Chromatography Ligands and Materials, and Uses Thereof

Abstract

The present disclosure provides compounds of formula I: JPEG2025536207000034.jpg41170 [wherein X1 is selected from CO and SO2; each of R1 to R5 is H, F, Cl, O, N, S, C 1~3 Alkyl, and C 1~3 alkyl-X2; any two adjacent moieties selected from R1-R5, together with the atoms to which they are attached, can form a 5- or 6-membered heterocyclic or carbocyclic ring; X2 is selected from O, S, NH(CO), (CO)NH, NH(SO2), and (SO2)NH; with the proviso that i. when each of R1-R5 is H, X1 is SO2; ii. when any two adjacent moieties selected from R1-R5, together with the atoms to which they are attached, form a 5-membered heterocyclic ring containing a ring containing two oxygen atoms in the ring, X1 is SO2; iii. when three of R1-R5 are CHO, X1 is CO. The present disclosure also relates to a chromatography material comprising a support and the chromatography ligand disclosed herein bound to the support. Also disclosed are uses of the chromatography material for separating one or more target molecules from impurities, as well as methods for separating one or more target molecules from impurities.

Description

[Technical Field]

[0001] The present disclosure is directed to chromatography ligands and materials, their use for separating one or more target molecules from impurities, and methods for separating one or more target molecules from impurities. In particular, the disclosure relates to the separation of target molecules, such as antibodies or antibody fragments, from impurities comprising aggregates of one or more target molecules. [Background technology]

[0002] Biopolymers, such as proteins, nucleic acids, and polysaccharides, often occur partially in the form of aggregates or multimers, e.g., dimers, trimers, or higher-order oligomers. In the field of biological production of recombinant proteins, when a desired polypeptide or protein is produced in a host organism and isolated from cells or cell extracts under conditions and concentrations completely different from those in its natural environment, the conditions may favor the formation of such aggregates through intermolecular disulfide bonds or other covalent bonds, or through non-covalent interactions. The presence of such aggregates of target macromolecules is often undesirable. Therefore, protein aggregation is a common problem encountered during bioprocess development and the production of biotherapeutics. Aggregated forms of macromolecules have lower biological activity than non-aggregated forms of the macromolecules and may even completely lose the desired biological activity or cause undesirable side effects. Therefore, the non-aggregated state of therapeutic proteins and the absence of molecular aggregates present in the final product are essential for therapeutic safety.

[0003] Preparative chromatography remains a leading technology for the purification of therapeutic proteins due to its advantages in resolution, scalability, and robustness. Monoclonal antibodies are one of the most powerful therapeutic tools for treating an increasing number of diseases. Continuous development of upstream processes has resulted in increased purification complexity, for example, in terms of the type and content of impurities due to high titers. Furthermore, the heterogeneity of monoclonal antibodies, including differences in charge distribution and size due to various molecular modifications throughout their lifespan from cell culture to polishing, increases challenges during the polishing process. Potential molecular modifications include charged acidic and basic variants, Fab fragments, and high molecular weight (HMW) aggregates, as well as glycosylation, deamidation, incomplete disulfide bond formation, oxidation, and isomerization, which result in the formation of additional product-related impurities. The biopharmaceutical industry also faces new purification challenges due to the higher molecular diversity of multispecific antibodies, including new and challenging product-related impurities that require more intensive polishing steps to achieve the required product quality. However, alongside the increasing challenges, critical quality attributes remain high, creating a high demand for chromatographic media with high resolution for polishing.

[0004] Multimodal (or mixed-mode) chromatography is an important tool for downstream processing of therapeutic proteins when small molecule ligands provide two or more types of interactions. Commercially available examples of multimodal chromatography include Capto™ MMC ImpRes (Cytiva Sweden AB, Uppsala, Sweden). This is a weak cation-exchange multimodal ligand that allows for high selectivity over a wide pH and salt window compared to conventional ion exchangers. It achieves effective removal of aggregates, viruses, and major contaminants in processes for the purification of monoclonal antibodies and is suitable for polishing antibody fragments.

[0005] However, there is a continuing need in the art for alternative chromatography ligands and their use in methods for improved separation of target molecules from impurities, e.g., aggregates such as high molecular weight aggregates of the target molecule. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US 6,602,990 [Patent Document 2] WO2018122089 [Patent Document 3] US20140296464A1 [Patent Document 4] US20160288089A1 [Patent Document 5] WO2018011600A1 [Patent Document 6] WO2018037244A1 [Patent Document 7] WO2013068741A1 [Patent Document 8] WO2015052465A1 [Patent Document 9] US7867784B2 [Patent Document 10] US 6,428,707 [Non-patent literature]

[0007] [Non-Patent Document 1] S Hjerten: Biochim Biophys Acta 79(2), pp. 393-398 (1964) [Non-patent document 2] "Styrene based polymer supports developed by suspension polymerization" (R Arshady: Chimica e L'Industria 70(9), pp. 70-75 (1988)) Summary of the Invention [Problem to be solved by the invention]

[0008] The above objective of providing alternative chromatography ligands and methods for improved separation of target molecules from impurities is achieved in the present disclosure, which is directed to novel chromatography ligands and uses thereof. [Means for solving the problem]

[0009] More specifically, the chromatography ligands of the present disclosure are defined by Formula I:

[0010] [ka]

[0011] [In the formula, X1 is selected from CO and SO2; Each of R1 to R5 is H, F, Cl, O, N, S, C 1~3 Alkyl, and C 1~3 alkyl-X2, any two adjacent moieties selected from R1 to R5 can form, together with the atoms to which they are attached, a 5- or 6-membered heterocyclic or carbocyclic ring; X2 is selected from O, S, NH(CO), (CO)NH, NH(SO2), and (SO2)NH; however, i. when each of R1 to R5 is H, X1 is SO2; ii. When any two adjacent moieties selected from R1 to R5, together with the atoms to which they are attached, form a 5-membered heterocycle containing a ring containing two oxygen atoms in the ring, X1 is SO2; iii. When three of R1 to R5 are CHO, X1 is CO.

