Hydrophobic interaction chromatography (HIC) compositions and methods for producing HIC compositions

Abstract

The hydrophobic interaction chromatography (HIC) composition includes a solid-phase substrate and a hydrophobically modified hydrophilic ligand covalently attached to the solid-phase substrate. The hydrophobic modified ligand includes a hydrophilic ligand moiety covalently attached to the solid-phase substrate, the hydrophilic ligand moiety including a polar group and multiple hydroxyl groups. The hydrophobic modified ligand also includes a peptide segment covalently attached to the hydrophilic ligand moiety and including 2 to 20 amino acid residues. The peptide segment is arranged linearly, and each of the amino acids is identical to or different from the other amino acid residues to promote HIC interactions.

Description

[Technical Field]

[0001] (Government support) This invention was made with government support under award GM140789 from the National Institutes of Health. The government has certain rights in this invention.

[0002] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 403,548, filed September 2, 2022, the entire text of which is incorporated herein by reference.

[0003] (Technical field) The present disclosure relates generally to chromatography compositions for use in hydrophobic interaction chromatography. [Background technology]

[0004] Hydrophobic interaction chromatography (HIC) is a chromatographic method that uses a salt solution, usually under aqueous conditions, to induce the reversible association of molecules with an appropriately modified surface. Traditional applications of HIC include biomolecular separations, achieving the isolation of a target biomolecule or class of biomolecules, or analyzing mixtures of such molecules. In recent years, this method has become favored for the separation of protein biomolecules because it can be performed under conditions considered mild or unlikely to disrupt native, biologically functional protein structures. HIC applications are not limited to proteins; they have also been applied to other biomolecules, including carbohydrates, nucleic acids, and complex molecular assemblies, including biomolecular complexes, complexes, intracellular organelles, and viruses. The characteristics of the method can be complicated by a variety of potential or known interactions between the target of interest, the chemically modified chromatographic surface, and the solvent or solvent additives used to operate the separation. In a broad sense, the purpose of chromatographic materials in separations is to facilitate the differential spatial and temporal migration of chemical species in response to material flow within a defined device or conditions. Thus, the composition of the flow stream, commonly referred to as the mobile phase, the rate of movement of the mobile phase, the structural and compositional characteristics of the sample, and the characteristics of the chromatographic surface, commonly referred to as the stationary phase, as well as external characteristics such as the operating temperature, all determine the nature of the separation process. The characteristics of the chromatographic surface of the stationary phase, including specific characteristics of the surface's chemical structure, determine the association of sample components with the surface versus the probability that the sample components will remain in the flow path. However, conventional stationary phases for HIC are known to have practical limitations in separating proteins and other biomolecules. Therefore, opportunities remain for developing improved compositions useful as stationary phases for HIC. Summary of the Invention [Means for solving the problem]

[0005] In one embodiment of the present disclosure, a hydrophobic interaction chromatography (HIC) composition includes a solid-phase substrate and a hydrophobic-modified hydrophilic ligand covalently attached to the solid-phase substrate. The hydrophobic-modified ligand includes a hydrophilic ligand moiety covalently attached to the solid-phase substrate, the hydrophilic ligand moiety including a polar group and multiple hydroxyl groups. The hydrophobic-modified ligand also includes a peptide segment covalently attached to the hydrophilic ligand moiety and including 2 to 20 amino acid residues. The amino acid residues can include both natural and non-natural amino acids. The peptide segment is arranged linearly, and each of the amino acid residues is the same as or different from the other amino acid residues to promote HIC interactions.

[0006] Another aspect of the present disclosure provides a method for preparing an HIC composition for hydrophobic interaction chromatography. The method includes providing a solid-phase substrate and a hydrophilic ligand comprising a polar group and multiple hydroxyl groups. The method also includes reacting the solid-phase substrate with the hydrophilic ligand to covalently bond the hydrophilic ligand to the solid-phase substrate to form a hydrophilically modified substrate. The method further includes providing an activated compound comprising a leaving group and reacting the activated compound with one of the multiple hydroxyl groups of the hydrophilically modified substrate to form the activated hydrophilically modified substrate. The method further includes providing a peptide segment comprising 2 to 20 amino acid residues derived from amino acids. The peptide segment is linearly arranged, and each amino acid residue is the same as or different from other amino acid residues to promote HIC interactions. The method further includes reacting the activated hydrophilically modified substrate with the peptide segment to release the leaving group of the activated compound and form the hydrophobically modified hydrophilic ligand and the HIC composition.

[0007] The balance and location of hydrophilic and hydrophobic interactions, coupled with minimized ionic interactions, make the HIC composition useful for HIC separations of proteins and various biomolecules. [Brief explanation 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 taken in conjunction with the accompanying drawings, in which:

[0009] [Figure 1] Overlay of chromatograms showing the separation of nucleobases by a control (3TPG) stationary phase and a stationary phase formed from the HIC composition. [Figure 2] Overlay of chromatograms showing the separation of lysozyme and trastuzumab on a control (3TPG) stationary phase and a stationary phase formed from an HIC composition. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present disclosure provides hydrophobic interaction chromatography (HIC) compositions. The HIC compositions are useful for HIC separations. For example, the HIC compositions are useful as stationary phases in HIC separations.

[0011] The HIC composition includes a solid-phase substrate and a hydrophobically-modified hydrophilic ligand covalently attached to the solid-phase substrate. The hydrophobically-modified hydrophilic ligand includes a hydrophilic ligand moiety covalently attached to the solid-phase substrate and a peptide segment directly or indirectly attached to the hydrophilic ligand moiety. In other words, the peptide segment modifies the hydrophilic nature of the hydrophilic ligand moiety.

[0012] The hydrophilic ligand moiety comprises a polar group and multiple hydroxyl groups. The polar group of the hydrophilic ligand moiety may be selected from carbonate, carbamate, amide, amine, ureide, ether, thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, or thiocarbamate. The above functional groups may be contained in a heterocyclic compound. For example, the polar group may be an aromatic ring containing an amine. In one embodiment, the polar group is selected from amide or carbamate. The multiple hydroxyl groups present on the hydrophilic ligand moiety may be two or more hydroxyl groups. Alternatively, the hydrophilic ligand moiety may contain 2 to 8, 2 to 7, or 3 to 5 hydroxyl groups.

[0013] First, regarding the solid-phase matrix, the solid-phase matrix is ​​typically, but not necessarily, silica. The silica used in the HIC composition is not limited to a specific grade. Both non-porous spherical silica and porous silica (including superficially porous silica) can be used. The average particle size of the silica particles is typically 0.5-100 μm, 1-50 μm, 1.5-10 μm, or 1.7-5 μm. The average pore size of the porous silica can be about 80 Å or greater, about 250 Å or greater, about 300 Å or greater, about 450 Å or greater, 200 to 1,000 Å, 250 to 900 Å, or 300 to 850 Å. Alternatively, pore sizes less than 70 Å are typically avoided, but average pore sizes of about 1 to about 50 Å, about 5 to about 40 Å, or about 10 to about 30 Å are expected. The surfaces of silica particles typically contain silica hydroxyl groups, so-called silanols, which are useful for covalently bonding various reagents, such as hydrophilic ligand moieties, to the silica surface. Most commonly, certain organosilane reagents are used to modify these silica surfaces to form covalently attached bonded phases. Suitable grades of silica are available from Advanced Materials Technologies, headquartered in Wilmington, Delaware, as Halo Fused-Core® Silica. RSilica), although many silica materials are widely available as commercial materials for a variety of useful applications. Alternative matrices include inorganic / organic hybrid materials. In the context of this disclosure, the term "inorganic / organic hybrid material" includes inorganic-based structures in which organic functional groups are integral to both the inner core (i.e., inorganic structure) and the surface of the hybrid material. The inorganic portion of the hybrid material can be, for example, alumina, silica, titanium, cerium, zirconium, or oxides thereof, or ceramic materials. Yet another matrix comprises a completely organic matrix containing hydroxyl groups on the surface of an organic matrix. In certain embodiments, the solid matrix is ​​formed from a carbohydrate. Alternatively, a carbohydrate may be included when covalently bound to the inorganic or inorganic / organic hybrid material. In other embodiments, the solid matrix is ​​not formed from a carbohydrate.

[0014] Although not required, the hydrophobically modified hydrophilic ligand may be of Formula I: [ka] It can be expressed as: (where, X is a polar group, Z is a linking group; Y is a peptide segment, n is 1-6, n' is 0-2, m is 2-8, p is 0 or 1, s is 1, R 1 , R 2 , R 3 are independently H or a linear or branched, substituted or unsubstituted, C1 to C18 alkyl group, and R 4 and R 5 are independently H or OH, and at least two m units contain at least one hydroxyl group.) It is understood that throughout this disclosure the phrase "m unit" refers merely for convenience to the repeat unit with the subscript "m" in Formula I.

