Hydrophobic interaction chromatography (HIC) composition and method of producing the HIC composition

Abstract

A hydrophobic interaction chromatography (HIC) composition includes a solid phase substrate and a hydrophobic-modified hydrophilic ligand covalently coupled to the solid phase substrate. The hydrophobic-modified ligand includes a hydrophilic ligand portion covalently bonded to the solid phase substrate with the hydrophilic ligand portion including a polar group and a plurality of hydroxyl groups. The hydrophobic-modified ligand further includes a hydrophobic ether-containing segment including at least one ether group, with the hydrophobic ether-containing segment directly or indirectly covalently coupled to the hydrophilic ligand portion.

Description

HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC) COMPOSITION AND METHOD OF PRODUCING THE HIC COMPOSITIONGOVERNMENT SUPPORT

[0001] This invention was made with government support under GM140789 awarded by the National Institutes of Health. The government has certain rights in this invention.CROSS-REFERENCE TO RELATED APPLICATION

[0002] The present application claims priority to and all the benefits of U.S. Provisional Patent Application No. 63 / 561,910 filed on March 16, 2024, which is hereby expressly incorporated herein by reference in its entirety.FIELD OF THE DISCLOSURE

[0003] The present disclosure generally relates to a chromatographic composition for use in hydrophobic interaction chromatography.BACKGROUND

[0004] Hydrophobic interaction chromatography (HIC) is a chromatographic method that employs salt solutions, generally in aqueous conditions, to bring about the reversible association of molecules with a suitably modified surface. A conventional application is to employ HIC for biomolecule separations, to effect isolation of a target biomolecule, or class of biomolecules, or to conduct analysis of a mixture of such molecules. Recently, the method has become favored for separations of protein biomolecules, as it can be conducted under conditions that are considered mild, or less likely to disrupt native, biologically functional, protein structures. In recent years, chemical modification of proteins has become a more common practice, yielding mixtures of proteins containing the native protein structure, as well as varying levels of chemically modified variants of the native protein starting material. In practice, it is sometimes simpler to analyze such variants under milder, native conditions, which to not disrupt the covalent or non-covalentstructures of native proteins or assemblies of biomolecules. Thus, the complexity of such mixtures requires increasingly sophisticated separations methods for analysis of components and impurities. The application of HIC is not limited to proteins, and has been applied to other biomolecules, including carbohydrates, nucleic acids, and complex molecular assemblies, including biomolecular complexes of proteins, nucleic acids, carbohydrates, and the like, as well as conjugates, subcellular organelles, viruses and the like. The features of the method can be complex, with a variety of potential or known interactions occurring between the targets of interest, the chemically modified chromatographic surface, and mobile phase solvent or solvent additives used to manipulate the separation. In broad terms, the purpose of the chromatographic material in a separation is to encourage the differential migration of chemical species in space and time, in response to a flow of material within a defined device or condition. Thus, the composition of the flowing stream, commonly referred to as the mobile phase, the rate of transfer of the mobile phase, structure and composition features of the sample, and features of the chromatographic surface, commonly referred to as the stationary phase, as well as external features, such as temperature of operation, all define the nature of the separative process. The features of the chromatographic surface of the stationary phase, including specific features of the chemical structure of the surface, define the associations of the sample elements with the surface, relative to the probability that the sample elements will remain in the flow stream. However, conventional stationary phases for HIC are known to have limitations in resolution of complex samples, as well as are challenged for the time required for the separation of sample components (speed or throughput). Chemical modifications of proteins can also complicate HIC separations, particularly when highly hydrophobic chemical modifiers are covalently attached to the protein structure, which may result in sample components that require stationary phases of greater or lesser retention properties in theuse of common mobile phases used in this mode of separation. Thus, there remains an opportunity to develop an improved composition useful as a stationary phase for HIC.SUMMARY OF THE DISCLOSURE AND ADVANTAGES

[0005] In one aspect of the present disclosure, a hydrophobic interaction chromatography (HIC) composition includes a solid phase substrate and a hydrophobic-modified hydrophilic ligand covalently coupled to the solid phase substrate. The hydrophobic-modified ligand includes a hydrophilic ligand portion covalently bonded to the solid phase substrate with the hydrophilic ligand portion including a polar group and a plurality of hydroxyl groups. The hydrophobic- modified ligand also includes a hydrophobic ether-containing segment directly or indirectly covalently coupled to the hydrophilic ligand portion.

[0006] In another aspect of the present disclosure, a method of producing a HIC composition for hydrophobic interaction chromatography is provided. The method includes providing a solid phase substrate and a hydrophilic ligand including a polar group and a plurality of hydroxyl groups with at least one hydroxyl group present at or near the terminus of the hydrophilic ligand. The method also includes reacting the solid phase substrate and the hydrophilic ligand to covalently couple the hydrophilic ligand to the solid phase substrate to form a hydrophilic-modified substrate. The method further includes providing an activation compound including a leaving group and reacting the activation compound with at least one hydroxyl group of at least some portion of the surfacebound hydrophilic-modified substrate to form an activated hydrophilic-modified substrate. The method further includes providing a hydrophobic ether-containing compound comprising an ether group and a nucleophile. The method further includes reacting the activated hydrophilic-modifiedsubstrate with the nucleophile of the hydrophobic ether-containing compound to release the leaving group of the activation compound and form the hydrophobic-modified hydrophilic ligand and the HIC composition.

[0007] The HIC composition is useful for HIC separations of proteins, modified proteins, and various biomolecules due to the balance and location of hydrophilic and hydrophobic interactions in combination with minimized ionic interactions.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Advantages of the present invention will be readily appreciated, as the same becomes better understood by reference to the following detailed description, when considered in connection with the accompanying drawings.

[0009] Separation of proteins by HIC can be a complicated process, conducted under conditions designed to preserve the native state of the proteins, while allowing for the separation of a complex mixture of proteins. The present invention concerns chromatographic materials that permit manipulation of protein separations to effect improved resolution of protein mixtures.

[0010] Figure 1A is an exemplary separation of a three-component protein mixture (lysozyme, Denosumab and Trastuzumab) using an ethyl-methoxide (C2OC1) ligand modified HIC column, with the surface modified at 0.30 pmol / m2of ligand, using a phosphate buffered mobile phase and gradient elution of ammonium sulfate.

[0011] Figure IB is an exemplary separation of a three-component protein mixture (lysozyme, Denosumab and Trastuzumab) using an ethyl -ethoxi de (C2OC2) ligand modified HIC column, with the surface modified at 0.30 pmol / m2of ligand, using a phosphate buffered mobile phase and gradient elution of ammonium sulfate.

[0012] Figure 1C is an exemplary separation of a three-component protein mixture (lysozyme, Denosumab and Trastuzumab) using a bis-ethyleneoxide-methoxide ((C2O)2C1) ligand modified HIC column, with the surface modified at 0.30 pmol / m2of ligand, using a phosphate buffered mobile phase and gradient elution of ammonium sulfate.

[0013] Figure ID is an exemplary separation of a three-component protein mixture (lysozyme, Denosumab and Trastuzumab) using a propyl-butoxide (C3OC4) ligand modified HIC column, with the surface modified at 0.30 pmol / m2of ligand, using a phosphate buffered mobile phase and gradient elution of ammonium sulfate.

[0014] Figure 2A is an exemplary separation of a three-component protein mixture (lysozyme, Denosumab and Trastuzumab) using an ethyl-ethoxide (C2OC2) ligand modified HIC column, with the surface modified at 0.45 pmol / m2of ligand, using a phosphate buffered mobile phase and gradient elution of ammonium sulfate.

[0015] Figure 2B is an exemplary separation of a three-component protein mixture (lysozyme, Denosumab and Trastuzumab) using an ethanol-ethoxide (C2OC2OH) ligand modified HIC column, with the surface modified at 0.45 pmol / m2of ligand, using a phosphate buffered mobile phase and gradient elution of ammonium sulfate.

[0016] Figure 3A is an exemplary separation of a three-component protein mixture (lysozyme, Denosumab and Trastuzumab) using an ethyl-ethoxide (C2OC2) ligand modified HIC column, with the surface modified at 0.30 pmol / m2of ligand, using a phosphate buffered mobile phase and gradient elution of ammonium sulfate.

[0017] Figure 3B is an exemplary separation of a three-component protein mixture (lysozyme,Denosumab and Trastuzumab) using an ethyl-ethoxide (C2OC2) ligand modified HIC column,with the surface modified at 0.79 pmol / m2of ligand, using a phosphate buffered mobile phase and gradient elution of ammonium sulfate.