[0012] The present disclosure further provides a method for preparing a chromatographic material, the method comprising the step of immobilizing a plurality of chromatographic ligands as defined above on a support.

[0013] Also provided is a chromatographic material comprising a chromatographic ligand as defined above attached to a support.

[0014] Also provided is the use of the chromatography materials disclosed herein to separate one or more target molecules from impurities.

[0015] The present disclosure also provides a method for separating one or more target molecules from impurities, comprising: a) applying a liquid sample containing one or more target molecules and impurities to a chromatographic material disclosed herein; b) eluting the target molecule from the chromatographic material; c) optionally eluting the impurities from the chromatographic material The present invention provides a method comprising:

[0016] Also provided is a method for separating one or more target molecules from impurities, comprising: a) applying a liquid sample containing one or more target molecules and impurities to a chromatographic material disclosed herein; b) obtaining the target molecules in a flow-through manner, wherein the target molecules pass through the chromatographic material essentially without binding to the chromatographic material; c) optionally eluting the impurities from the chromatographic material The method includes:

[0017] Preferred aspects of the present disclosure are set out in the following detailed description and dependent claims. [Brief explanation of the drawings]

[0018] [Figure 1] 1 is a flow chart of a first method for separating one or more target molecules from impurities according to the present disclosure. [Figure 2] 1 is a flow chart of a second alternative method for separating one or more target molecules from impurities according to the present disclosure. [Figure 3] 1 shows a comparison of separation resolution between HMW aggregates and monoclonal antibodies (mAbs) for different ligand prototypes for data obtained using Method 1 described in Example 1 herein. [Figure 4] 1 shows a comparison of separation resolution between HMW aggregates and mAbs for different ligand prototypes for data obtained using Method 2 described in Example 1 herein. [Figure 5] 1 shows retention data for three of the novel ligand prototypes described in Example 1 herein. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present disclosure is directed to chromatography ligands for the improved separation of one or more target molecules from impurities, which may include aggregates and / or fragments of said target molecules.

[0020] As described in the Examples herein, the known multimodal Capto MMC ImpRes ligand (Cytiva Sweden AB, Uppsala, Sweden) was chosen as the starting point for creating a large, chemically diverse virtual library of 100 Capto MMC analog ligand structures. Based on these structures, physicochemical properties were predicted in silico, yielding a matrix of numerical descriptors (e.g., pKa, cLogP, etc.). Principal component analysis (PCA) of these ligand descriptors yielded a large chemical diversity map, which was used to select ligands for synthesis and conjugation to agarose-based matrices. High-throughput plate-based screening and analysis of column retention and resolution then generated numerical descriptors of chromatographic resolution, which were connected to the chemical descriptors to guide further cycles of synthesis and evaluation of column separation resolution.

[0021] This disclosure describes the selection, synthesis, and chromatographic evaluation of a smaller library of novel multimodal ligands relative to a reference ligand, i.e., Capto MMC ImpRes. In the examples herein, it is shown that novel ligands that are more hydrophobic than the reference ligand achieved improved separation resolution between monoclonal antibodies and product-related impurities (i.e., aggregates and Fab fragments of the antibody) compared to the separation achieved with the reference ligand when using a linear salt gradient elution. More specifically, monoclonal antibodies were obtained in higher purity and yield with the use of the novel ligands. The humble role of secondary hydrophobic and hydrogen-bonding interactions, along with electrostatic interactions, provided by the ligand chemical structure of the more hydrophobic ligands results in better performance with respect to the removal of Fab fragment and aggregate impurities.

[0022] The present disclosure provides a chromatography ligand defined by Formula I:

[0023] [ka]

[0024] [In the formula, X1 is selected from CO and SO2; Each of R1 to R5 is H, F, Cl, O, N, S, C 1~3 Alkyl, and C 1~3 alkyl-X2, any two adjacent moieties selected from R1 to R5 can form, together with the atoms to which they are attached, a 5- or 6-membered heterocyclic or carbocyclic ring; X2 is selected from O, S, NH(CO), (CO)NH, NH(SO2), and (SO2)NH; however, i. when each of R1 to R5 is H, X1 is SO2; ii. When any two adjacent moieties selected from R1 to R5, together with the atoms to which they are attached, form a 5-membered heterocycle containing a ring containing two oxygen atoms in the ring, X1 is SO2; iii. When three of R1 to R5 are CHO, X1 is CO.

[0025] The 5- or 6-membered heterocyclic or carbocyclic ring can be unsaturated or saturated. Furthermore, the 5- or 6-membered heterocyclic or carbocyclic ring can be non-polar, aromatic, and / or aliphatic. The heterocyclic ring can contain up to three heteroatoms. When three heteroatoms are present, all of them are N. When up to two heteroatoms are present, each of them can be independently selected from N, O, and S.