[0015] When the hydrophobically modified hydrophilic ligand is represented by Formula I, the hydrophilic ligand moiety is represented by Formula Ia: [ka] It is expressed as: (X is a polar group, n is 1-6, and n' is 0-2, R 1 , R 2 , R 3 are independently H or a linear or branched, substituted or unsubstituted, C1 to C18 alkyl group; R 4 and R 5 are independently H or OH, and at least two m units contain at least one hydroxyl group.

[0016] Typically, but not necessarily, p is 1, such that the linking group Z is present in the hydrophobically modified hydrophilic ligand.

[0017] The polar group X is independently selected from carbonate, carbamate, amide, amine, ureido, ether, thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, or thiocarbamate, including heterocyclic compounds containing polar functional groups. For example, the polar group can be an aromatic ring containing an amine. In one embodiment, the polar group X is selected from amide or carbamate. In another embodiment, the polar group X is an amide. When the polar group X is an amide, the hydrophobically modified hydrophilic ligand has the formula Ib: [ka] It can be expressed as:

[0018] In certain embodiments of Formula I and Formula Ib, n is 2-4, m is 3-6, p is 1, and R 1 , R 2 , R 3are independently H or a straight or branched, substituted or unsubstituted C1 to C6 alkyl group. Although not required, when X is an amide, n' is typically 0. In other embodiments of Formula I, when X is a ureido, n' is 1 or 2. In one embodiment of Formula Ib, n is 3, X is an amide, m is 5, and four of the m units contain only one hydroxyl group. In one embodiment, the hydrophobically modified hydrophilic ligand has Formula Ic: [ka] It is expressed as:

[0019] In certain embodiments, p is 1 such that a linking group Z is included in the hydrophobically modified hydrophilic ligand. Although not required, when p is 1, the linking group Z is typically a carbamate group. In certain embodiments of Formula Ic where the polar group X is an amide, a linking group Z is also present, m is 5, four of the m units contain only one hydroxyl group, and the hydrophobically modified hydrophilic ligand has Formula II: [ka] It is expressed as:

[0020] When the linking group Z is a carbamate group, Formula II can be further represented by Formula IIa: [ka] It is expressed as:

[0021] Referring now to the peptide segment, as described above, the peptide segment modifies the hydrophilic ligand moiety. The peptide segment is directly or indirectly covalently attached to the hydrophilic ligand moiety. When the linking group Z is absent (i.e., when the subscript p is 0), the peptide segment is considered to be directly covalently attached to the hydrophilic ligand moiety. Conversely, when the linking group Z is present (i.e., when the subscript p is 1), the peptide segment is considered to be indirectly covalently attached to the hydrophilic ligand moiety. Typically, the peptide segment increases the hydrophobicity of the hydrophilic ligand.

[0022] The peptide segment is linearly arranged and contains 2 to 20 amino acid residues. The amino acids can be selected from natural or non-natural amino acids. Although not required, the peptide segment may contain one or more peptide nucleic acid (PNA) residues. The peptide segment generally consists of amino acid residues or a combination of amino acid residues and PNA residues. In other words, in these embodiments, the peptide segment does not contain any components other than amino acid residues and any PNA residues.

[0023] In the context of this disclosure, "linearly arranged" refers to a linear linkage of amino acid residues and any PNA residues that avoids circular and cyclic structures. For example, if a peptide segment consists of four amino acid residues, the first amino acid is covalently attached to the linking group Z, the second amino acid is attached to the carboxylic acid terminus of the first amino acid, the third amino acid is attached to the carboxylic acid terminus of the second amino acid, and the fourth amino acid is attached to the carboxylic acid terminus of the third amino acid. Those skilled in the art will understand that the carboxylic acid terminus of an amino acid participates in the reaction, which is one reason why amino acids are referred to as amino acid residues after the reaction. A peptide segment can contain two, three, four, five, six, or more amino acid residues. With current methods, peptides longer than about 20 amino acid residues are expensive to synthesize and purify, which is a current practical limitation. The cost and purity of synthetic or biosynthetic sequences may be addressed by future improvements in synthetic procedures. In principle, this is a practical limitation and does not theoretically constrain the use of longer peptides in constructing peptides for HIC interactions. Furthermore, in addition to the challenges of synthesis and purification, longer peptides or polypeptides are known to involve secondary structure considerations, as peptides longer than 5-7 amino acids can form a variety of stable or semi-stable structures, including helical segments, beta-turns, etc., depending on the solution environment and the amino acid sequence used. Such structures may be desirable or may interfere with the potential for desirable HIC interactions. In certain embodiments, peptide segments may be 3-5 amino acid residues.

[0024] Amino acid residues can be selected from the 20 standard amino acids (known as proteinogenic amino acids) that most commonly constitute protein structures, making them less expensive and more widely used in biochemical research. Various non-standard amino acids not commonly found in proteins can also be used in solid-phase peptide synthesis, solution-phase peptide synthesis, or a combination of these techniques. Peptides and polypeptides can also be produced by biosynthetic processes within cells or by cell-free translation using various biological components and enzymatic machinery, along with an appropriate RNA template. Such cellular processes, or subsystems derived from cellular processes, can incorporate modified amino acids, non-standard amino acids, and the like, to construct peptide or polypeptide chains. By way of example, such non-standard, or "unnatural," or synthetic amino acids may include those with large hydrophobic side chains (aliphatic, aromatic, or polyaromatic), those with side chains with extended or altered hydrocarbon extensions on the side chain (e.g., homoamino acids), or those with side chains with extended or altered hydrocarbon extensions between the carboxyl and amino groups (e.g., beta-homoamino acids), those with side chains protected with reactive groups, those with optical isomers (residues exhibiting D- versus L- optical rotation at the alpha carbon of the amino acid structure), and those with other side chains with altered structures (e.g., citrulline, norleucine, etc.). These amino acids may be formed by chemical or enzymatic processes as precursors in peptide synthesis, or may be formed post-synthetically by similar steps or chemical reactions. Similarly, there are abundant examples of chemically analogous approaches that can be used to construct polypeptide analogs with functional or sequence diversity and varied side chains, including peptide nucleic acids (PNAs), which have similar functional properties to nucleic acids (e.g., the ability to hybridize with nucleic acids) but share the characteristics of a polypeptide backbone with great structural diversity, can be synthesized using methods similar to peptides, and exhibit chemical properties that may be beneficial in promoting selective HIC interactions.

[0025] Typically, but not necessarily, at least half of the amino acid residues in a peptide segment are neutral at pH values ​​between 3 and 9. Individual amino acid residues that are neutral at pH values ​​between 3 and 9 promote hydrophobic interactions. Furthermore, individual amino acids that are neutral at pH values ​​between 3 and 9 not only promote hydrophobic interactions, but also minimize potential ionic interactions and, therefore, retention mechanisms in ion-exchange chromatography (IEX). The mixed-mode nature of HIC and IEX can also be exploited to manipulate chromatographic selectivity, although optimizing the separation can be more complex. By selecting a majority of individual amino acids in a peptide segment that are neutral at pH values ​​between 3 and 9, the entire peptide segment is believed to more uniformly promote hydrophobic interactions when used to modify the hydrophilic ligand moiety of a stationary phase.

[0026] Those skilled in the art will understand that a majority means that more than 50% of the amino acids present in a peptide segment are neutral at pH values ​​between 3 and 9. For example, if a peptide segment comprises four amino acid residues, at least three of the individual amino acid residues are neutral at pH values ​​between 3 and 9. In certain embodiments, more than 75% of the individual amino acid residues are neutral at pH values ​​between 3 and 9. In other embodiments, only one of the amino acid residues in a peptide segment is not neutral at pH values ​​between 3 and 9. In other embodiments, each of the amino acid residues in a peptide segment is neutral at pH values ​​between 3 and 9.