[0018] Figure 3C is an exemplary separation of a three-component protein mixture (lysozyme, Denosumab and Trastuzumab) using an ethyl-ethoxide (C2OC2) ligand modified HIC column, with the surface modified at 0.97 pmol / m2of ligand, using a phosphate buffered mobile phase and gradient elution of ammonium sulfate.

[0019] Figure 4A is an exemplary separation of an antibody drug conjugate (ADC) sample (enfortumab-vedotin) using a 3 -propoxy-ethoxide (C2OC3) ligand modified HIC column, with the surface modified at 0.17 pmol / m2of ligand, using a phosphate buffered mobile phase and gradient elution of decreasing ammonium sulfate and increasing isopropanol.

[0020] Figure 4B is an exemplary separation of an antibody drug conjugate (ADC) sample (enfortumab-vedotin) using a 3 -propoxy-ethoxide (C2OC3) ligand modified HIC column, with the surface modified at 0.26 pmol / m2of ligand, using a phosphate buffered mobile phase and gradient elution of decreasing ammonium sulfate and increasing isopropanol.

[0021] Figure 4C is an exemplary separation of an antibody drug conjugate (ADC) sample (enfortumab-vedotin) using a 3 -propoxy-ethoxide (C2OC3) ligand modified HIC column, with the surface modified at 0.47 pmol / m2of ligand, using a phosphate buffered mobile phase and gradient elution of decreasing ammonium sulfate and increasing isopropanol.

[0022] Figure 4D is an exemplary separation of an antibody drug conjugate (ADC) sample (enfortumab-vedotin) using a 3-propoxy-ethoxide (C2OC3) ligand modified HIC column, with the surface modified at 0.70 pmol / m2of ligand, using a phosphate buffered mobile phase and gradient elution of decreasing ammonium sulfate and increasing isopropanol.

[0023] Figure 5A shows overlaid chromatograms for the separations of four proteins (Ribonuclease, Lysozyme, Denosumab and Ipilimumab) using a mPEG(550) ligand modified HIC column, with the surface modified at 0.91 pmol / m2of ligand, using a phosphate buffered mobile phase and gradient elution of ammonium sulfate.

[0024] Figure 5B shows overlaid chromatograms for the separations of four proteins (Ribonuclease, Lysozyme, Denosumab and Ipilimumab) using an ePEG3 ligand modified HIC column, with the surface modified at 0.87 pmol / m2of ligand, using a phosphate buffered mobile phase and gradient elution of ammonium sulfate.DETAILED DESCRIPTION OF THE DISCLOSURE

[0025] The present disclosure provides a hydrophobic interaction chromatography (HIC) composition. The HIC composition is useful for HIC separations. For example, the HIC composition is useful as a stationary phase in HIC separations.

[0026] The HIC composition includes a solid phase substrate and a hydrophobic-modified hydrophilic ligand covalently coupled to the solid phase substrate. The hydrophobic-modified hydrophilic ligand includes a hydrophilic ligand portion covalently bonded to the solid phase substrate, and a hydrophobic ether-containing segment directly or indirectly covalently coupled to at least some portion of the hydrophilic ligand portion. In other words, the hydrophobic ether- containing segment modifies the hydrophilic nature of the hydrophilic ligand portion and provides at least one ether linkage extending from the hydrophilic ligand portion.

[0027] The hydrophilic ligand portion includes a polar group and a plurality of hydroxyl groups. The polar group of the hydrophilic ligand portion may be selected from a carbonate, a carbamate, an amide, an amine, a ureido, an ether, a thioether, a sulfinyl, a sulfoxide, a sulfonyl, a thiourea, a thiocarbonate, or a thiocarbamate. The aforementioned functionality may also be included in aheterocyclic compound. For example, the polar group may be an aromatic ring including an amine. In one aspect, the polar group X is selected from an amide or a carbamate. The plurality of hydroxyl groups present on the hydrophilic ligand portion may be 2 or more hydroxyl groups. Alternatively, the hydrophilic ligand portion may include 2 to 8, 2 to 7, or 3 to 5, hydroxyl groups.

[0028] Referring first to the solid phase substrate, although not required, the solid phase substrate is typically silica. The silica used for the HIC composition is not limited to any particular grade, or morphology. Both nonporous spherical silica and porous silica, including superficially porous silica, may be used. The silica particles typically have an average diameter particle size of from 0.5-100 pm, from 1-50 pm, from 1.5-10 pm, or from 1.7-5 pm. The porous silica may have an average pore diameter of greater than or equal to about 80 A, greater than or equal to about 250 A, greater than or equal to about 300 A, greater than or equal to about 450 A, from 200 to 1,000 A, from 250 to 900 A, or from 300 to 850 A. Alternatively, although pore diameters below 70 A are typically avoided, it is contemplated that the average pore diameter may be from about 1 to about 50 A, from about 5 to about 40 A, or from about 10 to about 30 A. The surface of the silica particles typically includes silica hydroxyl groups, so-called silanols, which are useful for covalent coupling of various reagents to the silica surface, such as the hydrophilic ligand portion. Mostly commonly, specific organosilane reagents are employed for these silica surface modifications to form a covalently-attached bonded phase. Suitable grades of silica are available under the tradename Halo Silica from Advanced Materials Technologies having a principal place of business in Wilmington, DE, but many silica materials are widely available as commercial materials for a variety of useful applications. Alternative substrates include hybrid inorganic / organic material. Within the context of this disclosure, the term “hybrid inorganic / organic material” includes inorganic-based structures wherein an organic functionality is integral to both internal core (i.e., inorganic structure) as wellas the hybrid material surface. The inorganic portion of the hybrid material may be, e.g., alumina, silica, titanium, cerium, or zirconium or oxides thereof, or ceramic material. Further alternative substrates include completely organic substrates that include hydroxyl groups at the surface of the organic substrate. For the purposes of this disclosure, the solid phase substrate is not formed from carbohydrates. However, carbohydrates could be included when covalently bonded to inorganic or hybrid inorganic / organic materials.

[0029] Although not required, the hydrophobic-modified hydrophilic ligand may be represented by Formula I:(R1O)3Si-[C(R2)(R3)]n-X-[C(R2)(R3)]n-[C(R4)(R5)]m-Zp-Y Formula I, wherein:X is the polar group;Z is a connecting group;Y is the hydrophobic ether segment; n is 1-6; n’ is 0-2; m is 1-8; p is 0 or 1;R1, R2, R3, is independently H or a straight or branched, substituted or unsubstituted, Cl to C18 alkyl group; andR4and R3is independently H or OH, with the proviso that the hydrophobic-modified hydrophilic ligand includes at least two hydroxyl groups. It is to be appreciated that the phrase “m units” throughout this disclosure is merely a convenient reference to the repeat unit with the subscript“m” in Formula I.

[0030] When the hydrophobic-modified hydrophilic ligand is represented by Formula I, the hydrophilic ligand portion is represented by Formula la:(R1O)3Si-[C(R2)(R3)]n-X-[C(R2)(R3)]1f-[C(R4)(R5)]m- Formula laWherein:X is the polar group; n is 1-6; and n’ is 0-2; m is 1-8R1, R2, R3, is independently H or a straight or branched, substituted or unsubstituted, Cl to C18 alkyl group; andR4and R5is independently H or OH, with the proviso that the hydrophilic ligand portion includes at least two hydroxyl groups.

[0031] Although not required, typically p is 1 such that the connecting group is present in the hydrophobic-modified hydrophilic ligand.

[0032] The polar group X is independently chosen from a carbonate, a carbamate, an amide, an amine, a ureido, an ether, a thioether, a sulfinyl, a sulfoxide, a sulfonyl, a thiourea, a thiocarbonate, or a thiocarbamate, including heterocyclic compounds including the polar functionality. For example, the polar group may be an aromatic ring including an amine. Or as another example, the polar group X may be bonded to or within in an aromatic ring with the polar group X being a thioether group. In one aspect, the polar group X is selected from an amide or a carbamate. In another aspect, the polar group X is an amide. When the polar group X is an amide, the hydrophobic-modified hydrophilic ligand may be represented by Formula lb:Formula lb.

[0033] In certain aspects of Formula I and Formula lb, n is 2-4, m is 3-6, p is 1, and R1, R2, R3is independently H or a straight or branched, substituted or unsubstituted, Cl to C6 alkyl group.Although not required, n’ is typically 0 when X is an amide. In other aspects of Formula I, when X is a ureido, n’ is 1 or 2. In one aspect of Formula lb, n is 3, X is an amide, m is 5, and four of the m units include only one hydroxyl group. In one aspect, the hydrophobic-modified hydrophilic ligand is represented by Formula Ic:Formula Ic.