[0026] In one embodiment, any two adjacent moieties selected from R1 to R5 can, together with the atoms to which they are attached, form a 5-membered heterocyclic ring containing up to one oxygen atom in the ring.

[0027] When the chromatography ligand is bound to a support, this can be represented according to the following (Formula Ia):

[0028] [ka]

[0029] The "support" portion of Formula Ia represents a support, such as a chromatography bead, to which a ligand can be attached. The ligand connects to the support through a covalent thioether bond formed with a thiol.

[0030] Formula Ia (support-bound chromatography ligand) may alternatively be

[0031] [ka]

[0032] where the wavy line represents the bond to the support. Hereinafter, the term "support" is used in formula Ia to indicate where the ligand can be attached to the support.

[0033] The term "chromatographic ligand" means a molecule with known or unknown affinity for a given analyte and capable of binding to the support of a chromatographic material, while "analyte" includes any specific binding partner for the ligand.

[0034] Analytes of interest for separation according to the present disclosure are so-called target molecules and impurities present in a liquid sample.

[0035] In this context, the term "target molecule" is intended to include macromolecules that are separated from a liquid sample and purified from impurities prior to use in an intended application, for example as a therapeutic substance.

[0036] The term "polymer" has its conventional meaning in the field of bioprocessing; however, polymers are produced (often recombinantly) from cells in cell culture and purified from the cell culture by any technique of separation and purification. Alternatively, polymers are present in biological solutions that do not necessarily originate from cell culture. Non-limiting examples of polymers are biopolymers, which are large biological polymers composed of monomers linked together, such as peptides and proteins (which may be natural or recombinant), including, but not limited to, enzymes, antibodies and antibody fragments, carbohydrates, and nucleic acid sequences, such as DNA and RNA. Polymers purified using chromatography ligands according to the present disclosure are typically proteins or polypeptides, particularly therapeutic proteins or polypeptides, such as antibodies. Alternatively, polymers can be nucleic acid sequences, e.g., used as vectors in therapeutic applications. Polymers or biopolymers can be, for example, biopharmaceutical, i.e., biological molecules, including, but not limited to, biological polymers intended for use as pharmaceutical compounds. While "polymer" is intended to mean one type of polymer, it should be understood that the singular form of the term can encompass multiple individual polymers of the same type, or analytes.

[0037] The term "liquid sample" (or simply "sample"), as used herein, should be understood to encompass any type of sample that can be obtained from a cell culture, or from a fluid derived from a cell culture, from which the fluid is at least partially purified, by any technique of separation and purification.

[0038] As used herein, the term "cell culture" refers to the cultivation of a cultured cell or group of cells, where the cells can be any type of cell, such as bacterial cells, viral cells, fungal cells, insect cells, or mammalian cells. Cell cultures can be unpurified, i.e., contain cells, or can be depleted, i.e., contain no cells or a small number of cells, but contain biomolecules released from the cells before cell removal. Furthermore, unpurified cell cultures used in the methods of the present disclosure can contain intact cells, cell debris, cell homogenates, and / or cell lysates.

[0039] The term "antibody," as used herein, refers to an immunoglobulin, which may be natural or partially or wholly synthetically produced. The term includes whole antibodies (intact antibodies), such as monospecific and polyspecific antibodies, but is not limited to these. The term also includes active antibody fragments, including Fab antigen-binding fragments, monovalent fragments, and bivalent fragments. The term also covers any protein having a binding domain that is homologous to an immunoglobulin binding domain. Such proteins may be derived from natural sources or partially or wholly synthetically produced. The term also includes fusion proteins comprising antibodies or antibody fragments, such as monoclonal antibodies or monoclonal antibody fragments covalently linked to other proteins. Exemplary antibodies include immunoglobulin isotypes and different types of fragments, such as Fab, Fab', F(ab'), Fv, dAb (single-domain antibody), and Fd (fragments obtained by papain hydrolysis of immunoglobulin molecules followed by reduction of disulfide bonds), as well as scFv (so-called single-chain variable region fragments, which are fusion proteins of the variable regions of the heavy and light chains of immunoglobulins), tandem scFv, BiTE (bispecific T cell-attracting molecule), DART (dual affinity retargeting molecule), and diabodies (single-chain and tandem bispecific antibodies). Bispecific monoclonal antibodies are an example of multispecific antibodies, which are artificial proteins capable of simultaneously binding to two different types of antigens or two different epitopes within the same antigen. The chromatography ligands according to the present disclosure can be used, for example, to purify therapeutic antibodies from impurities, such as aggregates or fragments of the therapeutic antibody, to obtain a high-quality final product. The presence of aggregates in therapeutic antibody preparations generally adversely affects patient safety and must be effectively removed during the manufacturing process.

[0040] The term "vector" is used herein to refer to a viral particle, usually a recombinant viral particle, intended for use in achieving gene transfer to modify specific cell types or tissues. Viral particles can be engineered, for example, to provide vectors that express therapeutic genes. Several viral types are currently being investigated for use in delivering genetic material (e.g., genes) to cells, resulting in either transient or permanent transgene expression. These include adenoviruses, retroviruses (gamma-retroviruses and lentiviruses), poxviruses, adeno-associated viruses (AAV), baculoviruses, and herpes simplex viruses.