[0027] HIC utilizes hydrophobic interactions that occur between analytes (whether preparative or analytical) and the stationary phase surface. These interactions are either enhanced by increased ionic strength, with the preferred use of certain salts, or diminished by the presence of polar organic solvents (e.g., isopropanol, methanol, ethanol, acetonitrile, etc.) added to the mobile phase. In some situations, both salt and organic solvent may be added to the mobile phase in combination. HIC is often initiated by introducing a sample into the HIC process at high ionic strength (high salt), which is then decreased over time as the volume of mobile phase increases, often with a controlled mixture of high and low ionic strength components. These gradient elution methods can induce separation of sample components by decreasing ionic strength and / or increasing organic solvent concentration over the course of the separation. In addition to manipulating the mobile phase characteristics in HIC, a key factor in determining the strength of the hydrophobic effect promoting analyte retention is the local or overall hydrophobicity of the ligands on the stationary phase in the separation process. Currently, this hydrophobicity is intended to be provided to the chromatographic surface by peptide segments covalently attached to the stationary phase via linking groups and hydrophilic ligands. Amino acid side chains, and peptides or polypeptides formed from such amino acids, can be described as hydrophobic or less hydrophobic based on relative measures of hydrophobicity well known in the scientific literature. Such relative hydrophobicity measures include a measure of the equilibrium partitioning of an amino acid, peptide, or polypeptide between water and octanol, a highly hydrophobic, immiscible organic solvent (known as the water / octanol partition coefficient). Individual amino acids, or congeners, can thus be ranked relative to one another or compared to reference compounds for which hydrophobicity can be described as a chemical property.Similarly, peptides composed of such amino acid blocks, etc., can be measured or calculated from the hydrophobicity of the constituent amino acids, adjusted for the formation of amide backbone structures in the peptide or polypeptide chain or the presence of free terminal charged groups (free amino or carboxy termini) of the peptide or polypeptide. While the water / octanol partition coefficient may not reveal specific biological meaning for amino acids or polypeptides composed of these components, a strong correlation is known to predict the desired hydrophobicity tendency resulting from such side chains or compositions in nonpolar biological environments, such as those occurring in protein segments that cross biological membranes, or polypeptide sequences occupying internal domains of protein molecules, or within hydrophobic compartments of residues that may occur on the surface of proteins. The tendency of hydrophobic side chains to be desired in hydrophobic environments (e.g., membrane interiors) correlates well with physicochemical measures such as the water / octanol partition coefficient. Thus, hydrophobicity in the context of a peptide sequence can be defined as a property of a given peptide sequence, or measured or calculated as a local peptide sequence property of a longer peptide, polypeptide, or protein sequence. As a result, discussions of the hydrophobicity of peptide sequences must be considered in the context of the global properties of the complete sequence or as a local property of shorter segments within the structure of a peptide or polypeptide sequence that may exist within a longer sequence. Longer sequences can be designed to have sections of highly hydrophobic sequences interspersed with less hydrophobic sequences formed by consecutive hydrophobic residues. The utility of such configurations can provide useful properties (such as HIC retention, separation selectivity, or eluting analyte band width) that are not apparent from considering only the overall hydrophobicity (averaged across all residues) of the entire peptide sequence, such as those used as covalently attached peptide segments in the present invention. Currently, we are unaware of any theory that predicts the local or nearest neighbor effects of stationary-phase-bound peptide amino acid sequences on HIC retention, separation selectivity, or band broadening relationships.A reasonable inference is that such chromatographic performance characteristics may be influenced by specific, or local, sequence arrangements in the context of longer peptide segment sequences used as peptide segment ligands attached to the stationary phase of HIC.

[0028] Typically, when the C-terminus is blocked, the entire peptide segment is neutral at pH values ​​between 3 and 9, and the N-terminus participates in covalent bond formation with the linking group. The C-terminal blocking agent is not particularly limited and can be any compound capable of reacting with the C-terminal carboxy acid to form a stable chemical bond. Typically, the C-terminus is blocked with an amide, resulting in a carboxyamide reaction product that is chemically stable, but not ionized, under typical mobile phase conditions for HIC separations. A more hydrophobic C-terminal carboxyamide, along with a longer aliphatic chain or aromatic composition, may be selected to enhance HIC separation performance.

[0029] The amino acid residues of the peptide segment are typically derived from amino acids selected from the group of glycine (Gly), leucine (Leu), alanine (Ala), isoleucine (Ile), valine (Val), methionine (Met), cysteine ​​(Cys), proline (Pro), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. In one embodiment, the amino acid residues are derived from amino acid units selected from the group of glycine (Gly), leucine (Leu), and combinations thereof.

[0030] In certain embodiments, the peptide segment is i. -Leu-Leu-Leu, ii. -Gly-Gly-Gly, iii. -Leu-Gly-Gly-Gly, iv. -Leu-Gly-Leu-Gly, v. -Leu-Leu-Gly-Gly, vi.-Leu-Leu-Leu-Gly, vii. -Gly-Gly-Gly-Leu, viii. -Gly-Leu-Gly-Leu, ix. -Gly-Gly-Leu-Leu, x. -Gly-Leu-Leu-Leu, and xi. -Gly-Gly-Leu-Leu-Gly-Gly-Leu-Leu-Gly-Gly-Leu-Leu is selected from the group: When the C-terminus of the peptide segment is blocked with an amide (AM), the peptide segment is ia. -Leu-Leu-Leu-AM, iia. -Gly-Gly-Gly-AM, iiia. -Leu-Gly-Gly-Gly-AM, iva. -Leu-Gly-Leu-Gly-AM, va.-Leu-Leu-Gly-Gly-AM, via. -Leu-Leu-Leu-Gly-AM, viia. -Gly-Gly-Gly-Leu-AM, viiia. -Gly-Leu-Gly-Leu-AM, ixa. -Gly-Gly-Leu-Leu-AM, xa. -Gly-Leu-Leu-Leu-AM, and xia. -Gly-Gly-Leu-Leu-Gly-Gly-Leu-Leu-Gly-Gly-Leu-Leu-AM is selected from the group:

[0031] In certain embodiments, the hydrophobically modified hydrophilic ligand is represented by formula IIa, and Y represents any one of the peptide segments i.-x. or ia.-xa. above. [ka]

[0032] Additional peptide segments different from i.-xi. are also contemplated and can be tailored based on the target molecule requiring separation. Specifically, based on the present disclosure, peptide segments can be selected with the following characteristics in mind: Peptide segments can be linear sequences of 2-20 amino acid residues, allowing for efficient synthesis and purification. Peptide segments of shorter length are generally less likely to favor stable, high-affinity biospecific interactions, such as with antibody recognition regions or specific protein-protein interaction sites. Amino acid compositions can be selected based on searching protein sequence databases readily available as public repositories of protein sequences or predicted protein sequences based on reverse translation of genome sequence databases, avoiding known protein-protein interaction sequences or common antigenic determinants used to direct immune responses or immunochemical associations. The HIC peptide segment composition is specifically designed for hydrophobic interaction chromatography, thereby avoiding undesirable biospecific interactions and sequences selected for such recognition properties. In addition, at the N-terminus, where the hydrophobic peptide segment reacts with a linking group or hydrophilic ligand moiety for immobilization, the side chain should not sterically hinder the reaction to form the binding site. While amino acids with "larger" side chains may be acceptable, this suggests that glycine (H-side chain) or alanine (CH3-side chain) are preferred. Hydrophobic amino acids include glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp). Met and Trp can be chemically modified, for example, by oxidation, and are less preferred as less stable building blocks. Neutral polar amino acids include asparagine (Asn), glutamine (Gln), cysteine ​​(Cys), serine (Ser), and threonine (Thr), which vary somewhat in their hydrophobicity scale characteristics.Cysteine ​​can be easily chemically modified by oxidation to form disulfide bridges, making it a less preferred amino acid as it is less chemically stable unless a specific structure is defined that is beneficial for HIC properties (e.g., disulfide-bridged polypeptides). Amino acids with side chains that can be protonated or deprotonated to form a charge may exhibit ionic interactions instead of the neutral interactions required in hydrophobic interaction chromatography. Therefore, it is preferable to avoid amino acids with side chains that can be protonated or deprotonated to form a charge unless such properties offer special advantages for separations using mixed-mode chromatography that combine HIC and IEX properties.

[0033] In these formulation guidelines, by convention, the following hydrophobic peptides are explicitly assumed to be written as n-terminus-X1-X2-X3-X4-X5···Xn-c-terminus. X1: gly or ala, or other choices. X2-Xn-c-1: hydrophobic, non-ionic, small or large amino acid residues. Xn-c: A hydrophobic, non-ionic, small or large amino acid residue in which the C-terminal carboxylic acid is blocked with an amide, such as an alkylamide group. As mentioned above, a mixture of hydrophobic and hydrophilic or even ionic amino acids may have separation advantages in some cases and is expected to be potentially useful. Similarly, a mixture of standard amino acids with one or more non-standard amino acids may have separation advantages, prevent undesired secondary structures, or provide resistance to chemical or enzymatic digestion if necessary. Similarly, a mixture of one or more PNAs may be used as interspersed or intervening residues with, for example, amino acids, or as sequences flanking the PNA residues, or vice versa.

[0034] Returning to the overall HIC composition, in addition to having hydrophobically-modified hydrophilic ligands bound to the solid-phase substrate, the HIC composition may have the described or similar hydrophilic ligands (i.e., hydrophilic ligands that are not hydrophobically-modified) bound and / or covalently attached to the solid-phase substrate. In other words, in certain embodiments, in various compositions or mixtures, both hydrophilic ligands and hydrophobically-modified hydrophilic ligands are covalently attached to the solid-phase substrate. This mixture may result from a deliberately intended reaction or may represent incomplete reaction of the hydrophilic ligands, producing a partially reacted collection of hydrophilic ligands hydrophobically modified by peptide segments.

[0035] The polar group of the hydrophilic ligand can be selected from carbonate, carbamate, amide, amine, ureide, ether, thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, or thiocarbamate, including heterocyclic compounds containing polar functional groups. For example, the polar group can be an aromatic ring containing an amine. In one embodiment, the polar group is selected from amide or carbamate. The multiple hydroxyl groups present on the hydrophilic ligand can be two or more hydroxyl groups. Alternatively, the hydrophilic ligand can contain 2 to 8, 2 to 7, or 3 to 5 hydroxyl groups.