[0034] In certain aspects, p is 1 such that the connecting group Z is included in the hydrophobic- modified hydrophilic ligand. Although not required, the connecting group Z is typically a carbamate group when p is 1. In certain aspects of Formula Ic, in which the polar group X is an amide, the connecting group Z is also present, m is 5 with four of the m units including only one hydroxyl group, and the hydrophobic-modified hydrophilic ligand is represented by Formula II:Formula II.

[0035] When connecting group Z is a carbamate group, Formula II is further represented byFormula Ila:

[0036] Referring now to the hydrophobic ether-containing segment, as described above, the hydrophobic ether-containing segment modifies the hydrophilic ligand portion. The hydrophobic ether-containing segment is directly or indirectly covalently coupled to the hydrophilic ligand portion. The hydrophobic ether-containing segment is considered to be directly covalently coupled to the hydrophilic ligand portion when the connecting group Z is not present (i.e., when subscript p is 0). Conversely, the hydrophobic ether-containing segment is considered to be indirectly covalently coupled to the hydrophilic ligand portion when the connecting group Z is present (i.e., when subscript p is 1).

[0037] The hydrophobic ether-containing segment includes at least one ether linkage. Within the context of this disclosure, “an ether linkage” means an organic molecule with an oxygen atom bonded to two segments of alkyl or aryl hydrocarbon groups. The groups on either side of the oxygen may be the same (symmetrical ethers), or different (mixed ethers). These ethers may be composed of segments that are repeated, or which have several unique ether segments, connected in a variety of ways, including branching, linear repeats, or the like. Thus, the “hydrophobic ether- containing segment” can be of a variety of segmented ethers, or ethers connected to a variety of organic groups, with a requirement of at least one ether linkage.

[0038] In certain embodiments, the hydrophobic ether-containing segment (Y) is represented byFormula VI:-[(R10)-O-(R10)]s-[(R10)-O-(R10)]s’-(O-R11)v Formula VI, wherein: R10is independently a C1-C8 alkyl or branched alkyl group or an aromatic hydrocarbon; R11is independently hydrogen, a C1-C8 alkyl or branched alkyl group, or an aromatic hydrocarbon; s is 1 to 250; s’ is 0 to 250, and v is 0 or 1. In other embodiments, R10is independently a C2-C4 alkyl or branched alkyl group; and the sum of s and s’ is from 1 to 10.

[0039] To reduce the complexity of the chromatographic separation of proteins by HIC, contributions of ion exchange mechanisms to retention need to be controlled, or even eliminated. In many cases, the hydrophobic ether-containing segment will be chosen to not contain functional groups which are ionizable at pH values of 3 to 9. In other words, the hydrophobic ether-containing segment is typically neutral at pH values of 3 to 9. Not only do the compositions that are neutral at pH values of 3 to 9 promote hydrophobic interaction, but the neutral compositions also minimize ionic interactions, resulting in more predictable HIC separations, which is desired especially for complex amphipathic molecules like proteins.

[0040] Persons of ordinary skill in the art will appreciate that some degree of ionizability of ether compositions could have advantage for certain separations, and that this could mean weak ion exchange capacity or interactions that may have practical benefits. Thus, ionized groups are not entirely excluded for potential utility, but, for practice of HIC, are of less critical value than the correct formation of hydrophobic properties that promote effective HIC separations, including good protein recovery and chromatographic properties, such as retention control, peak symmetry and chromatographic efficiencies. Similarly, polar, unionized groups, separate from ether oxygens, may be present in ether compositions, or may be produced from ether compositions after chemical treatment.

[0041] In certain embodiments, the hydrophobic-modified hydrophilic ligand is derived fromFormula VII:Formula VII, wherein v is 1 to 3.

[0042] In one embodiment, the hydrophobic-modified hydrophilic ligand is derived fromFormula Vila:Formula Vila.In other embodiments, the hydrophobic-modified hydrophilic ligand is derived from one or more of the following structures:

[0043] Additional hydrophobic ether-containing segments are also contemplated and can be customized based on the target molecules requiring separation. In particular, on the basis of this disclosure, hydrophobic ether-containing segments can be chosen considering the following characteristics. The hydrophobic ether-containing segments are of defined molecular weight, which permits certainty for design, and reproducibility of synthesis or commercial acquisition. Lower molecular weight compositions will be able to undergo favored reaction rates during synthesis of chromatographic compositions, with less limited diffusion of reactants for surface immobilization. Higher molecular weight compositions may form thicker coatings on the interactive surface, and in the case wherein porous solid phases structures are selected (such as may be the case for porous or superficially porous particles, or membranes), pore structures can be crowded or filled by such higher molecular weight ethers, limiting the ability of proteins or other large molecules to productively interact with the interactive surface, or producing a composition that suffers from restricted diffusion. Additionally, higher molecular weight reactants are often obtained or available as mixtures with a defined molecular weight distribution, rather than a singular discrete composition.

[0044] Referring back to the HIC composition as a whole, in addition to having the hydrophobic- modified hydrophilic ligand coupled to the solid phase substrate, the HIC composition may also have a hydrophilic ligand (i.e., a hydrophilic ligand without hydrophobic modification) coupled to and / or covalently bonded to the solid phase substrate. In other words, in certain aspects, both the hydrophilic ligand and the hydrophobic-modified hydrophilic ligand are covalently coupled to the solid phase substrate.

[0045] The polar group of the hydrophilic ligand may be selected from a carbonate, a carbamate, an amide, an amine, a ureido, an ether, a thioether, a sulfinyl, a sulfoxide, a sulfonyl, a thiourea, athiocarbonate, or a thiocarbamate, including heterocyclic compounds including the polar functionality. For example, the polar group may be an aromatic ring including an amine. In one aspect, the polar group is selected from an amide, or a carbamate. The plurality of hydroxyl groups present on the hydrophilic ligand may be 2 or more hydroxyl groups. Alternatively, the hydrophilic ligand may include 2 to 8, 2 to 7, or 3 to 5, hydroxyl groups.

[0046] In one aspect, the hydrophilic ligand is represented by Formula V:(R1O)3Si-[C(R2)(R3)]n-X-[C(R2)(R3)]n’-[C(R4)(R5)]m-[C(R8)(R9)]qFormula V, wherein:X is the polar group; n is 1-6; n’ is 0-2; m is 1-8; q is 1;R1, R2, R3, is independently H or a straight or branched, substituted or unsubstituted, Cl to C18 alkyl group;R4and R5is independently H or OH, with the proviso that at least two instances of R4and / or R5is OH; andR8and R9is independently H or OH provided that at least one of R8and R9is OH. In total, the hydrophilic ligand represented by Formula V includes at least three hydroxyl groups. At least two hydroxyl groups are provided from R4and / or R5and at least one hydroxyl group is provided by R8and / or R9.

[0047] The hydroxyl group of R8and / or R9included in unit q may also be referred to as a terminal hydroxyl group. Those having ordinary skill in the art will appreciate that the hydrophilic ligandportion of the hydrophobic-modified hydrophilic ligand and the hydrophilic ligand share a similar structure, with the exception that the hydrophilic ligand portion does not include unit q (i.e., - [C(R8)(R9)]q) and thus does not include a terminal hydroxyl group. Accordingly, the hydrophilic ligand may include each of the various structural configurations described above for of the hydrophilic ligand portion with the exception that the hydrophilic ligand further includes the q unit.

[0048] In one aspect, the hydrophilic ligand of Formula V is further represented by Formula Va:Formula Va.

[0049] When the HIC composition includes the hydrophilic ligand in addition to the hydrophobic-modified hydrophilic ligand, the relative amount of each ligand can be optimized based on the particular analyte that is the subject of the separation. For example, in certain aspects, the hydrophobic-modified hydrophilic ligand and the hydrophilic ligand are present in a molar ratio range of from of 1 :20 to 20: 1. Alternatively, the hydrophobic-modified hydrophilic ligand and the hydrophilic ligand may be present in a molar ratio range of from 1 : 10 to 10: 1, from 2:8 to 8:2, from 3:7 to 7:3, from 4:6 to 6:4, or about 1 :1. In certain aspects, the solid phase substrate is a superficially porous silica that is covalently bonded to the hydrophobic-modified hydrophilic ligand represented by Formula I and the hydrophilic ligand represented by Formula V. Alternatively, in one aspect, the solid phase substrate is a superficially porous silica and is covalently boned to the hydrophilic ligand represented by Formula Va and to the hydrophobic- modified hydrophilic ligand represented by Formula II.