[0041] The term "viral particle" is used herein to refer to a complete infectious virus particle. It comprises a core containing the viral genome (i.e., the viral genome) in the form of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), surrounded by a morphologically defined shell. The shell is called a capsid. The capsid and the enclosed viral genome together constitute the so-called nucleocapsid. The nucleocapsid of some viruses is surrounded by a lipoprotein bilayer envelope. In the field of bioprocessing, the genome of viral particles is modified to contain genetic inserts containing the genetic material of interest for the purpose of generating viral vectors for various applications, such as therapeutics. The modified viral particles are capable of infecting host cells in cell culture, where the viral particles are propagated, and then the viral particles are purified from the cell culture by any separation and purification technique.

[0042] As used herein, the term "impurity" is intended to mean any molecule or substance present in a liquid sample that is not the desired target molecule. The term "impurity" includes aggregates, e.g., aggregates of the target molecule, such as high molecular weight aggregates of the target molecule. The term "impurity" further includes fragments of the target molecule, for example, when the target molecule is an intact antibody and undesired fragments of said antibody are present in the liquid sample. The term "impurity" also includes host cell proteins (HCPs), and in the case of bispecific antibodies, homodimers.

[0043] The target molecules according to the present disclosure are typically non-aggregated macromolecules, whereas the impurities from which the target molecules are separated typically include aggregates and / or fragments of said macromolecules, typically proteins, e.g., antibodies.

[0044] As used herein, the term "unaggregated polymer" is intended to mean a polymer that has not been degraded. An unaggregated polymer may alternatively be referred to herein as an "unaggregated polymer" or an "intact polymer." In exemplary embodiments herein where the polymer is a protein or polypeptide, an unaggregated polymer may be described as having an essentially intact tertiary structure, typically comprising an essentially hydrophilic surface of the polymer, with hydrophobic moieties located in the interior of the polymer. Thus, an unaggregated polymer essentially has no hydrophobic moieties or groups exposed to the surface.

[0045] In contrast, in proteins or polypeptides that begin to degrade, the tertiary structure is gradually disrupted, exposing hydrophobic moieties to the environment surrounding the protein or polypeptide. Degraded or degraded protein or polypeptide polymers can form aggregates. A polymer in its non-aggregated form is in a monomeric state. Polymer aggregates can contain multimeric forms of the polymer, such as dimers, trimers, etc. of the polymer. Individual degrading polymers can form aggregates with other individual degrading specimens of the same type of polymer, and / or with individual degrading specimens of other types of degrading polymers, or combinations thereof. As polymer aggregates contain degrading polymers, the result is that the polymer aggregates have hydrophobic moieties exposed on their surfaces.

[0046] The term "high molecular weight (HMW) aggregates" is well known to those skilled in the art. Such aggregates are formed by the self-association of target molecules (e.g., monoclonal antibodies with a molecular weight of approximately 150 kDa) with each other through covalent and non-covalent bonds. This results in the formation of dimers (e.g., approximately 300 kDa for monoclonal antibody dimers) or higher-order aggregates, such as trimers (approximately 450 kDa for monoclonal antibody trimers). These aggregates can be either soluble or insoluble depending on the nature of the target molecule. Therefore, the term "high molecular weight aggregates" as used herein can refer to aggregates of target molecules in which the aggregates have a molecular weight that is approximately twice the molecular weight of the target molecule or more than twice the molecular weight of the target molecule.

[0047] As used herein, the term "hydrophobic moiety" is intended to mean the hydrophobic portion of a polymer or a hydrophobic group present on a polymer.

[0048] The term "hydrophobic group," as used herein, is defined as a molecular group having a logP value >0. The partition coefficient, abbreviated as P, is defined as a specific ratio of the concentrations of a solute, specifically a non-ionized solute, between two solvents (two liquid phases), so the logarithm of the ratio is logP. When one of the solvents is water and the other is a non-polar solvent, the logP value is a measure of lipophilicity or hydrophobicity. The defined precedents depend on the type of lipophilic and hydrophilic phases, which are always the numerator and denominator, respectively. For example, in a two-phase system of n-octanol (hereinafter simply "octanol (oct)") and water (wat), the following is true:

[0049]

number

[0050] A logP value <0 indicates a higher percentage of the solute is in the hydrophilic phase. Conversely, a logP value >0 indicates a higher percentage of the solute is in the lipophilic, or hydrophobic, phase.

[0051] As previously mentioned, modified polymers and aggregates of polymers are typically more hydrophobic than intact, non-modified, unagglomerated polymers. Therefore, aggregates bind to the hydrophobic groups of chromatography ligands to a greater extent than non-aggregated polymers. Target molecule aggregates also have a larger size than the target molecules themselves, providing a greater surface area for interaction with the chromatography ligand. Consequently, compared to target molecule binding to the multimodal ligands of the present disclosure, target molecule aggregates exhibit stronger binding to the ligand. As a result, aggregates generally elute from the chromatography device later than target molecules.

[0052] On the other hand, fragments of target molecules are smaller in size than intact target molecules and therefore have a smaller surface area for interacting with the ligand. Furthermore, for example, Fab fragments of monoclonal antibodies generally interact primarily with multimodal ligands via electrostatic interactions, whereas intact monoclonal antibodies generally interact with ligands via both hydrophobic and electrostatic interactions. As a result, intact monoclonal antibodies generally exhibit stronger binding to the multimodal ligands of the present disclosure than Fab fragments of monoclonal antibodies. As a result, intact antibodies generally elute from chromatography devices more slowly than intact antibody fragments.

