly to hydroxyl groups present on a surface of a substrate. The synthesis is summarized in Scheme 5 below.Scheme 5

[0151] To a three neck round bottom flask assembled with overhead stirring was added 10.0 g oven dried poly hydroxylated resin followed by sealing the reaction vessel with septum stoppers.

[0152] To the resin / reactor was added 70mL DMSO (10mL per gram of resin). The resin slurry was stirred (200rpm) until uniform. 0.34g of sodium methoxide was dissolved in 30 mL of DMSO and was added to the reaction slurry. The resin / base slurry continued stirring for 30 minutes at 30°C. 8.41g of succinic anhydride was dissolved 10 mL of DMSO then added in a single addition after which the reaction was heated to 60°C and allowed to stir at temperature for 18 hours.

[0153] After 18 hours, the reaction solid was isolated by filtration and washed (30 minutes per wash, 100 mL per wash) with a solution comprising 3 x MethanokD.I. water 1 :1 , 2 x D.l. Water, EthanokD.I. Water and Acetone. After a final wash, the substrate-bound multivalent cation-binding ligand was dried under vacuum at 60°C until at constant weight.Example 5

[0154] In this example, a multivalent cation-binding ligand was prepared wherein 2-aminoethyl dihydrogen phosphate groups were attached directly to epoxy groups present on a surface of a substrate. The synthesis is summarized in Scheme 6 below.Scheme 6

[0155] Prior to reaction, all reaction components and glassware exposed to reagents were oven dried at 100 °C for 24 hours. A three neck round bottom flask assembled with overhead stirring and an addition funnel sealed with septum stoppers was flushed with dry nitrogen. To the reactor was added 10.0 g of oven dried epoxy functionalized-polymer resin (an exemplary substrate) followed by sealing the reaction vessel with septum stoppers and flushing the reactor with dry nitrogen for 5 minutes. To the reactor was added 60mL of anhydrous toluene via cannula and stirred (200 rpm) until uniform. The reactor was charged triethylamine via syringe and continued to stir for 5 mins. 8 mL of phosphorus oxychloride was added to an addition funnel via syringe followed by 10 mL of anhydrous toluene. Phosphorus oxychloride solution was added dropwise to the resin / triethylamine / toluene solution over 2 minutes. After addition, the addition funnel was rinsed with an additional 10 mL of anhydrous toluene. Reaction contents continued to stir at ambient temperature over 18 hours.

[0156] After 18 hours, the reaction was isolated by filtration to give an off-white solid. The solid was then washed with washes consisting of 50-75mL of solvent, stirring for 10 mins / wash. Washes included a 1 :1mixture of D.L water and methanol. Washes continued until pH of reaction wash was equal to pH of virgin methanol :D.L water solution. After final wash, the substrate-bound multivalent cation-binding ligand was dried under vacuum at 60 °C until at constant weight.Example 6

[0157] In this example, a multivalent cation-binding co-polymer was prepared wherein vinyl phosphoric acid and divinyl benzene monomers were copolymerized via emulsion polymerization. The synthesis is summarized in Scheme 6 below.Scheme 6

[0158] Aqueous prep: To a single neck flask with magnetic stir bar was added 200ml of water followed by 1.76g polyvinyl alcohol (PVA). The flask was stirred while heating to 80°C. The solution was stirred until contents were fully dissolved. The solution was removed from heat and to the solution was added 12.88g of sodium chloride dissolved in 25mL water. The solution was stirred until uniform then transferred to a 500mL jacketed reactor with overhead stirring and stir shaft comprised of a lower PTFE, Turbine Agitator and upper four-Blade PTFE Agitator Stirrer Blades. The solution was stirred and brought to 20°C with external bath. The solution was stirred and degassed under N2 for20 minutes at 200rpm.

[0159] Organic prep: To a single neck 250mL RBF was added 10.50g of vinyl phosphoric acid (VPA) in a single portion. 16.15g of divinyl benzene (DVB) (previous stripped with Alumina) was added to the RBF followed by 25mL of Acetonitrile in 5mL portions until uniform.

[0160] Polymerization: The reaction vessel with aqueous suspension stir rate was increased to 400 rpm and the organic phase was added 0.574g azobisisobutyronitrile (AIBN) in a single addition. Once organic phase was uniform, a long thin funnel was added to the reaction vessel and the organic phase was added at a continuous rate until fully added to the aqueous phase. The reaction continued to stir at 400rpm while the internal temperature was ramped to 70 °C over 1 hour. Reaction continued heating at 70 °C over 18 hours.