[0050] The present disclosure also provides a method of producing the HIC composition. The method includes providing the solid phase substrate and providing the hydrophilic ligand including the polar group and the plurality of hydroxyl groups. At least one hydroxyl group is present at a terminus of the hydrophilic ligand, and typically only one hydroxyl group is present at the terminus. Both the solid phase substrate and the hydrophilic ligand are described above. The method further includes reacting the solid phase substrate and the hydrophilic ligand to covalently couple the hydrophilic ligand to the solid phase substrate to form a hydrophilic-modified substrate. The reaction between the solid phase substrate and the hydrophilic ligand is more specifically defined as a reaction between a functional group on the solid phase substrate, such as a silanol group, and at least one of the (R'O) units coupled to the Si atom of the hydrophilic ligand. In certain embodiments, one, two or three of the (R10) units coupled to the Si atom of the hydrophilic ligand react with surface functional groups (e.g., silanols) present on the solid phase substrate to covalently couple the hydrophilic ligand to the solid phase substrate. The method further includes providing an activation compound including a leaving group and reacting the activation compound preferentially with the terminus hydroxyl group of the hydrophilic-modified substrate to form an activated hydrophilic-modified substrate. The terminal hydroxyl group is by design a primary hydroxyl group, whereas in other locations, the hydrophilic ligand typically possesses secondary hydroxyl groups. This differentiation can permit selective reaction of the primary hydroxyl group relative to secondary hydroxyl groups. In other words, the method includes a first reaction between the solid phase substrate and the hydrophilic ligand and a second reaction between the reaction product of the first reaction (i.e., the hydrophilic-modified substrate) and the activation compound. Although not common, some degree of reaction may also occur between a secondary hydroxyl group of the hydrophilic ligand portion and the activation compound. The method further includesproviding a hydrophobic ether-containing compound including an ether group and a nucleophile. The method further includes reacting the activated hydrophilic-modified substrate with the nucleophile of the hydrophobic ether-containing compound to release the leaving group of the activation compound and form the hydrophobic-modified hydrophilic ligand and the HIC composition. In other words, the method also includes a third reaction between the reaction product of the second reaction (i.e., the reaction between the activation compound and the hydrophilic- modified substrate) and the hydrophobic ether-containing compound. The resulting reaction product of the third reaction is the HIC composition including the hydrophobic-modified hydrophilic ligand covalently coupled to the solid phase substrate.

[0051] The hydrophobic ether-containing compound may be represented by Formula VIII: W-[(R10)-O-(R10)]s-[(R10)-O-(R10)]s’-(O-R11)v Formula VIII, wherein:W is a nucleophileR10is independently a C1-C8 alkyl or branched alkyl group or an aromatic hydrocarbon;R11is independently hydrogen, a C 1-C8 alkyl or branched alkyl group or an aromatic hydrocarbon; s is 1 to 250; s’ is 0 to 250; and v is 0 or 1.

[0052] Although not required, the nucleophile (W) of the hydrophobic ether-containing compound is typically NH2. Other common nucleophiles can include thiols (R-SH), suitably substituted alcohols, halides, etc. Alternatively still, the nucleophile may be selected from amines and thiols. In certain embodiments, the nucleophile is NH2, R10is independently a C2-C4 alkyl or branched alkyl group, and the sum of s and s’ is from 1 to 10. In other embodiments, suitableexamples of the hydrophobic ether-containing compounds of the aromatic or aromatic amines include, but are not limited to, p-phenetidine, 2-phenoxyethylamine, 2-(2- methoxyphenoxy)ethylamine, homoveratrylamine, 4-amino-4'-methyldiphenyl ether, 4- phenoxybenzylamine, 4,4'-dimethoxybenzhydrylamine, N-aminoethylpropoxy, N- aminoethylethoxy, and combinations thereof. Such compounds can be variously branched, with mixed substitutions of aromatic, polynuclear aromatic, alkyl, or aromatic amines attached to the ether compounds. Similarly, the ether compounds can be branched, containing other functional groups, including ethers, alcohols, substituted carbonyls, etc.

[0053] Referring first to the first reaction between the solid phase substrate and the hydrophilic ligand, the reaction occurs between the surface hydroxyl groups present on the solid phase substrate and at least one of the three [(R'O)] units present in Formula V:(R1O)3Si-[C(R2)(R3)]n-X-[C(R2)(R3)]n’-[C(R4)(R5)]m-[C(R8)(R9)]qFormula V.The resulting reaction product produces the hydrophilic-modified substrate and preserves the hydroxyl group in the q unit represented by [C(R8)(R9)].

[0054] Typically, the reaction between the hydrophilic-modified substrate and the activation compound occurs under aprotic anhydrous solvent conditions, to limit hydrolytic loss of the activated conjugate. The activation compound may include a carbonyl group. Specific examples of the activating compound including the carbonyl group include, but are not limited to, phosgene (carbonyl dichloride), carbonyldiimidazole (CDI), or chloroformates, such as 4-nitrophenyl chloroformate (4-NPC), N-hydroxysuccinimdyl chloroformate, or other carbonates, such as N,N'- disuccinimidyl carbonate (DSC), or a combination thereof. An illustrative example of the second reaction product between the hydrophilic ligand of Formula Va and DSC is provided below, to form the N-hydroxysuccinimdyl (NHS) carbonate of the 3-TPG compound.

[0055] Alternative activation compounds include compounds having a tosylate group, such as, but not limited to, tosyl chloride (4-toluenesulfonyl chloride). Further suitable activation compounds include mesyl chloride (methanesulfonyl chloride), triphenylmethylene chloride (tritylchloride), phosphorus tribromide, or thionyl chloride. Selection of the alternatives for activation compounds will be, in part, governed by cost, solubility, toxicity, reactivity, commercial availability, and ease of handling. Although not typical, any of the reaction compounds can be used in combination with alternative activation compounds.

[0056] Without being bound to any particular theory, it is believed that under suitable conditions the activation compounds described herein can selectively react with the terminal hydroxyl group of the hydrophilic ligand. The selective reaction at the terminal hydroxyl group is also considered to be an important aspect of the present disclosure because uniformity and the general avoidance of multiple reaction products, cross-linked intermediates or cyclic carbonates and the like, is favorable to achieving consistent and reproducible chromatographic separations and / or separation materials. Once the hydrophilic ligand is reacted with the activation compound, the hydrophilic ligand portion of Formula la is established.

[0057] An illustrative example of the third reaction product obtained from reacting the hydrophobic ether-containing compound, in the form of 2-phenoxyethylamine, with the reaction product illustrated above is provided below.

[0058] As shown above, the reaction between the second reaction product and the hydrophobic ether-containing compound displaces the leaving group of the activation compound and creates a carbamate (urethane) linkage. The carbamate (urethane) linkage is representative of the connecting group Z in Formula I. In other words, the connecting group Z is collectively formed from a first reaction product between the terminal hydroxyl group of the hydrophilic ligand with the activation compound and from the subsequent reaction between the leaving group present within the first reaction product and the nucleophile of the hydrophobic ether-containing compound.

[0059] The method of producing the HIC composition may also include coupling both the hydrophobic-modified hydrophilic ligand and the hydrophilic ligand to the solid phase substrate by controlling the stoichiometry of the second reaction. Specifically, after the hydrophilic ligand has been covalently coupled to the surface of the solid phase substrate, the hydrophilic ligand inits current state may be preserved by including fewer moles of the activation compound than the number of moles of the hydrophobic ligand coupled to the substrate. Notably, because the hydrophobic ether-containing compound will preferentially react with the activated hydrophilic ligand and will generally disfavor reaction with the hydrophilic ligand (i.e., non-activated hydrophilic ligand) the remaining hydrophilic ligand is preserved in an unmodified state. As an alternative, the reactions of activation and modification of the hydrophilic ligand can occur in free solution, yielding a mixture, which can thereafter be covalently bonded to a solid phase carrier.