[0036] In one embodiment, the hydrophilic ligand has formula V: [ka] It is expressed as: (X is a polar group, n is 1-6, n' is 0-2, m is 2-8, q is 1, R 1 , R 2 , R 3 are independently H or a linear or branched, substituted or unsubstituted C1 to C18 alkyl group; R 4 and R 5is independently H or OH, at least two m units contain at least one hydroxyl group, and R 8 and R 9 are independently H or OH, with the proviso that R 8 and R 9 At least one of is OH.)

[0037] R contained in unit q 8 and / or R 9 The hydroxyl group in may also be referred to as a terminal hydroxyl group. Those skilled in the art will recognize that the hydrophilic ligand portion of the hydrophobically modified hydrophilic ligand and the hydrophilic ligand are defined as those in which the hydrophilic ligand portion is a unit q (i.e., -[C(R 8 )(R 9 It will be understood that the hydrophilic ligands share a similar structure except that they do not contain a q unit). Thus, the hydrophilic ligands can include each of the various structural configurations described above for the hydrophilic ligand moiety, except that the hydrophilic ligand further includes a q unit.

[0038] In one embodiment, the hydrophilic ligand of formula V can further be represented by formula Va: [ka] It is expressed as:

[0039] When an HIC composition includes a hydrophilic ligand in addition to a hydrophobically modified hydrophilic ligand, the relative amounts of each ligand can be optimized based on the individual analytes to be separated. For example, in certain embodiments, the hydrophobically modified hydrophilic ligand and the hydrophilic ligand are present in a molar ratio range of 1:10 to 10:1. Alternatively, the hydrophobically modified hydrophilic ligand and the hydrophilic ligand can be present in a molar ratio range of 2:8 to 8:2, 3:7 to 7:3, 4:6 to 6:4, or about 1:1. In certain embodiments, the solid substrate is superficially porous silica covalently bonded to a hydrophobically modified hydrophilic ligand represented by Formula I and a hydrophilic ligand represented by Formula V. Alternatively, in one embodiment, the solid substrate is superficially porous silica and covalently bonded to a hydrophobically modified hydrophilic ligand represented by Formula Va and a hydrophobically modified hydrophilic ligand represented by Formula II.

[0040] The present disclosure also provides a method for producing an HIC composition. The method includes providing a solid-phase substrate and a hydrophilic ligand comprising a polar group and multiple hydroxyl groups. In certain embodiments, the hydrophilic ligand has at least one terminal hydroxyl group, typically only one terminal hydroxyl group. Both the solid-phase substrate and the hydrophilic ligand are described above. The method further includes reacting the solid-phase substrate with the hydrophilic ligand to covalently bond the hydrophilic ligand to the solid-phase substrate and form a hydrophilically modified substrate. The method further includes providing an activating compound comprising a leaving group and reacting the activating compound with one of the multiple hydroxyl groups. In certain embodiments, reaction occurs preferentially with the terminal hydroxyl group of the hydrophilically modified substrate. Once reaction occurs between the activating compound and the hydroxyl groups, the new composition is referred to as an activated hydrophilically modified substrate. The terminal hydroxyl group is by design a primary hydroxyl group, while the hydrophilic ligand has secondary hydroxyl groups at other positions. This distinction allows for selective reaction of the primary hydroxyl groups over the secondary hydroxyl groups. In other words, this method involves a first reaction between the solid-phase substrate and the reaction product of the first reaction (i.e., the hydrophilically modified substrate) and a second reaction between the activated compound. Although not common, some reaction may also occur between the secondary hydroxyl group of the hydrophilic ligand moiety and the activated compound. This method further involves providing a peptide segment and reacting the free amino terminus of the peptide segment with the activated hydrophilically modified substrate to release the leaving group of the activator and form a hydrophobic-modified hydrophilic ligand covalently attached to the solid-phase substrate. In other words, this method also involves a third reaction between the reaction product of the second reaction (i.e., the reaction between the activated compound and the hydrophilically modified substrate) and the peptide segment. The resulting reaction product of the third reaction is an HIC composition comprising a hydrophobic-modified hydrophilic ligand covalently attached to the solid-phase substrate.

[0041] Referring first to the first reaction between the solid phase substrate and the hydrophilic ligand, this reaction occurs when a surface hydroxyl group present on the solid phase substrate reacts with a hydrophilic ligand of formula V: [ka] There are three [(R 1 O)] unit. The resulting reaction product produces a hydrophilically modified substrate and is [C(R 8 )(R 9 The hydroxyl group in the q unit represented by the formula (2) is protected.

[0042] Typically, the second reaction between the hydrophilically modified substrate and the activated compound occurs under aprotic, anhydrous solvent conditions to limit hydrolytic loss of the activated complex. The activated compound may contain a carbonyl group. Specific examples of activated compounds containing a carbonyl group include, but are not limited to, phosgene (carbonyl dichloride), carbonyldiimidazole (CDI), or a chloroformate (e.g., 4-nitrophenyl chloroformate (4-NPC)), or a carbonate (e.g., N,N'-disuccinimidyl carbonate (DSC) or a combination thereof). An example to aid in understanding the second reaction product between a hydrophilic ligand of Formula Va and DSC, in which the N-hydroxysuccinimidyl (NHS) carbonate of the 3-TPG compound is formed, is shown below. [ka]

[0043] Alternative activating compounds include, but are not limited to, compounds with a tosylate group, such as tosyl chloride (4-toluenesulfonyl chloride). Further suitable activating compounds include mesyl chloride (methanesulfonyl chloride), triphenylmethylene chloride (trityl chloride), phosphorus tribromide, or thionyl chloride. Although not typical, any of these reactive compounds may be used in combination with other activating compounds.

[0044] Without being bound by any particular theory, it is believed that under appropriate conditions, the activated compounds described herein can selectively react with the terminal hydroxyl groups of the hydrophilic ligands. Selective reaction at the terminal hydroxyl groups is also believed to be an important aspect of the present disclosure, as homogeneity and the general avoidance of multiple reaction products, crosslinked intermediates, or cyclic carbonates are favorable for achieving consistent chromatographic separation. When the hydrophilic ligand reacts with the activated compound, the hydrophilic ligand moiety of Formula Ia is established.

[0045] An example to aid in understanding the third reaction product resulting from the reaction of the blocked peptide segment -Leu-Gly-Gly-Gly-AM with the reaction product shown above is provided below. [ka]

[0046] As shown above, reaction of the second reaction product with the peptide segment displaces the leaving group of the activated compound and forms a carbamate (urethane) bond, which represents the linking group Z in Formula I.

[0047] The method for preparing the HIC composition can also include binding both the hydrophobically 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 is covalently bound to the surface of the solid-phase substrate, the hydrophilic ligand can be maintained in its current state by including fewer moles of activating compound than the number of moles of hydrophobic ligand bound to the substrate. In particular, the peptide segment reacts only with the activated hydrophilic ligand and not with the hydrophilic ligand (i.e., the unactivated hydrophilic ligand), leaving the remaining hydrophilic ligand unmodified. Alternatively, the activation and modification reactions of the hydrophilic ligand can occur in free solution to produce a mixture that can then be covalently bound to the solid-phase support.

[0048] (Example) Those skilled in the art will recognize that equivalents to the following instruments and suppliers exist, and therefore the instruments listed below should not be construed as limiting the invention.

[0049] Elemental analysis (%C, %H, %N) values ​​were determined by combustion analysis (Robertson Microlite Laboratories, Ledgewood, NJ). These values ​​were obtained based on known compound compositions and specific surface areas (m 2 The specific surface area (SSA), specific pore volume (SPY), and average pore diameter (APD) of these materials were measured using a multipoint N2 adsorption method (Micromeritics ASAP2400, Micromeritics Instruments, Norcross, GA). SSA was calculated using the BET method, SPY was a single-point value determined at P / P > 0.98, and APD was calculated from the desorption portion of the isotherm using the BJH method. Particle size was measured using a Beckman Coulter Multisizer 3 analyzer (30 μm aperture, 70,000 count, Miami, FL). Particle diameter (dp) was measured as the 50% cumulative diameter of the volumetric particle size distribution. The width of the distribution was measured as the 90% cumulative volume diameter divided by the 10% cumulative volume diameter (denoted as the 90 / 10 ratio). Generally, surface coverage values ​​are expressed as normalized values ​​to the elemental composition and SSA of the sample, where the molar surface coverage of the silica surface by ligands is μmol / m 2 is obtained.