[0053] In general, the use of chromatography ligands of the present disclosure is based on taking advantage of the differences in the binding of the ligands to non-aggregated macromolecules and to aggregates and / or fragments of macromolecules, respectively.

[0054] As further described above, Formula I is

[0055] [ka]

[0056] is.

[0057] Formula I meets the following criteria: X1 is selected from CO and SO2; Each of R1 to R5 is H, F, Cl, O, N, S, C 1~3 Alkyl, and C 1~3 alkyl-X2; Any two adjacent moieties selected from R1 to R5 can form, together with the atoms to which they are attached, a 5- or 6-membered heterocyclic or carbocyclic ring; and X2 is selected from O, S, NH(CO), (CO)NH, NH(SO2), and (SO2)NH; can be further defined as:

[0058] Formula I may be modified according to the following additional criteria: i. when each of R1 to R5 is H, X1 is SO2; ii. When any two adjacent moieties selected from R1 to R5, together with the atoms to which they are attached, form a five-membered heterocycle containing a ring containing two oxygen atoms in the ring, X1 is SO2; and iii. If three of R1 to R5 are CHO, X1 is CO can be further defined as:

[0059] Furthermore, the 5- or 6-membered heterocyclic or carbocyclic ring can be unsaturated or saturated. Furthermore, the 5- or 6-membered heterocyclic or carbocyclic ring can be non-polar, aromatic, and / or aliphatic. The heterocyclic ring can contain up to three heteroatoms. When three heteroatoms are present, all of them are N. When up to two heteroatoms are present, each of them can be independently selected from N, O, and S.

[0060] As used herein, the expression "lysozyme batch binding capacity" is intended to mean the binding capacity of a chromatographic material to lysozyme in batch mode, said chromatographic material comprising a chromatographic ligand bound to a support as described elsewhere herein.

[0061] Lysozyme was one of five model proteins used in a high-throughput, plate-based assay to test the chromatography ligands of the present disclosure when present in the material, as detailed below in Example 1. More specifically, binding capacity was tested by loading 60 μg of lysozyme onto 6 μL of chromatography material (i.e., support bound to the chromatography ligand) under binding conditions of pH 7.5 and 480 mM NaCl in batch mode.

[0062] The term "adsorption isotherm" has its conventional meaning in the art: it describes the relationship between the equilibrium concentration of a protein in solution and the amount bound to a chromatographic ligand at a particular temperature and solution conditions such as pH and ionic strength.

[0063] The reason for the "linear portion of the adsorption isotherm" is to understand the selectivity between protein and ligand under different binding conditions of pH and ionic strength. It also provides a measure of the amount of bound protein that is robust to variations in the concentration of protein loaded onto the chromatographic material.

[0064] Protein-ligand selectivity tests for screening pH and ionic strength windows are never performed at the saturated portion of the isotherm or under overload conditions. This is because protein-ligand selectivity can be affected by competitive binding of other impurities (it is impossible to obtain 100% pure monoclonal antibodies in these tests), and protein-protein interactions become problematic under overload conditions, which are characteristic of nonlinear isotherms. In this study, the maximum limit for the equilibrium binding capacity of lysozyme was 10 μg / μL of chromatographic material (60 μg of lysozyme divided by 6 μL of chromatographic material). The experimentally observed percentage equilibrium binding capacity can be calculated for each resin for each binding condition relative to the maximum equilibrium binding capacity (10 μg / μL of resin). Herein, ligands that exhibited at least 50%, e.g., 60%, of the maximum equilibrium binding capacity were selected, i.e., ligands that achieved an amount of lysozyme bound at equilibrium that was at least 50%, e.g., 60%, of the amount of lysozyme added.

[0065] The term "logS" has its conventional meaning in the art. LogS is directly related to the solubility of a compound in water and is defined as the common solubility unit, which corresponds to the base 10 logarithm of the molecule's solubility measured in mol / L. LogS is a measure of hydrophobicity; the more negative the logS value, the more hydrophobic the chromatography ligand.

[0066] The chromatography ligands of the present disclosure can also be further defined as having a log S of about -2.5 to about -5. The log S values ​​were calculated for a chromatography ligand structure that includes a support modeled as a methyl group. In other words, the log S values ​​were calculated for a methyl-S-ligand structure. More specifically, provided herein are chromatography ligands having a log S of about -2.5 to about -5 when the ligand is one in which the -SH of Formula I is replaced with a methyl thioether (-S-CH).

[0067] More specifically, the chromatography ligands of the present disclosure are (a) to (h):

[0068] [ka] [ka]

[0069] The compound may be defined by a chemical structure selected from any one of the following:

[0070] When the above chromatography ligands are bound to a support, they are:

[0071] [ka] [ka]

[0072] It can be shown that:

[0073] According to currently preferred embodiments, the chromatography ligands of the present disclosure comprise (a)-(f):

[0074] [ka]

[0075] The compound can be defined by a chemical structure selected from the group consisting of:

[0076] When the above chromatography ligands are bound to a support, they are:

[0077] [ka]

[0078] It can be shown that:

[0079] Table 1 describes the above-listed ligands (a)-(h) and reference ligands in terms of chemical structure, predicted log S values, and lysozyme batch binding capacity.

[0080] [Table 1A]

[0081] [Table 1B]

[0082] The present disclosure further provides a method for preparing a chromatographic material, the method comprising the step of immobilizing a plurality of chromatographic ligands as defined above on a support.