[0060] A variety of approaches can be employed to tether the ether chemical composition to the hydrophilic ligand segment. A variety of activated ether compositions are described in the literature, and some are commercially available, some of which may be directly coupled to the hydrophilic ligand hydroxylic groups. These compositions can be selected to possess linear or branched segments of connected alkyl ethers (for example polyethylene glycols, abbreviated as PEGs, or polypropylene glycols, abbreviated as PPGs). Such compositions of PEGs or PPGs, or other polyethers, often possess terminal hydroxyl groups, which can be further alkylated or functionalized, resulting in additional compositional complexity. Simple or complex etheramines, may be synthesized as organosilane reagent compositions. Mixtures of alkoxy-terminated PEG- functionalized with free terminal hydroxylic PEG organosilane compositions have been described as useful for size exclusion chromatography. Similarly, azidoethers are known, with a reactive azido (-N=N) functional group, and epoxyethers are relatively common, with a reactive oxirane functionality present in ether compounds (epoxides may also be considered a special reactive form of cyclic ethers). Although it may be possible to directly couple such ether compositions directly to solid phase surfaces, including silica surfaces, it is also feasible to couple to the hydroxy groups of the hydrophilic ligand. Indirect coupling of the ether chemical composition can be accomplishedby application of functional groups present on ether compositions, resulting in the formation of a connecting group, as described above in Formula Ila. In such cases, the Z group is a connecting group, and Y is an ether-containing segment.EXAMPLES

[0061] The elemental analysis (%C, %H, %N) values reported below were measured by combustion analysis (Robertson Microlit Laboratories, Ledgewood, NJ). These values were employed to establish ligand coverage measures based on known composition of compounds and Specific Surface Areas (SSA) (m2 / g). The SSA, specific pore volumes (SPY) and the average pore diameters (APD) of these materials were measured using the multi-point N2 sorption method (Micromeritics ASAP 2400; Micromeritics Instruments Inc., Norcross, Ga.). The SSA was calculated using the BET method, the SPY was the single point value determined for P / Po>0.98 and the APD was calculated from the desorption portion of the isotherm using the BJH method. Particle sizes were measured using a Beckman Coulter Multisizer 3 analyzer (30 pm aperture, 70,000 counts; Miami, Fla.). The particle diameter (dp) was measured as the 50% cumulative diameter of the volume-based particle size distribution. The width of the distribution was measured as the 90% cumulative volume diameter divided by the 10% cumulative volume diameter (denoted 90 / 10 ratio). Generally, values of surface coverage are expressed as normalized to the elemental composition and SSA of samples, to yield molar surface coverage of the silica surface with ligand in pmol / m2.

[0062] Commercially available 2.7 pm diameter fully hydroxylated superficially porous silica particles (25 g of Halo Silica, Advanced Materials Technologies, Wilmington, DE, SSA=22 m2 / g; APD=645 A) were dispersed while under a blanket of nitrogen, refluxed in toluene (250 mL, Millipore / Sigma, St. Louis, NJ) using a Dean-Stark trap for 1 hour, to collect a small quantity ofadsorbed water. After brief cooling to about 65°C, a quantity of 22 mmol of diisopropylethylamine (DIPEA, Sigma- Aldrich, St. Louis, MO) was added with stirring, followed by 56 66 mmol of N- (3-triethoxysilylpropyl)gluconamide, (3-TPG, 30% in ethanol, Gelest Inc., Morrisville, PA). The resulting mixture was heated to 78°C, to remove the bulk of ethanol, then brought to reflux overnight, with occasional collection of about 5 mL portions of solvent to aid removal of the ethanol evolved during bonding of the ethoxy-silane to the surface of the silica particles. After cooling, the resulting silica particles were collected by fdtration on a sintered glass funnel, washed with 200 mL of warm toluene, DMF, acetonitrile, followed by dispersion into 50% acetonitrile / water heated to 60°C, then collection by filtration and washing with acetonitrile and methanol (all solvents from Sigma-Millipore). The filter dried silica was further dried in a vacuum oven at 110°C for at least 1 hour. The resulting 3-TPG bonded silica then underwent an additional bonding reaction in 250 mL of dimethylformamide (DMF, Sigma-Aldrich, St. Louis, MO), using 6 mmol of DIPEA, and 18 mmol of 3-TPG, at a temperature of 85°C overnight, with occasional removal of about 5 mL of solvent through the Dean-Stark trap. After cooling, the solids are recovered by filtration, washing with 200mL of warm DMF, then acetonitrile, followed by dispersion into 50% acetonitrile / water heated to 60°C, then collection by filtration and washing with acetonitrile and methanol. The silica was dried as before under vacuum at 110°C. The resulting 3-TPG bonded silica particles are densely bonded with 3-TPG, with elemental analysis typically revealing 3.5-3.8 pmol / m2on the silica surface, based on carbon and nitrogen analysis. Li and Modification

[0063] 3 -TPG bonded silica particles described above are dried in a vacuum oven for 2 hours. A suitable portion of 5-40 g of the material is dispersed in volume of 10 mL per g, using dry acetonitrile (Sigma-Aldrich, St. Louis, MO), then a quantity of 0.2 mmol / g of 4-dimethylaminopyridine (DMAP, Sigma-Aldrich) is added, with stirring at room temperature, followed by a quantity of disuccinimidylcarbonate (DSC, Oakland Chemicals), which was typically or 20-600 pmol / g, added with stirring and dispersion in an ultrasonic bath. The reaction to form NHS activated intermediates proceeds for 0.5-3 hours at room temperature under nitrogen, after which the NHS-activated silica particles are collected on filter, washed with 2 volumes of 10 mL / g of dry acetonitrile and 2 volumes of 10 mL / g of anhydrous isopropanol. After drying under nitrogen on filter, then transfer to drying under vacuum at room temperature, the small sample of NHS activated 3-TPG silica is dispersed at 50 mg / mL in 0.25 M NH4OH for hydrolysis, and spectrophotometric assay of NHS content, using the approach described by Li and Vanderah (2021). Activated 3-TPG silicas are dispersed in a solution of anhydrous acetonitrile reaction medium, at 10 mL / g silica solid with stirring. The etheramine compound is added to the reaction mixture in a quantity of at least lOx the calculated quantity of NHS reactive groups bound to the surface. Most of the etheramines were used as purchased from Tokyo Chemical Industries (Tokyo, JP), or from Millipore / Sigma (St. Louis, MO, USA). mPEG amines were commercially obtained from Broadpharm (San Diego, CA, USA) or Biopharma PEG (Watertown, MA, USA), and if solid at room temperature, were melted at 55°C for 15 minutes before use. For small quantities of etheramines, the liquids are dispersed into 1 mL of anhydrous acetonitrile, then added to the silica reaction mixture. The etheramines and activated silica slurry is allowed react by stirring overnight at room temperature, under a nitrogen gas blanket.

[0064] On completion of reaction, the etheramine-modified 3-TPG silica is collected by centrifugation (2500 x g for 5 minutes), dispersed in acetonitrile, then washed by dispersion and collection using centrifugation in 10 mL / g of acetonitrile. Hydrolysis of remaining unreacted NHS modified sites was conducted by dispersing the silica particles in a solution of 0.2 M TrisHClbuffer (pH 8.5), with mixing for 30 minutes. The modified silicas were then washed twice by dispersion and centrifugation in water, dispersed in 10 ml / g of acetonitrile, and the particles collected by vacuum filtration, washing on filter with about 10 mL / g of acetonitrile and methanol, before drying on filter, followed by vacuum oven drying at 110°C. The completed HIC column packing materials are assessed for ligand coverage by elemental analysis.

[0065] Assay of the NHS modification on the surface of 3-TPG by the DSC reagent showed 0.12-1.4 pmol / m2, determined by the quantity of DSC employed for surface activation. Reproducible densities of the reaction are controlled by accurate assessment of the SSA of the silica particles, reaction times for the activation, and by correction for the degree of reaction by the 3-TPG silane to the surface. NHS surface activation, as determined by spectrophotometric assay, agreed well with results from elemental analysis of ligand modified surface silica.Chromatographic Properties of Ligand-Modified 3-TPG Silica

[0066] 3 -TPG and etheramines modified 3-TPG silicas were employed to load stainless steel HPLC columns of 2.1 or 4.6 mm internal diameter x 100 mm, 50 mm in length. These materials were applied to chromatographic separations of a variety of protein mixtures, using HIC, examples of which are as shown in Figures 1-5. Separation was accomplished using the Shimadzu Nexera LC instrument, at a flow rate of 0.25 mL / min, or as otherwise specified, at a column temperature of 25 °C or 30°C, using mobile phases and gradient elution conditions are specified in the examples shown in Figures 1-5. Samples of proteins, including the antibody-drug conjugate mimic MSQ- C8, were obtained from Sigma / Millipore, or in the case of monoclonal antibodies (trastuzumab, denosumab, ipilimumab) or antibody-drug conjugate (enfortumab-vedotin) were pharmaceutical preparations obtained as commercial formulations. All protein solutions were prepared at known concentrations of about 5-25 mg / mL in 25 mM sodium phosphate buffer (lysozyme, ribonuclease),or for the pharmaceuticals, using the formulation buffer and conditions recommended in the product insert, prior to aliquoting into seal polypropylene tubes, and storage at -80°C. Immediately before use, the samples are thawed, mixed and diluted as required before analyses, then maintained in a cold environment.EXAMPLE 1