[0161] Reaction quench: After 18 hours, the reaction solid was isolated by filtration. The off-white solid was then washed with 75mL of solvent and stirring for 10 mins / wash. Washes included a 1 :1 mixture of D.L water and methanol. Washes continued until pH of reaction wash was equal to pH of virgin methanol:D.L water solution.Example 7

[0162] In this example, substrate-bound multivalent cation-binding ligands according to the examples above were evaluated for binding of different positively-charged species.

[0163] The substrate-bound multivalent cation-binding ligands (referred to herein as “sample resin”) are suspended in a 20mM solution of a positively-charged species, such as an M2+metal (10 mL per gram of sample resin). The sample resin is slurried for 20-30 minutes then isolated by filtration. Solids are then washed with D.L water until the filtrate wash reaches a pH of 7. The resulting chelate comprising the substrate-bound multivalent cation-binding ligand associated with the positively-charged species can then be used for multivalent metal retention separations.

[0164] The following M2+metal ion chelates were effectively chelated with the substrate-bound multivalent cation-binding ligand examples described above: magnesium2-, calcium2-, iron2-, colbalt2-, copper2-.

[0165] Particular results are summarized below in Table 2 for examples comprising the sample resin of Example 1 (referred as “Comp A” in Table 2) using the following protocol: 0.75g of polyphosphate sample resin was added to 9.0 mL of 0.1 M M2+solution at ambient temperature. The solids were slurried for 2 minutes by inversion then allowed to settle by gravity. Once settled, a 2mL sample of the reaction liquor was removed and analyzed using a Thermo Scientific Nanodrop 2000 spectrophotometer (available from Thermo Fisher Scientific, Waltham, MA) with a 1cm pathlength.

[0166] Initial ionic capacity testing determined Comp A to be 365 umol / mL (1.46 mmol / g) for 3 PO groups. Preliminary chelating results, above, estimate M2+capacity between 0.2-0.3 mmol / g. Chelating with two equivalents per M2+group, maximum capacity estimates to 0.73 mmol / g. Non optimized M2- chelate efficiency (27-41 %). Differences between calculated and gravimetric M2+concentration values are a result of atmospherically absorbed moisture. Cobalt(ll)chloride was made with a previously unopened container.Example 8

[0167] In this example, various compositions according to the present disclosure were evaluated, wherein the substrates were bound directly or indirectly to at least one phosphate group. The compositions were evaluated for their ability to selectively separate different target species (i.e., proteins including myoglobin, ovalbumin, chymotripsinogen A, and cytochrome C).

[0168] First, dynamic binding capacity (DBC) with lysozyme was evaluated and compared with results obtained using a commercial hydroxyapatite separation medium (CHT, a ceramic hydroxyapatite resin) (see Table 3). Several examples exhibited higher lysozyme binding capacity as compared to the commercial CHT resin.

[0169] Separations were then performed with certain examples and the different target species, with the resulting chromatograms being shown in FIGS. 2-6, wherein FIG. 2 shows results for composition 4; FIG. 3 shows results for composition 7 (as compared against CHT); FIG. 4 shows results for composition 3 (as compared against CHT); FIG. 5 shows results for composition 4 (as compared against CHT); and FIG. 6 shows results for composition 8 (as compared against CHT). These figures illustrate the ability of the compositions to facilitate separating different target species from one another.Example 9

[0170] In this example, the ability of compositions disclosed herein to separate a target (e.g., myoglobin) from a sample was evaluated as well as the ability of the composition to be regenerated. Table 4 provides results wherein no regeneration was conducted and Table 5 provides results with Ca2+regeneration with Ca(OH)2, demonstrating that the composition can incorporate calcium and facilitate quantitative elutions of myoglobin from the composition.Example 10

[0171] In this example, an mRNA static binding assay was conducted with four different mRNA species having 40, 1000, 2500, and 4000 nucleotides using compositions according to Examples 8 and / or 11 . Theresults are shown in FIG. 7, which shows that compositions of the disclosure are able to exhibit higher mRNA binding than a commercial hydroxyapatite medium, even over a variety of mRNA sizes.Example 11