[0083] The term "separation matrix" is used herein to refer to a material comprising a support to which one or more ligands containing functional groups are attached. The functional groups of the ligands bind to compounds, also referred to herein as analytes, that are to be separated from a liquid sample and / or from other compounds present in the liquid sample. A separation matrix may further comprise compounds to which the ligands are attached to the support. The terms "linker," "extender," and "surface extender" can be used to describe such compounds, as further described below. The term "resin" is sometimes used in the art for a separation matrix. The terms "chromatographic material" and "chromatographic matrix" are used herein to refer to types of separation matrices.

[0084] The term "surface" as used herein means all external surfaces, which in the case of a porous support includes the outer surface and the pore surfaces.

[0085] The separation matrix can be contained in any type of separation device, as further defined elsewhere herein. As a non-limiting example, the chromatographic material can be packed into a chromatographic column prior to adding the liquid sample to the chromatographic material contained in the chromatographic column.

[0086] The chromatography materials disclosed herein include a support to which a ligand is attached. The term "support" has its conventional meaning in the field of bioprocessing and may alternatively be referred to as a "support material" or a "solid phase," other terms conventionally used in the field.

[0087] In formula Ia, the portion of the ligand that is intended to be bound to the support is shown.

[0088] [ka]

[0089] In this regard, the term "support" at the top left of Formula Ia indicates that the ligand can be attached to a support. If a linker or extender is used to attach the ligand to the support (discussed in more detail below), such linker or extender can also be included in the term "support."

[0090] Also disclosed herein are compounds defined in Formula I, and further defined when referring to chromatography ligands.

[0091] The disclosed chromatography materials include supports to which ligands are attached. The supports can be composed of different types of materials and have different shapes or forms, as described in more detail below.

[0092] The support can be made from organic or inorganic materials and can be porous or non-porous. In one embodiment, the support is prepared from natural polymers, such as cross-linked carbohydrate substances, such as agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, pectin, starch, and the like. Natural polymer supports are easily prepared and are optionally cross-linked according to standard methods, such as inverse suspension gelation (S. Hjerten: Biochim Biophys Acta 79(2), pp. 393-398 (1964)). In a particularly advantageous embodiment, the support is a type of relatively rigid, yet porous, agarose, prepared by methods that enhance its flowability; see, e.g., U.S. Pat. No. 6,602,990 (Berg). In an alternative embodiment, the support is prepared from a synthetic polymer or copolymer, such as a cross-linked synthetic polymer, such as styrene or a styrene derivative, divinylbenzene, acrylamide, acrylic acid esters, methacrylic acid esters, vinyl esters, vinylamides, etc. Such synthetic polymers are readily prepared and, optionally, cross-linked according to standard methods; see, for example, "Styrene-based polymer supports developed by suspension polymerization" (R. Arshady: Chimica e L'Industria 70(9), pp. 70-75 (1988)). Natural or synthetic polymer supports are also available, for example, in the form of porous particles, from commercial sources, such as Cytiva, Sweden. In yet another alternative embodiment, the support is prepared from an inorganic polymer, such as silica. Inorganic porous and non-porous supports are well known in the art and are readily prepared according to standard methods.

[0093] The support for the chromatographic material can be in the form of particles, for example substantially spherical, elongated or irregularly shaped particles.

[0094] Capto ImpRes-based chromatography matrix (Cytiva, Uppsala, Sweden) comprises a support in the form of a substantially spherical particle or bead, approximately 40 μm in diameter, which is a non-limiting example of a particle suitable for incorporating the ligands of the present disclosure by binding the ligand to the support.

[0095] Suitable particle sizes for the chromatographic materials of the present disclosure can range from 5 to 500 μm, e.g., 10 to 200 μm, e.g., 20 to 100 μm in diameter. In specific embodiments, the average particle size ranges from about 20 μm to about 50 μm, e.g., about 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, or 50 μm, preferably from about 25 μm to about 40 μm.

[0096] Suitable average pore sizes of the chromatographic materials of the present disclosure in the form of particles can be any size larger than the target molecules and impurities to be separated, including, but not limited to, average pore sizes of about 9 nm (e.g., suitable for separating monoclonal antibodies) to about 80 nm, such as about 9 nm, 10 nm, 11 nm, 12 nm, 15 nm, 20 nm, 30 nm, 50 nm, 75 nm, or 80 nm.

[0097] Those skilled in the art can readily select suitable particle size and porosity depending on the process being used.

[0098] The chromatographic material can be dry, e.g., dry particles that are immersed in a liquid to retain their original shape during use. For example, such dry chromatographic material can include dry agarose particles.

[0099] Alternatively, the support for the chromatographic material may be in the form of magnetic particles. The term "magnetic particles" is defined herein as particles that can be attracted to a magnetic field. At the same time, magnetic particles for use in the methods of the present disclosure do not aggregate in the absence of a magnetic field. In other words, the magnetic particles behave like superparamagnetic particles. The particles may have any symmetrical shape, such as a sphere or a cube, or any asymmetrical shape. Spherical magnetic particles are often referred to as magnetic beads. It should be understood that the terms "magnetic particles," "magnetic beads," "Mag particles," "Mag beads," "magparticles," and "magbeads" can be used interchangeably herein without limiting the scope to magnetic particles having a spherical shape. Magnetic particles suitable for use in the methods of the present disclosure are described in WO2018122089, which is incorporated herein by reference in its entirety.