[0067] Four packing materials were prepared from a single portion of NHS-activated 3-TPG silica, which was present at 0.30 pmol / m2NHS on the 3-TPG modified silica surface. Etheramine ligands were formed by reaction in dry acetonitrile with the activated silicas for >16 hours, using methoxy -ethylamine (C2OC1), 2-ethoxy-ethylamine (C2OC2), ethoxy-2-(ethoxy)-ethylamine [(C2O)2C2], and 3 -butoxy -propylamine (C3OC4) to produce the hydrophobic ligand modified surface column packing materials. The packing materials were recovered as described previously, for loading 2.1 mm x 50 mm length test columns. Samples of 2 uL (1 ug each) mixtures of proteins were injected using a diluent of 1.0 M ammonium sulfate in phosphate buffer. Separations were conducted by linear gradient elution, using a decreasing ammonium sulfate salt concentration to develop the separation. The mobile phases were delivered at 0.4 mL / min., with a column temperature of 30°C, with a gradient of 0-100%B over 4 minutes, and an isocratic hold at 100%B for a further 2 minutes, before returning to starting conditions. Mobile phase A was 2.0 M ammonium sulfate / 0.02 M sodium phosphate (pH 7.0), and buffer B was 0.02 M sodium phosphate (pH 7.0). Figure 1 presents chromatograms showing the separation of the mixture of lysozyme and two monoclonal antibodies, trastuzumab, and denosumab. All three proteins are poorly retained by 3-TPG modified silica, eluting early in the chromatograms. After surface modification with hydrophobic ligand, retention was least for the least hydrophobic ligand, C2OC1, and highest for the more hydrophobic C3OC4 ligand. Intermediate retention is observed for the C2OC2monoether, which is intermediate in carbon number between these two ligands (C2OC1 has 3 and C3OC4 has 7 alkyl carbons). The diether, (C2O)2C2, is similar in retention to the intermediate 4 carbon monoether ligand, but these two ligands exhibit notable differences in band spacing for the eluted proteins. The least hydrophobic ligand shows poor retention for lysozyme, whereas the most retentive ligand is, for these conditions, excessive, with the trastuzumab eluting past the end of the gradient, in the isocratic hold at Buffer B. At the ligand density shown (0.30 pmol / m2), the intermediate hydrophobic ligands (C2OC2 and (C2O)2C2) result in desired elution within the gradient linear elution window for HIC salt concentration.EXAMPLE 2

[0068] Two column packing materials were prepared using NHS-activated 3-TPG silica, which was present at 0.45 pmol / m2NHS on the 3-TPG modified silica surface. 2-Ethoxy-ethylamine (C2OC2) and 2-(2-aminoethoxy)ethanol (C2OC2OH) ligands were formed by reaction in acetonitrile with the activated silicas for >16 hours, using the free base compounds. The packing materials were recovered as described previously, for loading 2.1 mm x 50 mm length test columns. Samples of 2 uL (1 ug each) mixtures of proteins were injected using a diluent of 1.0 M ammonium sulfate in phosphate buffer. Separations conditions are as described in Example 1. Figure 2A shows good retention, resolution and peak shapes of all three proteins, (C2OC2), whereas comparison to Figure 2B indicates that the addition of the polar alcohol group at the terminal position of the hydrophobic ligand causes a decreased retention of all three proteins, with poor peak shape for lysozyme, and lower resolution of denosumab and trastuzumab.EXAMPLE 3

[0069] Column packing materials were prepared using NHS-activated 3-TPG silica, which was present at 0.30, 0.79 and 0.97 pmol / m2NHS on the 3-TPG modified silica surface. 2-Ethoxy-ethylamine (C2OC2) ligands were formed by reaction with the activated silicas for >16 hours, using the free base compound. The packing materials were recovered as described previously, for loading 2.1 mm x 50 mm length test columns. Samples of 2 uL (1 ug each) mixtures of proteins were injected using a diluent of 1.0 M ammonium sulfate in phosphate buffer. Separations conditions are as described in Example 1. Comparing Figure 3A-3C shows a progressive HIC retention increase of all three proteins, with increasing density of the hydrophobic ligand. Using this hydrophobic ligand shows useful separations for this protein mixture for all three selected ligand densities, with subtle, but potentially useful selectivity and band width differences between these prototype materials. The highest density material, at 0.97 pmol / m2NHS on the 3-TPG modified silica surface, exhibits retention of trastuzumab at or near the end of the linear gradient elution profile.EXAMPLE 4

[0070] Column packing materials were prepared using NHS-activated 3-TPG silica, which was present at 0.17, 0.26, 0.47 and 0.70 pmol / m2NHS on the 3-TPG modified silica surface. 3- Propoxy-ethylamine (C2OC3) ligands were formed by reaction with the activated silicas for >16 hours, using the free base compound. The packing materials were recovered as described previously, for loading 2.1 mm x 50 mm length test columns. Samples of 4 uL (10 ug) of an antibody drug conjugate (ADC) enfortumab-vedotin (Seattle Genetics) were injected using a diluent of 1.0 M ammonium sulfate in phosphate buffer. Separations were conducted by linear gradient elution, using a decrease in salt concentration and increase in isopropanol concentration, similar to methods described by Lyon, Meyer, Setter and Senter and Hamblett, et al (Methods Enzymology vol 502, 2012). (Clinical Cancer Research vol 10, 2004). The mobile phases were delivered at 0.4 mL / min., with a column temperature of 30°C, with a gradient of 5-100%B over10 minutes, and an isocratic hold at 100%B for a further 2 minutes, before returning to starting conditions. Mobile phase A was 1.5 M ammonium sulfate / 0.02 M sodium phosphate (pH 7.0) / with 5% isopropanol ((v / v), and mobile phase B was 0.02 M potassium phosphate (pH 7.0) / with 25% isopropanol (v / v). Absorbance values measured in the chromatograms of Figure 4 show results at 220 nm (10 nm bandwidth). As noted previously in the scientific literature many ADCs formed by reaction at antibody sulfhydryl groups can exhibit multiple reaction products, varying in the degree of conjugation of the drug, relative to the antibody. The description of stoichiometry is often referred to as the drug-antibody ratio (DAR). In Figure 4 apparent DAR are denoted as DO, D2, D4 and D6, representing the mAb without drug attached (DO), and increasing number of drug conjugates of 2, 4, and 6 per IgG molecule. Minor variants of these ADCs are also present in such samples, including those eluting late in HIC separations. Since the drug entity (vedotin) is comparably hydrophobic, it is necessary for the strong eluting mobile phase (B), to be supplemented with a strong organic solvent (isopropanol), to elute higher ADCs with higher DARs. This has been observed by many examples in the literature, with the use of compound gradients of decreasing ionic strength and increasing organic solvent now being broadly employed in the biopharmaceutical industry for ADC separations, and for DAR analysis. Such conditions are employed in the analysis shown in Figure 4A-D, comparing varying prototype HIC materials for the separation of this ADC biopharmaceutical. Highly similar results have been obtained using other ADCs, including the Millipore / Sigma ADC mimic MSQ-C8. As shown by comparison of retention of the ADC components in Figure 4, the density of hydrophobic ligand (C2OC3) strongly affects HIC retention of all protein variants in this ADC preparation. Increasing density of the ligand increases gradient elution time. Low ligand density (Figure 4A, 0.17 pmol / m2) exhibits comparably early elution, and somewhat broader protein peaks in this HIC separation. Comparablyhigh hydrophobic ligand density (Figure 4D, 0.70 pmol / m2) shows strong retention, sufficient to place the D4 at the end of the linear gradient elution range, and enough to prevent elution of the D6 variant (and minor higher DAR variants) from eluting even in the highest organic concentration used for this experiment. The intermediate ligand density prototype materials (Figure 4B, 4C), exhibit elution of the main ADC components (DO, D2, D4, D6), as well as later eluting components, which are assumed to be higher DAR variants. This example illustrates that separation of protein mixtures, and protein variants that may result from purposeful or inadvertent modifications, can be usefully manipulated by the use of specific hydrophobic ligands at specific ligand densities.EXAMPLE 5