[0172] In this example, an IgG static binding assay was conducted with monoclonal antibodies. The results are summarized in Table 6 below, which shows that compositions of the disclosure are able to exhibit higher mAb yields and percent recovery than a commercial hydroxyapatite medium.Example 12

[0173] In this example, a binding assay was performed to evaluate the effect of pH and salt on the efficacy of a composition comprising multivalent cation-binding ligands according to the present disclosure. The multivalent cation-binding ligands used in this example are illustrated below (denoted as (550) Vinyl, (R6)PO3, and (150)P020P03), with the resin to which they are bound being illustrated as a circle. A control (denoted as “CHT”), was also used as a comparison example. O3,

[0174] For the binding assay, the evaluations were conducted using 96-well plates, with each well containing 20 pL of a 50% slurry of resins comprising a multivalent cation-binding ligand according to the present disclosure. The resins were equilibrated with buffers of either 10 mM or 25 mM sodium phosphate at pH levels of 5.0, 5.8, 6.8, and 9.2, respectively. Concentrated Herceptin monoclonal antibodies (mAbs),containing 10% aggregates, were loaded onto the resins at loading densities of 20, 40, 60, or 80 gg / gL of resin. The plates were then shaken at 400 rpm for 30 minutes. The mAbs were eluted using buffers containing either 0.2 M or 0.5 M NaCI at various pH levels. The eluates were collected in 1 mL deep 96-well plates. To measure protein concentration, 100 gL of each sample was transferred to a UV-transparent plate, and absorbance at 280 nm was read using a Varioskan plate reader. Protein concentrations were determined based on a standard curve for the monoclonal antibodies. Results are summarized in Tables 7- 10 for examples at pH 6.8 using 25 mM NaPC at 0.2 M NaCI (Table 7) or 0.5 M NaCI (Table 8), or using 10 mM NaPC>4 at 0.2 M NaCI (Table 9) or 0.5 M NaCI (Table 10). Results are summarized in Tables 11 -14 for examples at pH 5.8 using 25 mM NaPC at 0.2 M NaCI (Table 11 ) or 0.5 M NaCI (Table 12), or using 10 mM NaPO at 0.2 M NaCI (Table 13) or 0.5 M NaCI (Table 14). Results are summarized in Tables 15 and 16 for examples at pH 9.2 using 10 mM NaPO4 at 0.2 M NaCI (Table 15) or 0.5 M NaCI (Table 16). Results are summarized in Tables 17 and 18 for examples at pH 5.0 using 10 mM NaPC>4 at 0.2 M NaCI (Table 17) or 0.5 M NaCI (Table 18).Example 13

[0175] In this example, an aggregation separation assay also was performed with these compositions. For the aggregation separation assay, Monomer purity and aggregate levels were analyzed by Size- Exclusion Chromatography using a Thermo Scientific MAbPac-1 column on a Thermo Fisher HPLC 3000 system. Twenty micrograms of loading materials and elution collections were loaded with sodium phosphate and sodium chloride mobile phase. Peak areas were used to calculate monomer recovery and aggregate removal percentages.

[0176] Results for monomer and aggregation purifications based on 10 mM NaPC , with elutions using 0.5M NaCI, and pH 6.8 are summarized in Table 19, below. Results for monomer and aggregation purifications based on 10 mM NaPC>4, with elutions using 0.5M NaCI, and pH 5.8 are summarized in Table 20, below. Results for monomer and aggregation purifications based on 10 mM NaPO4, with elutions using 0.5M NaCI, and pH 9.2 are summarized in Table 21 , below. And, results for monomer and aggregation purifications based on 10 mM Sodium Acetate, with elutions using 0.5M NaCI, and pH 5.0 are summarized in Table 22, below.

[0177] As can be seen with data in the tables above, the multivalent cation-binding ligands tested for MAb binding capacity performed well at lower phosphate buffer solutions (10mM NaPC ). The multivalent cationbinding ligands tolerated buffers >pH 5.0 and had successful Mab binding capacity. MAb recovery percentage varied between 25% to 85% for the different multivalent cation-binding ligands that were evaluated, monomer percent recovery was >90% and aggregate percent recovery ranged from 10 to 20.5% depending on the multivalent cation-binding ligand used and the differences in buffer composition and buffer pH.Example 14