[0050] Commercially available 2.7 µm diameter fully hydroxylated superficially porous silica particles (Halosilica 25 g, Advanced Materials Technologies, Wilmington, DE, USA; SSA = 22 m 2SiO2 ( / g, APD = 645 Å) was dispersed in toluene (250 mL, Millipore / Sigma, St. Louis, NJ) under a nitrogen atmosphere at reflux for 1 h using a Dean-Stark trap to collect a small amount of adsorbed water. After briefly cooling to approximately 65 °C, 22 mmol of diisopropylethylamine (DIPEA, Sigma-Aldrich, St. Louis, MO) was added with stirring, followed by 66 mmol of N-(3-triethoxysilylpropyl)gluconamide (3TPG, 30% ethanol solution, Gelest, Morrisville, PA) (36 (strikeout)). The resulting mixture was heated to 78 °C to remove most of the ethanol and refluxed overnight, during which approximately 5 mL of solvent was removed periodically to aid in the removal of ethanol generated during the binding of ethoxysilane to the silica particle surface. After cooling, the resulting silica particles were collected by filtration through a sintered glass funnel, washed with 200 mL of warm toluene, DMF, and acetonitrile, and then dispersed in 50% acetonitrile / water heated to 60 °C. The particles were then collected by filtration and washed with acetonitrile and methanol (all solvents were from Sigma-Millipore). The filtered and dried silica was further dried in a vacuum oven at 110 °C for at least 1 hour. The resulting 3-TPG-bonded silica was subjected to an additional coupling reaction using 6 mmol of DIPEA and 18 mmol of 3-TPG in 250 mL of dimethylformamide (DMF, Sigma-Aldrich, St. Louis, MO) at 85 °C overnight, during which approximately 5 mL of solvent was removed periodically through a Dean-Stark trap. After cooling, the solid was collected by filtration, washed with 200 mL of warm DMF and then acetonitrile, and then dispersed in 50% acetonitrile / water heated to 60 °C. The solid was then collected by filtration and washed with acetonitrile and methanol. The silica was dried under vacuum at 110° C. as before. The resulting 3-TPG-bound silica particles had a high density of 3-TPG bound to the silica surface, typically 3.5–3.8 μmol / m 2 It became clear that...

[0051] (peptide modification) The 3-TPG-bonded silica particles from Example 1 were dried in a vacuum oven for 2 hours. An appropriate amount of material (5-20 g) was dispersed in dry acetonitrile (Sigma-Aldrich, St. Louis, MO) at a volume of 10 mL per gram. Then, 0.2 mmol / g of 4-dimethylaminopyridine (DMAP, Sigma-Aldrich) was added with stirring at room temperature, followed by a large amount of disuccinimidyl carbonate (DSC, Oakland Chemicals) (normal or 0.96 mmol / g (Rx 3c)) with stirring and dispersion in an ultrasonic bath. The reaction to form the NHS-activated intermediate proceeded for 1.5 hours at room temperature under a nitrogen atmosphere. After that, each reaction mixture was separated, and the silica particles were collected on a filter and washed with 25 mL / g of dry acetonitrile, THF, 20% cold THF in 5 mM aqueous HCl, followed by THF, acetonitrile, and methanol. After drying under vacuum at room temperature, a small sample of the NHS-activated TPG silica was dispersed at 50 mg / mL in 0.25 M NH4OH for hydrolysis and spectrophotometric determination of NHS content, as described by Li and Vanderah (2021). The activated 3-TPG silica was dispersed in a peptide solution in acetonitrile reaction medium at 10 mL / g of silica solid in acetonitrile with stirring. Peptide solutions were prepared by dissolving various peptides obtained from Biomatic (Kitchener, Ontario, Canada). An appropriate amount of peptide (1.2-1.5 molar equivalents relative to the NHS present on the silica) was added to an appropriate volume of acetonitrile to form a 10 mL / g silica slurry. The peptide solution was made basic by adding 1.1 molar equivalents of DIPEA relative to the amount of peptide (usually obtained as the trifluoroacetate or hydrochloride salt). The peptide and activated silica slurry was stirred overnight at room temperature under a nitrogen atmosphere.

[0052] After the reaction was complete, the peptide-modified TPG silica was recovered by centrifugation (1500 x g, 5 min), dispersed in acetonitrile, and washed twice by repeated dispersion in 10 mL / g acetonitrile and centrifugation. Hydrolysis of remaining unreacted NHS modification sites was performed by dispersing the silica particles in a 0.2 M carbonate buffer (pH 9.5) / 10% acetonitrile solution, mixing for 30 minutes, and then dispersing in 0.5 M Tris buffer (pH 7.8) for 30 minutes. The modified silica was then washed twice by dispersion in water and centrifugation, dispersed in 10 mL / g water, vacuum filtered, and washed on the filter with acetonitrile and methanol (approximately 10 mL / g), followed by vacuum oven drying at 110 °C before drying on the filter. In this example, peptides of specific sequences were selected, all of which were modified at the C-terminus by amidation.

[0053] Quantification of NHS modification on the 3-TPG surface by DSC reagent was 0.52 μmol / m 2 The peptide-modified 3-TPG silica was acid hydrolyzed and then quantified using amino acid analysis (Creative Proteomics, Shirley, NY). Quantitative analysis revealed the correct AA composition and high coverage (approximately 0.5 μmol / m of peptide on the surface of the packing material). 2 ) was revealed. Based on amino acid analysis, all of the peptide-modified 3-TPG silicas reacted similarly. The ability to monitor activation density (UV), combined with post-reaction amino acid analysis, allowed us to monitor this synthetic sequence.

[0054] (Chromatographic properties of peptide-modified 3-TPG silica) 3-TPG silica and peptide-modified 3-TPG silica were used to pack stainless steel HPLC columns with an internal diameter of 2.1 mm and a length of 100 mm. These materials were applied to the chromatographic separation of small polar nucleobase compounds using hydrophobic interaction chromatography, as shown in Figure 1. Separation was performed using a Shimadzu Nexera® LC system at a flow rate of 0.25 mL / min, a column temperature of 25°C, and an aqueous mobile phase consisting of 92% acetonitrile and 8% 0.1 M ammonium formate (pH 3.0). Nucleobases were detected by absorbance at 260 nm. Retention of these polar compounds was highest with unmodified 3-TPG silica and decreased with increasing hydrophobic side chains in the amino acid residues present in the peptide sequence. Nucleobase separation was performed using columns packed with 3-TPG silica and peptide-modified 3-TPG silica. HILIC mode analysis of the separation of these three polar compounds demonstrates lower retention for peptide sequences with increasing hydrophobic amino acid residue composition.

[0055] These same columns were used to test the separation of known proteins by hydrophobic interaction chromatography by injecting 1-2 μL of an appropriately diluted protein mixture into a high ionic strength mobile phase. Separation was performed by gradient elution, using decreasing salt concentrations to drive the separation. The mobile phase was pumped at 0.4 mL / min, the column temperature was 30 °C, and a gradient of 0-100% B was used in 8 min, where mobile phase A was 2.0 M ammonium sulfate / 0.02 M potassium phosphate (pH 7.0) and buffer B was 0.02 M potassium phosphate (pH 7.0). Figure 2 shows a chromatogram showing the separation of a mixture of two proteins, lysozyme and trastuzumab (Herceptin), in HIC mode. Notably, retention of both proteins was lowest on unmodified 3-TPG silica, and was low for simple GGG peptide segments. However, retention of both proteins increased on peptide-modified 3-TPG silica, most notably for peptides rich in hydrophobic amino acids. Hydrophobic peptides, like those with intermediate hydrophobic compositions (eg, GLGL and GGLL), showed moderate retention, but also showed subtle differences in the separation of these two proteins.

[0056] It should be understood that the appended claims are not limited to describing any particular compound, composition, or method described in the specification, but may vary among specific embodiments falling within the scope of the appended claims. With respect to any Markush group relied upon herein to describe particular features or aspects of various embodiments, different, special, and / or unexpected results may be obtained from each element of the respective Markush group, independently of all other Markush elements. Each element of a Markush group may be relied upon individually or in combination to provide adequate support for specific embodiments within the scope of the appended claims.

[0057] Furthermore, all ranges and subranges relied upon in describing various embodiments of the present disclosure are understood to be individually and collectively encompassed within the scope of the appended claims and to describe and contemplate all ranges, including integers and / or decimal values, even if not explicitly set forth herein. Those skilled in the art will readily recognize that the recited ranges and subranges fully describe and enable various embodiments of the present disclosure, and that such ranges and subranges may be further subdivided into related halves, thirds, quarters, fifths, etc. As an example, the range "0.1 to 0.9" may be further divided into a lower third, i.e., 0.1 to 0.3, a middle third, i.e., 0.4 to 0.6, and an upper third, i.e., 0.7 to 0.9, each of which, individually and collectively, are within the scope of the appended claims and may be relied upon individually or collectively to fully support particular embodiments within the scope of the appended claims. Furthermore, with respect to words defining or modifying a range, such as "at least," "greater than," "less than," "less than or equal to," etc., it should be understood that such words include subranges and / or upper or lower limits. As another example, the range "at least 10" inherently includes the subranges of at least 10 to 35, at least 10 to 25, 25 to 35, etc., each of which may be relied upon individually and / or collectively to provide adequate support for particular embodiments within the scope of the appended claims. Finally, individual numbers within a disclosed range may be relied upon to provide adequate support for particular embodiments within the scope of the appended claims. For example, the range "1 to 9" includes various individual integers, such as 3, and individual numbers including decimal points (or fractions), such as 4.1, which may be relied upon to provide adequate support for particular embodiments within the scope of the appended claims.