[0100] The support of the chromatographic material can alternatively be in any other form conventionally used in separations, such as a monolith, a filter or membrane, a capillary, a chip, a nanofiber, a flat surface, etc.

[0101] When the support of the chromatographic material comprises a monolith, suitable average pore sizes of the monolith for purposes of separating target molecules from impurities range from a minimum average pore size of about 9-12 nm (e.g., suitable for separating monoclonal antibodies) to a maximum pore size of up to about 5 μm, e.g., about 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 μm.

[0102] When the support of the chromatographic material comprises nanofibers, such nanofibers may comprise, for example, electrospun polymeric nanofibers. When used, such nanofibers form a stationary phase comprising a plurality of pores through which the mobile phase can permeate.

[0103] The support of the chromatographic material can comprise a membrane structure, e.g., a single membrane, a stack of membranes, or a filter. The membrane can be an adsorbent membrane. When the support of the chromatographic material comprises a membrane structure, suitable pore sizes of the membrane structure for purposes of separating target molecules from impurities range from a minimum average pore size of about 9-12 nm (e.g., suitable for separating monoclonal antibodies) to a maximum pore size of up to about 5 μm, e.g., about 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 μm. When the chromatographic material comprises a membrane structure, such a membrane structure can comprise, for example, a nonwoven web of polymeric nanofibers.

[0104] In the context of membrane structures, non-limiting examples of suitable polymers can be selected from polysulfone, polyamide, nylon, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, and polyethylene oxide, and mixtures thereof.

[0105] Alternatively, the polymer can be a cellulosic polymer, such as selected from the group consisting of cellulose and partial derivatives of cellulose, particularly cellulose esters, crosslinked cellulose, grafted cellulose, or ligand-conjugated cellulose. Cellulose fiber chromatography (also known as Fibro chromatography; Cytiva, Sweden) is an ultrafast chromatographic purification method with short processing times and high productivity, taking advantage of the high flow rates and large capacity of cellulose fibers. When the support of a chromatographic material includes cellulose fibers such as Fibro, suitable pore sizes for separating target molecules from impurities range from a minimum average pore size of about 9-12 nm (e.g., suitable for separating monoclonal antibodies) to a maximum pore size of up to about 5 μm, e.g., about 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 μm.

[0106] The term "membrane chromatography" has its conventional meaning in the field of bioprocessing. In membrane chromatography, there is binding of components of a fluid, such as individual molecules, aggregates, or particles, to the surface of a solid phase with which the fluid contacts. The active surface of the solid phase is accessible by convective transport. The advantage of membrane adsorbers over packed chromatography columns is their suitability for running at higher flow rates. This is also called convection-based chromatography. Convection-based chromatography matrices include any matrix in which the application of a hydraulic pressure difference between the inlet and outlet of the matrix forces perfusion of the matrix, achieving substantial convective transport of substances into or out of the matrix, which is performed very rapidly at high flow rates. Convection-based chromatography and membrane adsorber are described, for example, in US20140296464A1, US20160288089A1, WO2018011600A1, WO2018037244A1, WO2013068741A1, WO2015052465A1, US7867784B2, which are incorporated herein by reference in their entireties.

[0107] Binding of a ligand to the support of the chromatographic material of the present disclosure can be achieved by introducing a linker between the support and the ligand. Binding can be carried out according to any conventional covalent bonding methodology, such as the use of epichlorohydrin; epibromohydrin; allyl glycidyl ether; bisepoxides, such as butanediol diglycidyl ether; halogen-substituted aliphatic substances, such as dichloropropanol; and divinyl sulfone. Non-limiting examples of suitable linkers include polyethylene glycols (PEGs) having 2 to 6 carbon atoms, carbohydrates having 3 to 6 carbon atoms, and polyalcohols having 3 to 6 carbon atoms. All of these methods are well known in the art and readily implemented by those skilled in the art.

[0108] Ligands can be attached to the support via longer linker molecules, also known as "surface extenders" or simply "extenders." Extenders are well known in the art and are commonly used to sterically increase the distance between the ligand and the support. Extenders are sometimes referred to as tentacles or flexible arms. For a more detailed description of possible chemical structures, see, for example, US Pat. No. 6,428,707, which is incorporated herein by reference. Briefly, extenders can be in the form of polymers, such as homopolymers or copolymers. Hydrophilic polymer extenders can be biopolymers of synthetic origin, i.e., with a synthetic skeleton, or of biological origin, i.e., with a naturally occurring skeleton. Typical synthetic polymers are polyvinyl alcohol, polyacrylic and polymethacrylamide, polyvinyl ethers, etc. Typical biopolymers are polysaccharides, such as starch, cellulose, dextran, and agarose.

[0109] The term "eluent" is used in its conventional sense in the art, ie, a buffer of pH and / or ionic strength suitable for releasing one or more compounds from a separation matrix.

[0110] The term "eluate" is used in its conventional sense in the art, ie, the portion of a liquid sample that elutes from a chromatography column after the liquid sample has been loaded onto the chromatography column.

[0111] In the methods disclosed herein for preparing a chromatography material, the density of the multiple ligands immobilized on the support can be about 15 to about 50 μmol / mL, for example, about 15, 20, 25, 30, 35, 40, 45, or 50 μmol / mL, preferably about 20 to about 35 μmol / mL.

[0112] Also provided in this disclosure is a chromatographic material comprising the above-defined chromatographic ligand attached to a support. The chromatographic material and support are defined and exemplified above.