[0178] In this example, RNA binding to multivalent cation-binding ligands was evaluated. RNAs with various sizes (1000-4000 base pairs) were incubated with ten different multivalent cation-binding ligands, then were eluted by low pH according to final step of previously developed protocol. All phosphoric prototypes demonstrated higher efficacy of binding over commercially available CHT resin. Six out of ten resin prototypes demonstrated three times higher efficacy of binding / elution over CHT resin. Newly developed phosphoric resins can be recommended to companies which are looking for more efficient and faster RNA purifications from IVT (In Vitro Translation) culture. Results are summarized in Table 23, below.Example 15

[0179] Calf thymus DNA binding to phosphoric resins prototypes. Commercially available calf thymus DNA (13.000 base pairs) were exposed to four newly synthesized phosphoric resin prototypes. Two of four demonstrated 50% binding after ten minutes of incubation. This approach can be used to collect very low concentration of template DNAs from any buffers / media. Results are shown in summarized in Table 24, below, wherein compositions 12, 13, and 14 comprised a multivalent cation-binding ligand having a structure as illustrated below.Compositions 12 and 13 Composition 14Example 16

[0180] In this example, residual DNA clearance of AAV samples using compositions according to the present disclosure were evaluated. Results are summarized in Table 25, below.

[0181] During AAV production, DNA impurities, such as templates (5-10%), are typically present, which can mislead the estimation of actual AAV concentration based on UV absorbance measurement at 260nm. As established with this example, a procedure wherein a final product is allowed to flow through a composition according to the present disclosure can be used to thereby eliminate this issue including scale- up manufacturing. DNA molecules incorporated into viral particle cannot be affected as viral DNA / RNA are protected by capsid structure.

[0182] In view of the many possible aspects to which the principles of the present disclosure may be applied, it should be recognized that the illustrated aspects are only preferred examples of the disclosure and should not be taken as limiting the scope of the present disclosure. Rather, the scope of the disclosure is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A composition, comprising: a substrate; and a multivalent cation-binding ligand covalently bound to a surface of the substrate, wherein the multivalent cation-binding ligand has a structure according to one of Formulas I or II- -Y-Z1Formula IFormula II wherein each Y, independently for each occurrence, is a heteroaliphatic group, a bond, or an aliphatic group; each Z1is selected from -O[X(A)s(A’)mA”]q-Hr or -[X(A)s(A’)mA”]q-Hr, wherein each X, independently for each occurrence, is P, S, B, N, or C; each A, independently for each occurrence, is selected from O or S; each A', independently for each occurrence, is selected from -OH, -SH, -O', or -S'; each A”, independently for each occurrence, is selected from O, S, O', or S'; each s, independently for each occurrence, is an integer selected from 0 or 1 ; each m, independently for each occurrence, is an integer selected from 0 or 1 ; q is an integer selected from 1 to 10; and r is an integer selected from 0 or 1 ; each Z2, independently for each occurrence, is selected from(I) -O[X(A)s(A’)mA”]q-Hr or -[X(A)s(A')mA”]q-Hr, wherein each X, independently for each occurrence, is P, S, N, B, or C; each A, independently for each occurrence, is selected from O or S; each A’, independently for each occurrence, is selected from -OH, -SH, -O', or -S'; each A”, independently for each occurrence, is selected from O, S, O', or S'; each s, independently for each occurrence, is an integer selected from 0 or 1 ; each m, independently for each occurrence, is an integer selected from 0 or 1 ; q is an integer selected from 1 to 10; and r is an integer selected from 0 or 1 ; or(ii) an R3group, wherein R3is hydrogen, an aliphatic group, a heteroaliphatic group, or an aromatic group, provided that if an R3group is present, then at least one Z2is -O[X(A)s(A’)mA”]q-HrOr -[X(A)s(A’)mA"]q-Hr; each of R1and R2, independently for each occurrence, is selected from hydrogen, aliphatic, heteroaliphatic, or aromatic;TG is a terminating group selected from hydrogen, hydroxyl, or alkoxy; n is an integer selected from 1 to 10,000 or higher; and u is an integer selected from 0 or 1 .

2. The composition of claim 1 , wherein m is 1 when X is C, B, N, or S, and m is 2 when X is P.

3. The composition of claim 1 or claim 2, wherein each Y independently for each occurrence is selected from a bond, an alkyl group, an alkenyl group, an alkynyl group, a heteroalkyl group, a heteroalkenyl group, or a heteroalkynyl group.