[0058] The present disclosure has been described in an easy-to-understand manner, and it is understood that the terminology used is intended to be terms 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 other than as specifically described. The subject matter of all combinations of independent and dependent claims (both singly and multiple dependent) is expressly contemplated herein.

[0059] (Addendum) (Appendix 1) 1. A hydrophobic interaction chromatography (HIC) composition comprising: a solid phase substrate, and a hydrophobically modified hydrophilic ligand covalently attached to said solid phase substrate; Including, the hydrophobic modified ligand comprises a hydrophilic ligand portion covalently attached to the solid phase substrate and a peptide segment; the hydrophilic ligand portion comprises a polar group and a plurality of hydroxyl groups; the peptide segment is directly or indirectly covalently attached to the hydrophilic ligand moiety and comprises 2 to 20 amino acid residues, including natural or unnatural amino acid residues; the peptide segments are linearly arranged, and each of the amino acid residues is the same as or different from the other amino acid residues to promote HIC interaction; Hydrophobic interaction chromatography (HIC) compositions.

[0060] (Appendix 2) 2. The HIC composition of claim 1, wherein at least a majority of the amino acid residues of the peptide segment are neutral at a pH value of 3 to 9 to promote hydrophobic interactions.

[0061] (Appendix 3) 3. The HIC composition of claim 1 or 2, wherein the peptide segment comprises 2 to 7 amino acid residues.

[0062] (Appendix 4) 4. The HIC composition of any one of claims 1 to 3, wherein the peptide segment further comprises one or more peptide nucleic acid residues.

[0063] (Appendix 5) 5. The HIC composition of any one of claims 1 to 4, wherein the peptide segment consists of residues selected from the group consisting of natural amino acid residues, unnatural amino acid residues, and peptide nucleic acid residues.

[0064] (Appendix 6) 6. The HIC composition of any one of claims 1 to 5, wherein the C-terminus of the peptide segment is blocked to minimize ionic interactions.

[0065] (Appendix 7) 7. The HIC composition of claim 6, wherein the C-terminus is blocked with an amide.

[0066] (Appendix 8) 8. The HIC composition of any one of claims 1 to 7, wherein the peptide segment extends to a terminal amino acid residue having an amide-blocked carboxyl terminus.

[0067] (Appendix 9) 9. The HIC composition of any one of claims 1 to 8, wherein each of the amino acid residues of the peptide segment is neutral at a pH value of 3 to 9.

[0068] (Appendix 10) The hydrophobically modified hydrophilic ligand has Formula I: [ka] 10. The HIC composition according to any one of claims 1 to 9, (where, X is a polar group, Z is a linking group; Y is a peptide segment, n is 1-6, n' is 0-2, m is 2-8, p is 0 or 1, R 1 , R 2 , R 3 are independently H or a straight or branched, substituted or unsubstituted C1 to C18 alkyl group, and R 4 and R 5 are independently H or OH, and at least two m units contain at least one hydroxyl group.

[0069] (Appendix 11) The hydrophilic ligand moiety has the formula Ia: [ka] 11. The HIC composition according to any one of claims 1 to 10, (where, X is a polar group, n is 1-6, n' is 0-2, R 1 , R 2 , R 3 are independently H or a linear or branched, substituted or unsubstituted, C1 to C18 alkyl group, and R 4 and R 5 are independently H or OH, and at least two m units contain at least one hydroxyl group.

[0070] (Appendix 12) 12. The HIC composition of claim 10 or 11, wherein the polar groups X are independently selected from carbonate, carbamate, amide, amine, ureido, ether, thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, or thiocarbamate.

[0071] (Appendix 13) 13. The HIC composition of claim 12, wherein the polar group X is selected from an amide, a carbamate, or a ureido group.

[0072] (Appendix 14) 14. The HIC composition of claim 13, wherein the polar group X is an amide.

[0073] (Appendix 15) 15. The HIC composition of any one of claims 10 to 14. (where, n is 2-4, m is 3-6, p is 1, R 1 , R 2 , R 3 are independently H or a straight or branched, substituted or unsubstituted C1 to C6 alkyl group.

[0074] (Appendix 16) 16. The HIC composition according to any one of claims 10 to 15, wherein the linking group Z is a carbamate group and p is 1.

[0075] (Appendix 17) The hydrophobically modified hydrophilic ligand has Formula II: [ka] 17. The HIC composition according to any one of claims 10 to 16,

[0076] (Appendix 18) 18. The HIC composition of any one of claims 1 to 17, wherein the amino acid residues of the peptide segment are derived from amino acids selected from the group consisting of glycine (Gly), leucine (Leu), alanine (Ala), isoleucine (Ile), valine (Val), methionine (Met), cysteine ​​(Cys), proline (Pro), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), and combinations thereof.

[0077] (Appendix 19) 19. The HIC composition of claim 18, wherein the peptide segment comprises amino acid residues derived from amino acids selected from the group consisting of glycine (Gly), leucine (Leu), and combinations thereof.

[0078] (Appendix 20) The peptide segment is i. -Leu-Leu-Leu, ii. -Gly-Gly-Gly, iii. -Leu-Gly-Gly-Gly, iv. -Leu-Gly-Leu-Gly, v. -Leu-Leu-Gly-Gly, vi.-Leu-Leu-Leu-Gly, vii. -Gly-Gly-Gly-Leu, viii.-Gly-Leu-Gly-Leu, ix. -Gly-Gly-Leu-Leu, and x.-Gly-Leu-Leu-Leu 20. The HIC composition of any one of claims 1 to 19, selected from the group consisting of:

[0079] (Appendix 21) The peptide segment is -Gly-Gly-Leu-Leu-Gly-Gly-Leu-Leu-Gly-Gly-Leu-Leu The HIC composition of Appendix 1, represented by:

[0080] (Appendix 22) 21. The HIC composition of claim 20, wherein each of the amino acid residues is neutral at a pH value of 3 to 9.

[0081] (Appendix 23) The hydrophobically modified hydrophilic ligand has formula III: [ka] 23. The HIC composition according to any one of claims 10 to 22, wherein

[0082] (Appendix 24) 24. The HIC composition of any one of claims 1 to 23, further comprising a hydrophilic ligand covalently bound to the solid phase substrate, the hydrophilic ligand comprising a polar group and a plurality of hydroxyl groups.

[0083] (Appendix 25) The hydrophilic ligand has the formula V: [ka] 25. The HIC composition of claim 24, (where, X is a polar group, n is 1-6, n' is 0-2, m is 2-8, q is 1, R 1 , R 2 , R 3 are independently H or a straight or branched, substituted or unsubstituted, C1 to C18 alkyl group; R 4 and R 5 are independently H or OH, and at least two m units contain at least one hydroxyl group; and R 8 and R 9 are independently H or OH, with the proviso that R 8 and R 9 at least one of which is OH)

[0084] (Appendix 26) The hydrophilic ligand of formula V may further be represented by formula Va: [ka] 26. The HIC composition of claim 25,

[0085] (Appendix 27) 27. The HIC composition of any one of claims 24 to 26, wherein the hydrophobically modified hydrophilic ligand and the hydrophilic ligand are present in a molar ratio ranging from 1:10 to 10:1.

[0086] (Appendix 28) 28. The HIC composition of any one of claims 1 to 27, wherein the solid phase substrate is a silica material or an inorganic / organic hybrid material.

[0087] (Appendix 29) 29. The HIC composition of claim 28, wherein the solid phase substrate is a silica material.

[0088] (Appendix 30) A kit comprising the HIC composition of any one of appendices 1 to 29.

[0089] (Appendix 31) 30. Use of the HIC composition according to any one of claims 1 to 29 for HIC.

[0090] (Appendix 32) 1. A method for producing a HIC composition for hydrophobic interaction chromatography comprising a hydrophobically modified hydrophilic ligand, the method comprising: providing a solid phase substrate; providing a hydrophilic ligand comprising a polar group and a plurality of hydroxyl groups; reacting the solid phase substrate with the hydrophilic ligand to covalently bond the hydrophilic ligand to the solid phase substrate to form a hydrophilically modified substrate; providing an activated compound containing a leaving group; reacting said activating compound with one of said plurality of hydroxyl groups to form an activated hydrophilically modified substrate; providing a peptide segment comprising 2 to 20 amino acid residues, including natural or unnatural amino acid residues; reacting the activated hydrophilically modified substrate with the peptide segment to release the leaving group of the activated compound and form the hydrophobically modified hydrophilic ligand and the HIC composition; Including, the peptide segments are linearly arranged, and each of the amino acid residues is the same as or different from the other amino acid residues to promote HIC interaction; method.

[0091] (Appendix 33) 33. The method of claim 32, wherein at least a majority of the amino acid residues of the peptide segment are neutral at a pH value of 3 to 9 to promote hydrophobic interactions.

[0092] (Appendix 34) 34. The method of claim 32 or 33, wherein the peptide segment comprises 2 to 7 of the amino acid residues.