[0071] A column packing material was prepared using NHS-activated 3-TPG silica, which was present at 0.91 pmol / m2NHS on the 3-TPG modified silica surface. Methoxy-terminated- (polyethylene glycol)-amine of molecular weight average 550 (mPEG(550)-NH2, BLD Pharmatech Co., Cincinnati, OH, USA) was added at 10% (wt / wt silica) in 10 mL acetonitrile per g silica particles. The mPEG(55O) ligand was formed by reaction with the activated silicas for >24 hours, and based on average molecular weight present 12 ethylene glycol subunits in the ligand chain. The packing materials were recovered as described in Example 1, for loading 2.1 mm x 100 mm length test columns. Samples of 2 uL (2 ug each) of proteins were injected using a diluent of 1.0 M ammonium sulfate in phosphate buffer. Separations were conducted by linear gradient elution, using a decrease in salt concentration to develop the separation. The mobile phases were delivered at 0.25 mL / min., with a column temperature of 25°C, with a gradient of 0-100%B over 20 minutes, and an isocratic hold at 100%B for a further 2 minutes, before returning to starting conditions. Mobile phase A was 2.0 M ammonium sulfate / 0.02 M sodium phosphate (pH 7.0), andMobile phase B was 0.02 M potassium phosphate (pH 7.0). The proteins included bovine Ribonuclease A and chicken hen white Lysozyme (Millipore Sigma), denosumab, and ipilimumab. Figure 5A presents chromatograms showing the overlaid chromatograms for HIC separations of ribonuclease, lysozyme and these two monoclonal antibodies, denosumab and ipilimumab. All proteins eluted within the linear gradient range for HIC elution, although ribonuclease (Rna) is poorly retained (c. 3 min), showing a somewhat broader peak, eluting nearer the column void volume (about 1.0 min). Ribonuclease is considered a comparably hydrophilic protein, usually eluting at or nearer the void volume in HIC separations (at higher ionic strength in HIC). The other protein peaks are well retained, with lysozyme (Lys) minor impurities resolved before and after the main peak at about 7.5 min. denosumab (Den) and ipilimumab (Ipi) show acceptable peak shapes. The ipilimumab peak elutes at about 14 min. This monoclonal antibody is a highly hydrophobic human mAb, based on previous reports in the scientific literature, and as judged by retention on other commercially available HIC materials.EXAMPLE 6

[0072] A column packing material was prepared using NHS-activated 3-TPG silica, which was present at 0.87 pmol / m2NHS on the 3-TPG modified silica surface. The Ethoxy-terminated- (polyethylene glycol)-amine of three PEG units [2-(2-(2-Ethoxyethoxy)ethoxy)ethanamine, Millipore Sigma) was added at 10% (wt / wt silica) in 10 mL acetonitrile per g silica particles. This resulting ePEG3 ligand was formed by reaction with the activated silicas for >24 hours. The packing material was recovered as described in Example 1, for loading the 2.1 mm x 100 mm length test column. Samples of 2 uL (2 ug each) of proteins were injected using a diluent of 1.0 M ammonium sulfate in phosphate buffer. Separations were conducted by linear gradient elution, using a decrease in salt concentration to develop the separation. The mobile phases were deliveredat 0.25 mL / min., with a column temperature of 25°C, with a gradient of 0-100%B over 20 minutes, and an isocratic hold at 100%B for a further 2 minutes, before returning to starting conditions. Mobile phase A was 2.0 M ammonium sulfate / 0.02 M sodium phosphate (pH 7.0), and Mobile phase B was 0.02 M potassium phosphate (pH 7.0). The proteins included bovine Ribonuclease A and chicken hen white Lysozyme (Millipore Sigma), denosumab, and ipilimumab. Figure 5B presents chromatograms showing the overlaid chromatograms for HIC separations of ribonuclease, lysozyme and these two monoclonal antibodies, denosumab and ipilimumab. Of note, ribonuclease (Rna) is retained more than observed for mPEG(550) ligand material (c. 4 min), with a sharper peak. The other protein peaks are well retained, with greater retention ePEG3 than observed with the mPEG(550) ligand material. Lysozyme minor impurities are resolved before and after the main peak at about 8.8 min. Denosumab and ipilimumab show acceptable peak shapes. The ipilimumab peak elutes at about 17 min., well before the end of the linear gradient elution, and is thus not excessively retained as a relatively hydrophobic human mAb. Comparison of the retention and separations of proteins in Figure A and Figure B show that overall PEG unit number may not dominate for retention of proteins in HIC materials, however the effect of the chain terminal substitution effects may need to be understood to have full appreciation of the effects of structure of the ligand on protein retention in HIC.

[0073] It is to be understood that the appended claims are not limited to express any particular compounds, compositions, or methods described in the detailed description, which may vary between particular embodiments which fall within the scope of the appended claims. With respect to any Markush groups relied upon herein for describing particular features or aspects of various embodiments, different, special, and / or unexpected results may be obtained from each member of the respective Markush group independent from all other Markush members. Each member of aMarkush group may be relied upon individually and or in combination and provides adequate support for specific embodiments within the scope of the appended claims.

[0074] Further, any ranges and subranges relied upon in describing various embodiments of the present disclosure independently and collectively fall within the scope of the appended claims, and are understood to describe and contemplate all ranges including whole and / or fractional values therein, even if such values are not expressly written herein. One of skill in the art readily recognizes that the enumerated ranges and subranges sufficiently describe and enable various embodiments of the present disclosure, and such ranges and subranges may be further delineated into relevant halves, thirds, quarters, fifths, and so on. As just one example, a range “of from 0.1 to 0.9” may be further delineated into a lower third, i.e., from 0.1 to 0.3, a middle third, i.e., from 0.4 to 0.6, and an upper third, i.e., from 0.7 to 0.9, which individually and collectively are within the scope of the appended claims, and may be relied upon individually and / or collectively and provide adequate support for specific embodiments within the scope of the appended claims. In addition, with respect to the language which defines or modifies a range, such as “at least,” “greater than,” “less than,” “no more than,” and the like, it is to be understood that such language includes subranges and / or an upper or lower limit. As another example, a range of “at least 10” inherently includes a subrange of from at least 10 to 35, a subrange of from at least 10 to 25, a subrange of from 25 to 35, and so on, and each subrange may be relied upon individually and / or collectively and provides adequate support for specific embodiments within the scope of the appended claims. Finally, an individual number within a disclosed range may be relied upon and provides adequate support for specific embodiments within the scope of the appended claims. For example, a range “of from 1 to 9” includes various individual integers, such as 3, as well as individual numbersincluding a decimal point (or fraction), such as 4.1 , which may be relied upon and provide adequate support for specific embodiments within the scope of the appended claims.

[0075] The present disclosure has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation. Many modifications and variations of the present disclosure are possible in light of the above teachings. The present disclosure may be practiced otherwise than as specifically described. The subject matter of all combinations of independent and dependent claims, both singly and multiply dependent, is herein expressly contemplated.

Claims

CLAIMS1. A hydrophobic interaction chromatography (HIC) composition comprising: a solid phase substrate; and a hydrophobic-modified hydrophilic ligand covalently coupled to the solid phase substrate with the hydrophobic-modified ligand comprising; a hydrophilic ligand portion covalently bonded to the solid phase substrate with the hydrophilic ligand portion including a polar group and a plurality of hydroxyl groups, and a hydrophobic ether-containing segment, the hydrophobic ether-containing segment directly or indirectly covalently coupled to the hydrophilic ligand portion.

2. The HIC composition of claim 1 wherein the hydrophobic-modified hydrophilic ligand is derived from Formula I:(R1O)3Si-[C(R2)(R3)]n-X-[C(R2)(R3)]n-[C(R4)(R5)]m-Zp-Y Formula I, wherein:X is the polar group;Z is a connecting group;Y is the hydrophobic ether-containing segment; n is 1-6; n’ is 0-2; m is 1-8; p is 0 or 1;R1, R2, R3, is independently H or a straight or branched, substituted or unsubstituted, C 1 to Cl 8 alkyl group; andR4and R5is independently H or OH, with the proviso that the hydrophobic-modified hydrophilic ligand includes at least two hydroxyl groups.

3. The HIC composition of claim 2 wherein the hydrophilic ligand portion is derived from Formula la:(R1O)3Si-[C(R2)(R3)]n-X-[C(R2)(R3)]n-[C(R4)(R5)]mFormula la wherein:X is the polar group; n is 1-6;n’ is 0-2; m is 1-8;R1, R2, R3, is independently H or a straight or branched, substituted or unsubstituted, C 1 to Cl 8 alkyl group; andR4and R5is independently H or OH and at least two m units include at least one hydroxyl group.