4. The composition of any one of claims 1 -3, wherein each Y is a bond, -Ph-CH2-N(H)-Ph, -Ph- CH2-, or -[NH2]+[CH2CH2]t-, or -[NH2]+[CH2CH2Y']t-, wherein Y’ is O, S, or NH, and t is an integer ranging from 1 to 8.

5. The composition of any one of claims 1 -4, wherein Z1is selected from -OP(O)(OH)2, - P(O)(OH)2, -O-C(O)OH, -C(O)(OH), -O-S(O)2(OH), -S(O)2(OH), -OP(S)(OH)2, -P(S)(OH)2, -O-C(S)OH, - C(S)(OH), -O-S(S)2(OH), -S(S)2(OH), -OP(S)(SH)2, -P(S)(SH)2, -O-C(S)SH, -C(S)(SH), -O-S(S)2(SH), - S(S)2(SH), -OP(S)(OH)(SH), -P(S)(OH)(SH), -OP(O)(O )2, -P(O)(Oj2, -O-C(O)O-, -0(0)0’, -O-S(O)2(O ), - S(O)2(O ), -OP(S)(O )2, -P(S)(O )2, -O-C(S)O , -C(S)O , -O-S(S)2O-, -S(S)2O-, -OP(S)(S )2, -P(S)(S 2, -o- C(S)S-, -C(S)S-, -O-S(S)2S-, -S(S)2S-, -OP(S)(O )(S ), or -P(S)(O )(Sj; optionally wherein Z1is selected from - P(O)(OH)2, -C(O)(OH), -S(O)2(OH), -P(S)(OH)2, -C(S)(OH), -S(S)2(OH), -P(S)(SH)2, -C(S)(SH), -S(S)2(SH), - P(S)(OH)(SH), -P(O)(O-)2, -C(O)O-, -S(O)2(O ), -P(S)(O )2, -C(S)O’, -S(S)2O’, -P(S)(Sj2, -C(S)S-, or -P(S)(O’ )(S ); further optionally wherein Z1is selected from -P(O)(OH)O-P(O)(OH)OH, -P(O)(OH)O-P(O)(OH)O- P(O)(OH)OH, -P(O)(OH)O-P(O)(OH)O-P(O)(OH)O-P(O)(OH)OH, -P(O)(O O-P(O)(O )2, -P(O)(OH)O- P(O)(OH)O-P(O)(O-)2, or -P(O)(OH)O-P(O)(OH)O-P(O)(OH)O-P(O)(O’)2.

6. The composition of any one of claims 1 -4, wherein at least one Z2is selected from - OP(O)(OH)2, -P(O)(OH)2, -O-C(O)OH, -C(O)(OH), -O-S(O)2(OH), -S(O)2(OH), -OP(S)(OH)2, -P(S)(OH)2, -O- C(S)OH, -C(S)(OH), -O-S(S)2(OH), -S(S)2(OH), -OP(S)(SH)2, -P(S)(SH)2, -O-C(S)SH, -C(S)(SH), -O- S(S)2(SH), -S(S)2(SH), -OP(S)(OH)(SH), -P(S)(OH)(SH), -OP(O)(O-)2, -P(O)(O-)2, -O-C(O)O-, -C(O)O-, -o- S(O)2(O ), -S(O)2(O ), -OP(S)(O2, -P(S)(O-)2, -O-C(S)O-, -C(S)O-, -O-S(S)2O-, -S(S)2O-, -OP(S)(S2, - P(S)(S2, -O-C(S)S-, -C(S)S-, -O-S(S)2S-, -S(S)2S-, -OP(S)(O )(S ), -P(S)(Oj(Sj, -OB(OH)2, -B(OH)2, -OB(O- )2, -B(O )2, -ON(O)O’, or -N(O)O’; optionally wherein at least one Z2is selected from -P(O)(OH)2, -C(O)(OH), -S(O)Z(OH), -P(S)(OH)2, -C(S)(OH), -S(S)Z(OH), -P(S)(SH)2, -C(S)(SH), -S(S)Z(SH), -P(S)(OH)(SH), -P(O)(O- )2, -C(O)O-, -S(O)2(O ), -P(S)(O-)2, -C(S)O-, -S(S)2O-, -P(S)(S-)2, -C(S)S-, or -P(S)(O )(S ), -OB(OH)2, -B(OH)2, -OB(O )2, -B(O )2J-ON(O)O‘, or -N(O)O_; further optionally wherein at least one Z2is selected from - P(O)(OH)O-P(O)(OH)OH, -P(O)(OH)O-P(O)(OH)O-P(O)(OH)OH, -P(O)(OH)O-P(O)(OH)O-P(O)(OH)O- P(O)(OH)OH, -P(O)(O-)O-P(O)(O-)2, -P(O)(OH)O-P(O)(OH)O-P(O)(O )2, or -P(O)(OH)O-P(O)(OH)O- P(O)(OH)O-P(O)(O )2, -OP(O)(OH)O-P(O)(OH)OH, -OP(O)(OH)O-P(O)(OH)O-P(O)(OH)OH, -OP(O)(OH)O- P(O)(OH)O-P(O)(OH)O-P(O)(OH)OH, -OP(O)(OjO-P(O)(O )2, -OP(O)(OH)O-P(O)(OH)O-P(O)(O )2, or - OP(O)(OH)O-P(O)(OH)O-P(O)(OH)O-P(O)(O-)2.