[0093] (Appendix 35) 35. The method of any one of claims 32 to 34, wherein the peptide segment further comprises one or more peptide nucleic acid residues.

[0094] (Appendix 36) 36. The method of any one of claims 32 to 35, wherein the peptide segment consists of residues selected from the group consisting of natural amino acid residues, unnatural amino acid residues, and peptide nucleic acid residues.

[0095] (Appendix 37) 37. The method of any one of claims 32 to 36, wherein the C-terminus of the peptide segment is blocked to minimize ionic interactions.

[0096] (Appendix 38) 38. The method of any one of claims 32 to 37, wherein the C-terminus of the peptide segment is blocked with an amide.

[0097] (Appendix 39) 39. The method of any one of claims 32 to 38, wherein the peptide segment extends to a terminal amino acid residue having a carboxyl terminus blocked with an amide to form a carboxamide.

[0098] (Appendix 40) 40. The method of any one of claims 32 to 39, wherein each of the amino acid residues of the peptide segment is neutral at a pH value of 3 to 9.

[0099] (Appendix 41) The hydrophilic ligand has the formula V: [ka] 41. The method of any one of Appendices 32 to 40, (where, X is a polar group, n is 1-6, n' is 0-2, m is 2-8, q is 1, R 1 , R 2 , R 3 are independently H or a straight or branched, substituted or unsubstituted C1 to C18 alkyl group; R 4 and R 5 are independently H or OH, and at least two m units contain at least one hydroxyl group; and R 8 and R 9 are independently H or OH, with the proviso that R 8 and R 9 at least one of which is OH and is present at the terminus of the hydrophilic ligand

[0100] (Appendix 42) 42. The method of claim 41, wherein the polar groups X are independently selected from carbonate, carbamate, amide, amine, urea, ether, thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, or thiocarbamate (including heterocyclic compounds containing polar functional groups).

[0101] (Appendix 43) 43. The method of claim 42, wherein the polar group X is selected from an amide, carbamate, or ureido group.

[0102] (Appendix 44) 44. The method of claim 43, wherein the polar group X is an amide.

[0103] (Appendix 45) 45. The method of any one of appendices 41 to 44. (n is 2-4, m is 3-6, p is 1, and R 1 , R 2 , R 3 are independently H or a straight or branched, substituted or unsubstituted C1 to C6 alkyl group.

[0104] (Appendix 46) The hydrophilic ligand has the formula Va: [ka] 46. ​​The method of any one of Appendices 41 to 45,

[0105] (Appendix 47) 47. The method of any one of claims 32 to 46, wherein the amino acid residues of the peptide segment are derived from amino acids selected from the group consisting of glycine (Gly), leucine (Leu), alanine (Ala), isoleucine (Ile), valine (Val), methionine (Met), cysteine ​​(Cys), proline (Pro), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), and combinations thereof.

[0106] (Appendix 48) 48. The method of claim 47, wherein the peptide segment comprises amino acid residues derived from amino acids selected from the group consisting of glycine (Gly), leucine (Leu), and combinations thereof.

[0107] (Appendix 49) The peptide segment is i. -Leu-Leu-Leu, ii. -Gly-Gly-Gly, iii. -Leu-Gly-Gly-Gly, iv. -Leu-Gly-Leu-Gly, v. -Leu-Leu-Gly-Gly, vi.-Leu-Leu-Leu-Gly, vii. -Gly-Gly-Gly-Leu, viii. -Gly-Leu-Gly-Leu, ix. -Gly-Gly-Leu-Leu, and x.-Gly-Leu-Leu-Leu 49. The method of any one of claims 32 to 48, wherein the compound is selected from the group consisting of:

[0108] (Appendix 50) The hydrophobically modified hydrophilic ligand has formula III: [ka] 50. The method of any one of Appendices 41 to 49, wherein

[0109] (Appendix 51) 51. The method of any one of claims 32 to 50, wherein the activated compound contains a carbonate group and is represented by 4-nitrophenyl chloroformate (4-NPC), N,N'-disuccinimidyl carbonate (DSC), carbonyldiimidazole (CDI), or a combination thereof.

[0110] (Appendix 52) 51. The method of any one of claims 32 to 50, wherein the activated compound comprises a tosylate group.

[0111] (Appendix 53) 52. The method of claim 51, wherein the activating compound is tosyl chloride.

[0112] (Appendix 54) 51. The method of any one of claims 32 to 50, wherein the activating compound is mesyl chloride, phosphorus tribromide, thionyl chloride, or a combination thereof.

[0113] (Appendix 55) The hydrophobically modified hydrophilic ligand has Formula I: [ka] 55. The method of any one of Appendices 32 to 54. (where, X is a polar group, Z is a linking group; Y is a peptide segment, n is 1-6, n' is 0-2, m is 2-8, p is 1, R 1 , R 2 , R 3 are independently H or a straight or branched, substituted or unsubstituted C1 to C18 alkyl group, and R 4 and R 5 are independently H or OH, and at least two m units contain at least one hydroxyl group.

[0114] (Appendix 56) 56. The method of claim 55, wherein the carboxyl terminus of the peptide segment is blocked with an amide.

[0115] (Appendix 57) 57. The method of any one of claims 32 to 56, wherein reacting the activating compound with one of the plurality of hydroxyl groups to form the activated hydrophilically modified substrate is further defined as reacting the activating compound with the hydroxyl group present at a terminus of the hydrophilically modified substrate.

Claims

1. 1. A hydrophobic interaction chromatography (HIC) composition comprising: a solid phase substrate, and a hydrophobically modified hydrophilic ligand covalently attached to said solid phase substrate; Including, the hydrophobic modified ligand comprises a hydrophilic ligand portion covalently attached to the solid phase substrate and a peptide segment; the hydrophilic ligand portion comprises a polar group and a plurality of hydroxyl groups; the peptide segment is directly or indirectly covalently attached to the hydrophilic ligand moiety and comprises 2 to 20 amino acid residues, including natural or unnatural amino acid residues; the peptide segments are linearly arranged, and each of the amino acid residues is the same as or different from the other amino acid residues to promote HIC interactions; Hydrophobic Interaction Chromatography (HIC) Compositions.

2. 2. The HIC composition of claim 1, wherein at least a majority of the amino acid residues of the peptide segment are neutral at pH values ​​between 3 and 9 to promote hydrophobic interactions.

3. The HIC composition of claim 1 or 2, wherein the peptide segment comprises 2 to 7 amino acid residues.

4. The HIC composition of claim 1 , wherein the peptide segment further comprises one or more peptide nucleic acid residues.

5. 5. The HIC composition of claim 1, wherein the peptide segment consists of residues selected from the group consisting of natural amino acid residues, unnatural amino acid residues, and peptide nucleic acid residues.

6. 6. The HIC composition of claim 1, wherein the C-terminus of the peptide segment is blocked to minimize ionic interactions.

7. The HIC composition of claim 6 , wherein the C-terminus is blocked with an amide.

8. 8. The HIC composition of claim 1, wherein the peptide segment extends to a terminal amino acid residue having an amide-blocked carboxyl terminus.

9. 9. The HIC composition of claim 1, wherein each of the amino acid residues of the peptide segment is neutral at a pH value of 3 to 9.

10. The hydrophobically modified hydrophilic ligand has Formula I: 【Chemical 1】 The HIC composition according to any one of claims 1 to 9, wherein (where, X is a polar group, Z is a linking group; Y is a peptide segment, n is 1-6; n' is 0-2; m is 2-8; p is 0 or 1; R １ , R ２ , R ３ are independently H or a straight or branched, substituted or unsubstituted C1 to C18 alkyl group; R ４ and R ５ are independently H or OH, and at least two m units contain at least one hydroxyl group.

11. The hydrophilic ligand moiety has the formula Ia: 【Chemistry 2】 The HIC composition according to any one of claims 1 to 10, wherein (where, X is a polar group, n is 1-6; n' is 0-2; R １ , R ２ , R ３ are independently H or a linear or branched, substituted or unsubstituted C1 to C18 alkyl group; R ４ and R ５ are independently H or OH, and at least two m units contain at least one hydroxyl group.

12. 12. The HIC composition of claim 10 or 11, wherein the polar groups X are independently selected from carbonate, carbamate, amide, amine, ureido, ether, thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, or thiocarbamate.

13. 13. The HIC composition of claim 12, wherein the polar group X is selected from an amide, carbamate, or ureido group.

14. 14. The HIC composition of claim 13, wherein the polar group X is an amide.

15. 15. The HIC composition of any one of claims 10 to 14. (where, n is 2-4, m is 3-6, p is 1, R １ , R ２ , R ３ are independently H or a straight or branched, substituted or unsubstituted C1 to C6 alkyl group.

16. 16. The HIC composition of claim 10, wherein the linking group Z is a carbamate group and p is 1.