4. The HIC composition of claim 2 or 3 wherein the polar group X is independently chosen from a carbonate, a carbamate, an amide, an amine, an ureido, an ether, a thioether, a sulfinyl, a sulfoxide, a sulfonyl, a thiourea, a thiocarbonate, or a thiocarbamate.

5. The HIC composition of claim 4 wherein the polar group X is selected from an amide, a carbamate, or a ureido.

6. The HIC composition of claim 5 wherein the polar group X is an amide.

7. The HIC composition of any one of claims 2 to 6 wherein: n is 2-4; m is 3-6; p is 1 ; andR1, R2, R3, is independently H or a straight or branched, substituted or unsubstituted, C 1 to C6 alkyl group.

8. The HIC composition of any one of claims 2 to 7 wherein the connecting group Z is a carbamate and p is 1.

9. The HIC composition of any one of claims 2 to 8 wherein the hydrophobically-modified hydrophilic ligand is derived from Formula II:Formula II.

10. The HIC composition of any one of claims 2 to 9 wherein the hydrophobic ether-containing segment is represented by Formula VI:-[(R10)-O-(R10)]s-[(R10)-O-(R10)]s’-(O-R11)v Formula VI, wherein:R10is independently a C1-C8 alkyl or branched alkyl group or an aromatic hydrocarbon;R11is independently hydrogen, a C1-C8 alkyl or branched alkyl group or an aromatic hydrocarbon; s is 1 to 250; s’ is 0 to 250; and v is 0 or 1.

11. The HIC composition of claim 10 wherein:R10is independently a C2-C4 alkyl or branched alkyl group; and the sum of s and s’ is from 1 to 10.

12. The HIC composition of any one of claims 2 to 11 wherein the hydrophobic-modified ligand is derived from Formula Vll:Formula VII, wherein v is 1 to 3.

13. The HIC composition of any one of claims 2 to 11 wherein the hydrophobic-modified ligand is derived from Formula Vila:Formula Vila.

14. The HIC composition of any one of claims 1 to 13 further comprising a hydrophilic ligand covalently bonded to the solid phase substrate with the hydrophilic ligand including a polar group and a plurality of hydroxyl groups.

15. The HIC composition of claim 14 wherein the hydrophilic ligand is derived from Formula V:(R1O)3Si-[C(R2)(R3)]11-X-[C(R2)(R3)]n-[C(R4)(R5)]m-[C(R8)(R9)]qFormula V wherein:X is the polar group; n is 1-6; n’ is 0-2; m is 2-8; q is 1;R1, R2, R3, is independently H or a straight or branched, substituted or unsubstituted, C 1 to C18 alkyl group;R4and R5is independently H or OH and at least two m units include at least one hydroxyl group; andR8and R9is independently H or OH provided that at least one of R8and R9is OH.

16. The HIC composition of claim 15 wherein the hydrophilic ligand of Formula V is further represented by Formula Va:Formula Va.

17. The HIC composition of any one of claims 14 to 16 wherein the hydrophobic-modified hydrophilic ligand and the hydrophilic ligand are present in a molar ratio range of from 1 :20 to 20: 1.

18. The HIC composition of any one of claims 1 to 17 wherein the solid phase substrate is a silica material or a hybrid inorganic / organic material.

19. The HIC composition of claim 18 wherein the solid phase substate is a silica material.

20. The HIC composition of any one of claims 1 to 19 wherein the hydrophobic ether- containing segment is neutral at pH values of 3 to 9 for promoting hydrophobic interaction.

21. A kit comprising the HIC composition of any one of claims 1 to 20.

22. A method of producing a HIC composition for hydrophobic interaction chromatography including a hydrophobic-modified hydrophilic ligand, the method comprising: providing a solid phase substrate; providing a hydrophilic ligand including a polar group and a plurality of hydroxyl groups with at least one hydroxyl group present at a terminus of the hydrophilic ligand; reacting the solid phase substrate and the hydrophilic ligand to covalently couple the hydrophilic ligand to the solid phase substrate to form a hydrophilic-modified substrate; providing an activation compound including a leaving group; reacting the activation compound with the terminus hydroxyl group of the hydrophilic- modified substrate to form an activated hydrophilic-modified substrate; providing a hydrophobic ether-containing compound comprising an ether group and a nucleophile; and reacting the activated hydrophilic-modified substrate with the nucleophile of the hydrophobic ether-containing compound to release the leaving group of the activation compound and form the hydrophobic-modified hydrophilic ligand and the HIC composition.

23. The method of claim 22 wherein the hydrophilic ligand is derived from Formula V:(R1O)3Si-[C(R2)(R3)]n-X-[C(R2)(R3)]n’-[C(R4)(R5)]m-[C(R8)(R9)]qFormula V, wherein:X is the polar group; n is 1-6; n’ is 0-2; m is 1-8; q is 1;R1, R2, R3, is independently H or a straight or branched, substituted or unsubstituted, C 1 to C18 alkyl group;R4and R5is independently H or OH, with the proviso that the hydrophilic ligand includes at least two hydroxyl groups from R4and / or R5; andR8and R9is independently H or OH provided that at least one of R8and R9is OH to represent the hydroxyl group present at the terminus of the hydrophilic ligand.

24. The method of claim 23 wherein the polar group X is independently chosen from a carbonate, a carbamate, an amide, an amine, a urea, an ether, a thioether, a sulfinyl, a sulfoxide, a sulfonyl, a thiourea, a thiocarbonate, or a thiocarbamate, including heterocyclic compounds including the polar functionality.

25. The method of claim 24 wherein the polar group X is selected from an amide, a carbamate, or ureido.

26. The method of claim 25 wherein the polar group X is an amide.

27. The method of any one of claims 23 to 26 wherein: n is 2-4; m is 3-6; q is 1; andR1, R2, R3, is independently H or a straight or branched, substituted or un substituted, C 1 to C6 alkyl group.

28. The method of any one of claims 23 to 27 wherein the hydrophilic ligand is represented by Formula Va:

29. The method of any one of claims 22 to 28 wherein the activation compound includes a carbonate and is represented by 4-nitrophenyl chloroformate (4-NPC), N,N'-disuccinimidyl carbonate (DSC), carbonyldiimidazole (CDI), or a combination thereof.

30. The method of any one of claims 22 to 28 wherein the activation compound includes atosyl ate group.

31. The method of claim 30 wherein the activation compound is tosyl chloride.

32. The method of any one of claims 22 to 29 wherein the activation compound is mesyl chloride, phosphorus tribromide, thionyl chloride, or a combination thereof.

33. The method of any one of claims 22 to 32 wherein the hydrophobic ether-containing compound is represented by Formula VIII:W-[(R10)-O-(R10)]s-[(R10)-O-(R10)]s -(O-R11)v Formula VIII, wherein:W is a nucleophile;R10is independently a C1-C8 alkyl or branched alkyl group or an aromatic hydrocarbon;R11is independently hydrogen, a C 1-C8 alkyl or branched alkyl group or an aromatic hydrocarbon; s is 1 to 250; s’ is 0 to 250; and v is 0 or 1.

34. The method of claim 33 wherein W is NH2.

35. The method of claim 33 or 34 wherein:R10is independently a C2-C4 alkyl or branched alkyl group; and the sum of s and s’ is from 1 to 10.

36. The method of any one of claims 22 to 28 wherein the hydrophobically-modified hydrophilic ligand is derived from Formula I:(R1O)3Si-[C(R2)(R3)]n-X-[C(R2)(R3)]n -[C(R4)(R5)]m-Zp-Y Formula I, wherein:X is the polar group;Z is a connecting group derived from the activation compound;Y is a segment derived from the hydrophobic ether-containing compound; n is 1-6; n’ is 0-2; m is 2-8;p is 1 ;R1, R2, R3, is independently H or a straight or branched, substituted or unsubstituted, C 1 to Cl 8 alkyl group; andR4and R5is independently H or OH and at least two m units include at least one hydroxyl group.

37. The method of claim 36 wherein the hydrophobic-modified ligand is derived from FormulaVII:Formula VII, wherein v is 1 to 3.

38. The method as set forth in claim 36 wherein the hydrophobic-modified ligand is derived from Formula Vila:Formula Vila.

39. The method as set forth in any one of claims 22 to 38 wherein a segment of the hydrophobic-modified hydrophilic ligand derived from the hydrophobic ether-containing compound is neutral at pH values of 3 to 9 for promoting hydrophobic interaction.