7. The composition of any one of claims 1 -4 or 6, wherein at least one Z2is R3, wherein3is hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a heteroalkyl group, a heteroalkenyl group, a heteroalkynyl group, an aryl group, or a heteroaryl group.

8. The composition of any one of claims 1-4, 6, or 7, wherein each of R1and R2independently, for each occurrence, is selected from hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a heteroalkyl group, a heteroalkenyl group, a heteroalkynyl group, an aryl group, or a heteroaryl group.

9. The composition of any one of claims 1 -4 or 6-8, wherein the TG group is hydrogen or hydroxyl.

10. The composition of claim 1, wherein the multivalent cation-binding ligand has a structure according to Formula IAFormula IA.11 . The composition of claim 1 , wherein the multivalent cation-binding ligand has a structure according to one of Formulas IIA-IIEFormula IIC Formula HDFormula HE.

12. The composition of any one of claims 1-11 , wherein the multivalent cation-binding ligand is selected from13. The composition of any one of claims 1-12, wherein the substrate is a porous material, a non-porous material, a microporous material, a woven material, a non-woven material, a polymeric material, a non-polymeric material, or a fibrous material.

14. The composition of any one of claims 1-13, wherein the substrate is in the form of a resin, a bead, a sphere, a particle, a microcarrier, a membrane, a web, a bag, a bioreactor, a tube, a plate, an array, a filter, a fiber, a fabric, or any combination thereof.

15. The composition of any one of claims 1-14, wherein the substrate comprises a ceramic material, a glass material, a metal material, a silica material, a synthetic or natural polymeric material, or any combination thereof.

16. The composition of any one of claims 1-15, wherein the substrate comprises a functional group that extends from the surface of the substrate to which the multivalent cation-binding ligand according to Formula I is bound and wherein the multivalent cation-binding ligand is bound to the functional group via a direct covalent bond between Z1and the heteroatom.

17. The composition of any one of claims 1-5, 10, or 12-16, wherein the multivalent cationbinding ligand and the substrate are bound as illustrated in Formula III, wherein X’ is O; S; NR’ wherein R’ is hydrogen, aliphatic, or aromatic; Se; Te; Po; methyl; or phenylSubstrate— X'—Y—Z1Formula III.

18. The composition of any one of claims 1-4, 6-9, or 11-16, wherein the substrate comprises a functional group that extends from the surface of the substrate to which the multivalent cation-binding ligand according to Formula II is bound and wherein the multivalent cation-binding ligand is bound to the functional group via a covalent bond formed between the heteroatom and a carbon atom to which Z2is bound, directly or indirectly.

19. The composition of any one of claims 1-4, 6-9, 11 -16, or 18, wherein the multivalent cationbinding ligand and the substrate are bound as illustrated in Formula IV wherein X' is O; S; NR' wherein R’ is hydrogen, aliphatic, or aromatic; Se; Te; Po; methyl; or phenylFormula IV.

20. The composition of any one of claims 1-19, wherein the functional group is a heteroatomcontaining functional group that comprises a heteroatom selected from oxygen, sulfur, or nitrogen.21 . The composition of any one of claims 1 -20, wherein the functional group is a heteroatomcontaining functional group and is selected from a thiol group, a hydroxyl group, or an amine group.