17. The hydrophobically modified hydrophilic ligand has Formula II: 【Chemistry 3】 17. The HIC composition according to any one of claims 10 to 16, wherein

18. 18. The HIC composition of any one of claims 1 to 17, wherein the amino acid residues of the peptide segment are derived from amino acids selected from the group consisting of glycine (Gly), leucine (Leu), alanine (Ala), isoleucine (Ile), valine (Val), methionine (Met), cysteine ​​(Cys), proline (Pro), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), and combinations thereof.

19. 20. The HIC composition of claim 18, wherein the peptide segment comprises amino acid residues derived from amino acids selected from the group consisting of glycine (Gly), leucine (Leu), and combinations thereof.

20. The peptide segment is i. -Leu-Leu-Leu, ii. -Gly-Gly-Gly, iii. -Leu-Gly-Gly-Gly, iv. -Leu-Gly-Leu-Gly, v. -Leu-Leu-Gly-Gly, vi. -Leu-Leu-Leu-Gly, vii. -Gly-Gly-Gly-Leu, viii. -Gly-Leu-Gly-Leu, ix. -Gly-Gly-Leu-Leu, and x. -Gly-Leu-Leu-Leu 20. The HIC composition of any one of claims 1 to 19, selected from the group consisting of:

21. The peptide segment is -Gly-Gly-Leu-Leu-Gly-Gly-Leu-Leu-Gly-Gly-Leu-Leu The HIC composition of claim 1 ,

22. 21. The HIC composition of claim 20, wherein each of the amino acid residues is neutral at a pH value of 3 to 9.

23. The hydrophobically modified hydrophilic ligand has the formula III: 【Chemistry 4】 23. The HIC composition according to any one of claims 10 to 22, wherein

24. 24. The HIC composition of any one of claims 1 to 23, further comprising a hydrophilic ligand covalently bound to the solid phase substrate, the hydrophilic ligand comprising a polar group and a plurality of hydroxyl groups.

25. The hydrophilic ligand has the formula V: 【Chemistry 5】 25. The HIC composition of claim 24, wherein: (where, X is a polar group, n is 1-6; n' is 0-2; m is 2-8; q is 1, R １ , R ２ , R ３ are independently H or a straight or branched, substituted or unsubstituted C1 to C18 alkyl group; R ４ and R ５ are independently H or OH, and at least two m units contain at least one hydroxyl group; R ８ and R ９ are independently H or OH, with the proviso that R ８ and R ９ at least one of which is OH

26. The hydrophilic ligand of formula V may further be represented by formula Va: 【Chemistry 6】 26. The HIC composition of claim 25, wherein:

27. 27. The HIC composition of any one of claims 24 to 26, wherein the hydrophobically modified hydrophilic ligand and the hydrophilic ligand are present in a molar ratio ranging from 1:10 to 10:

1.

28. 28. The HIC composition of any one of claims 1 to 27, wherein the solid phase substrate is a silica material or an inorganic / organic hybrid material.

29. 30. The HIC composition of claim 28, wherein the solid phase substrate is a silica material.

30. 30. A kit comprising the HIC composition of any one of claims 1 to 29.

31. 30. Use of the HIC composition of any one of claims 1 to 29 in HIC.

32. 1. A method for preparing a HIC composition for hydrophobic interaction chromatography comprising a hydrophobically modified hydrophilic ligand, the method comprising: providing a solid phase substrate; providing a hydrophilic ligand comprising a polar group and a plurality of hydroxyl groups; reacting the solid phase substrate with the hydrophilic ligand to covalently bond the hydrophilic ligand to the solid phase substrate to form a hydrophilically modified substrate; providing an activated compound containing a leaving group; reacting said activating compound with one of said plurality of hydroxyl groups to form an activated hydrophilically modified substrate; providing a peptide segment comprising 2 to 20 amino acid residues, including natural or unnatural amino acid residues; reacting the activated hydrophilically modified substrate with the peptide segment to release the leaving group of the activated compound and form the hydrophobically modified hydrophilic ligand and the HIC composition; Including, the peptide segments are linearly arranged, and each of the amino acid residues is the same as or different from the other amino acid residues to promote HIC interactions; method.

33. 33. The method of claim 32, wherein at least a majority of the amino acid residues of the peptide segment are neutral at pH values ​​between 3 and 9 to promote hydrophobic interactions.

34. 34. The method of claim 32 or 33, wherein the peptide segment comprises 2 to 7 of the amino acid residues.

35. 35. The method of any one of claims 32 to 34, wherein the peptide segment further comprises one or more peptide nucleic acid residues.

36. 36. The method of any one of claims 32 to 35, wherein the peptide segment consists of residues selected from the group of natural amino acid residues, unnatural amino acid residues, and peptide nucleic acid residues.

37. 37. The method of any one of claims 32 to 36, wherein the C-terminus of the peptide segment is blocked to minimize ionic interactions.

38. 38. The method of any one of claims 32 to 37, wherein the C-terminus of the peptide segment is blocked with an amide.

39. 39. The method of any one of claims 32 to 38, wherein the peptide segment extends to a terminal amino acid residue having a carboxyl terminus blocked with an amide to form a carboxamide.

40. 40. The method of any one of claims 32 to 39, wherein each of the amino acid residues of the peptide segment is neutral at a pH value of 3 to 9.

41. The hydrophilic ligand has the formula V: 【Chemistry 7】 41. The method of any one of claims 32 to 40, wherein (where, X is a polar group, n is 1-6; n' is 0-2; m is 2-8; q is 1, R １ , R ２ , R ３ are independently H or a straight or branched, substituted or unsubstituted C1 to C18 alkyl group; R ４ and R ５ are independently H or OH, and at least two m units contain at least one hydroxyl group; and R ８ and R ９ are independently H or OH, with the proviso that R ８ and R ９ at least one of which is OH and is present at the terminus of the hydrophilic ligand

42. 42. The method of claim 41, wherein the polar groups X are independently selected from carbonate, carbamate, amide, amine, urea, ether, thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, or thiocarbamate (including heterocyclic compounds containing polar functional groups).

43. 43. The method of claim 42, wherein the polar group X is selected from an amide, carbamate, or ureido group.

44. 44. The method of claim 43, wherein the polar group X is an amide.

45. 45. The method of any one of claims 41 to 44. (where, n is 2-4, m is 3-6, p is 1, and R １ , R ２ , R ３ are independently H or a straight or branched, substituted or unsubstituted C1 to C6 alkyl group.

46. The hydrophilic ligand has the formula Va: 【Chemistry 8】 46. ​​The method of any one of claims 41 to 45, wherein

47. 47. The method of any one of claims 32 to 46, wherein the amino acid residues of the peptide segment are derived from amino acids selected from the group consisting of glycine (Gly), leucine (Leu), alanine (Ala), isoleucine (Ile), valine (Val), methionine (Met), cysteine ​​(Cys), proline (Pro), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), and combinations thereof.

48. 48. The method of claim 47, wherein the peptide segment comprises amino acid residues derived from amino acids selected from the group consisting of glycine (Gly), leucine (Leu), and combinations thereof.

49. The peptide segment is i. -Leu-Leu-Leu, ii. -Gly-Gly-Gly, iii. -Leu-Gly-Gly-Gly, iv. -Leu-Gly-Leu-Gly, v. -Leu-Leu-Gly-Gly, vi. -Leu-Leu-Leu-Gly, vii. -Gly-Gly-Gly-Leu, viii. -Gly-Leu-Gly-Leu, ix. -Gly-Gly-Leu-Leu, and x. -Gly-Leu-Leu-Leu 49. The method of any one of claims 32 to 48, wherein the compound is selected from the group consisting of:

50. The hydrophobically modified hydrophilic ligand has the formula III: 【Chemistry 9】 50. The method of any one of claims 41 to 49, wherein

51. 51. The method of any one of claims 32 to 50, wherein the activating compound comprises a carbonate group and is represented by 4-nitrophenyl chloroformate (4-NPC), N,N'-disuccinimidyl carbonate (DSC), carbonyldiimidazole (CDI), or a combination thereof.

52. 51. The method of any one of claims 32 to 50, wherein the activating compound comprises a tosylate group.

53. 52. The method of claim 51, wherein the activating compound is tosyl chloride.

54. 51. The method of any one of claims 32 to 50, wherein the activating compound is mesyl chloride, phosphorus tribromide, thionyl chloride, or a combination thereof.

55. The hydrophobically modified hydrophilic ligand has Formula I: 【Chemistry 10】 55. The method of any one of claims 32 to 54, wherein (where, X is a polar group, Z is a linking group; Y is a peptide segment, n is 1-6; n' is 0-2; m is 2-8; p is 1, R １ , R ２ , R ３ are independently H or a straight or branched, substituted or unsubstituted C1 to C18 alkyl group; R ４ and R ５ are independently H or OH, and at least two m units contain at least one hydroxyl group.

56. 56. The method of claim 55, wherein the carboxyl terminus of the peptide segment is blocked with an amide.

57. 57. The method of any one of claims 32 to 56, wherein reacting the activating compound with one of the plurality of hydroxyl groups to form the activated hydrophilically modified substrate is further defined as reacting the activating compound with the hydroxyl group present at the terminus of the hydrophilically modified substrate.

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