22. The composition of any one of claims 1 -21 , further comprising a positively-charged species.

23. The composition of claim 22, wherein the positively-charged species is associated with the multivalent cation-binding ligand.

24. The composition of any one of claims 22 or 23, wherein the positively-charged ion species is associated with the multivalent cation-binding ligand through coordination bonds formed between the positively-charged ion species and (i) negatively charged oxygen atoms of the multivalent cation-bindingligand, (ii) lone pair electrons of oxygen atoms of the multivalent cation-binding ligand, or (iii) a combination thereof.

25. The composition of any one of claims 22-24, wherein the positively-charged ion species is a multivalent cationic species having a 2+, 3+, or 4+ oxidation state.

26. The composition of claim 24 or 25, wherein the multivalent cationic species has a 2+ ox id at ion state.

27. The composition of any one of claims 22-26, wherein the positively-charged ion species is selected from Ca2+, Mg2+, Fe2+, Zn2+, Cu2+, Cr2+, Ag2+, Co2+, Ni2+, Mn2+, V2+, or any combination thereof.

28. The composition of any one of claims 22-26, comprising a plurality of positively-charged ion species, wherein each positively-charged ion species of the plurality is the same or different.

29. The composition of any one of claims 1 -28, further comprising a target, a contaminant, or a combination thereof.

30. The composition of claim 29, wherein the composition comprises the target and the target is associated with the substrate through interactions between the target and the positively-charged ion species.31 . The composition of claim 29 or claim 30, wherein the target is a biological molecule of interest.

32. The composition of claim 31 , wherein the biological molecule of interest is selected from antibodies, proteins, peptides, glycoproteins, lipoproteins, enzymes, nucleic acids, nucleoproteins, viruses, virus-like particles, viral fragments, viral capsids, viral antigens, antigenic proteins, cellular markers, cells or particular cell types, a cellular component or cell parts, organelles, receptor proteins, vaccines, or any combinations thereof.

33. The composition of claim 29, wherein the composition comprises the contaminant and the contaminant is associated with the substrate through interactions between the contaminant and the positively-charged ion species.

34. A kit, comprising: the composition according to any one of claims 1 -28; and a container configured to house the composition during use of the composition.

35. The kit of claim 34, wherein the container is a chromatography column, a reactor, a wellplate, or a cartridge.

36. The kit of claim 34 or claim 35, further comprising instructions for use, buffers, or reagents for use with the composition.

37. A separation method, comprising: exposing a sample to the composition according to any one of claims 22-28 to form a mixture; and exposing the mixture to a solution to facilitate separating a target from the sample.

38. The method of claim 37, wherein the target is a biological molecule and the sample comprises the target and one or more contaminants.

39. The method of claim 38, wherein the biological molecule of interest is selected from antibodies, proteins, peptides, glycoproteins, lipoproteins, enzymes, nucleic acids, nucleoproteins, viruses, viral fragments, viral capsids, viral antigens, antigenic proteins, cellular markers, cells or particular cell types, a cellular component or cell parts, organelles, receptor proteins, vaccines, or any combinations thereof.

40. The method of any one of claims 37-39, wherein the solution is an elution solution formulated to dissociate the target from the multivalent cation-binding ligand so as to isolate the target from the sample.41 . The method of any one of claims 37-30, further comprising exposing the composition to a stripping solution after separating the target from the sample.

42. The method of claim 41 , wherein the stripping solution comprises an acidic species or a basic species.

43. The method of any one of claims 37-42, further comprising forming the composition prior to exposing the sample to the composition, wherein forming the composition comprises combining a composition according to any one of claims 1-24 with a positively-charged ion species.

44. The method of any one of claims 37-43, wherein the composition is used as an anion exchange resin, a cation exchange resin, or a zwitterionic resin.

45. Use of a composition according to any one of claims 1-28 for isolating an antibody and / or an antibody-derived product, wherein isolating includes removing aggregates, homodimers, fragments, DNA, host cell proteins, viral agents, or any combination thereof.

46. Use of a composition according to any one of claims 1-28 for capturing a recombinant protein, peptide, or enzyme; a virus or a virus-like particle; an adeno-associated virus; a nucleic acid; a plasma-derived protein; or any combination thereof.

47. A composition comprising: a solid substrate; and an associating means for associating a positively-charged ion species with the solid substate.

48. The composition of claim 47, wherein the associating means is a multivalent cation-binding ligand.

Images