[0113] In a currently preferred embodiment, the support comprises beads having a diameter of from about 25 μm to about 50 μm, preferably from about 30 μm to about 45 μm.

[0114] The chromatography materials disclosed herein can have a density of multiple ligands immobilized on the support of about 15 to about 50 μmol / mL, for example, about 15, 20, 25, 30, 35, 40, 45, or 50 μmol / mL, preferably about 20 to about 35 μmol / mL.

[0115] A chromatography material comprising a chromatography ligand disclosed herein can be further defined as having a lysozyme batch binding capacity in the linear portion of the adsorption isotherm at binding conditions of pH 7.5 and 480 mM NaCl, and further defined as the amount of lysozyme bound to the chromatography material at equilibrium being at least 50%, e.g., 60%, of the amount of lysozyme added to the chromatography material. The terms "lysozyme batch binding capacity" and "linear portion of the adsorption isotherm" are further defined above.

[0116] As described elsewhere herein, chromatography ligands can be defined as having a log S of about -2.5 to about -5.

[0117] The ligand of the chromatographic material is defined by formula I, as further detailed above.

[0118] Also provided is the use of the chromatography materials disclosed herein to separate one or more target molecules from impurities.

[0119] In the context of said use, the one or more target molecules may be one or more antibodies, as detailed elsewhere herein. Preferably, the antibody is a monoclonal antibody. Optionally, the monoclonal antibody is a multispecific monoclonal antibody, e.g., a bispecific monoclonal antibody. Alternatively, the one or more target molecules may be one or more antibody fragments. Optionally, the one or more antibody fragments may be selected from antigen-binding fragments, such as Fab, Fab', F(ab')2, scFv, Fv, dAb, or Fd, as detailed and exemplified elsewhere herein.

[0120] Furthermore, in the context of said use, the impurities may comprise aggregates of one or more target molecules, e.g., high molecular weight aggregates of target molecules, as detailed elsewhere herein. For example, if the target molecule is an antibody, the impurities may comprise aggregates of said antibodies, e.g., high molecular weight aggregates of one or more antibody fragments. Alternatively, if the target molecule is an antibody fragment, the impurities may comprise aggregates of one or more antibody fragments, e.g., high molecular weight aggregates of one or more antibody fragments.

[0121] The present disclosure also provides a method for separating one or more target molecules from impurities, as shown in FIG. 1, comprising: a) applying a liquid sample containing one or more target molecules and impurities to a chromatographic material disclosed herein; b) eluting the target molecule from the chromatographic material; c) optionally eluting the impurities from the chromatographic material The present invention solves or at least alleviates problems associated with existing methods for separating one or more target molecules from impurities by providing a method comprising:

[0122] In the method, the target molecule, and optionally impurities, can be eluted from the chromatographic material by applying an elution buffer comprising (i) a salt gradient, (ii) a pH gradient, or a combination of (i) and (ii). Elution buffers suitable for the separation of various types of target molecules, such as monoclonal antibodies, are well known in the art and can be readily selected by one skilled in the art.

[0123] It should be understood that the term "gradient," when used in the context of elution conditions, encompasses both continuous and step gradients. Continuous gradients can be linear or non-linear, or a combination thereof.

[0124] The present disclosure provides an alternative method for separating one or more target molecules from impurities, as shown in FIG. 2, comprising: a) applying a liquid sample containing one or more target molecules and impurities to a chromatographic material disclosed herein; b) obtaining the target molecule in a flow-through mode, wherein the target molecule passes through the chromatographic material essentially without binding to the chromatographic material; c) optionally eluting the impurities from the chromatographic material The present invention further provides a method comprising:

[0125] The flow-through method described above may be particularly suitable when using chromatographic materials that include supports in the form of cellulose fibers (eg, Fibro) or nanofibers, as further detailed above.

[0126] The above-described methods for separating target molecules from impurities are based on multimodal interactions, i.e., electrostatic interactions, hydrophobic interactions, hydrogen bonding, etc., between chromatographic ligands and molecules present in a liquid sample, as shown in Figures 1 and 2.

[0127] In the context of the method, the one or more target molecules may be one or more antibodies, as described in detail elsewhere herein. Preferably, the antibody is a monoclonal antibody. Optionally, the monoclonal antibody is a multispecific monoclonal antibody, e.g., a bispecific monoclonal antibody. Alternatively, the one or more target molecules may be one or more antibody fragments. Optionally, the one or more antibody fragments may be selected from antigen-binding fragments, e.g., Fab, Fab', F(ab')2, scFv, Fv, dAb, or Fd, as described and exemplified in detail elsewhere herein.

[0128] Furthermore, in the context of the method, impurities may comprise aggregates of one or more target molecules, e.g., high molecular weight aggregates of target molecules, as detailed elsewhere herein. For example, if the target molecule is an antibody, the impurities may comprise aggregates of said antibodies, e.g., high molecular weight aggregates of one or more antibody fragments. Alternatively, if the target molecule is an antibody fragment, the impurities may comprise aggregates of one or more antibody fragments, e.g., high molecular weight aggregates of one or more antibody fragments.

[0129] The above-disclosed method for separating a target molecule from impurities may further comprise a step (a1) preceding step (a), which comprises pretreating the liquid sample. Optionally, the pretreating step may comprise subjecting the cell culture harvest containing the target molecule to cell lysis, clarification, and / or filtration.

[0130] The above-disclosed method for separating target molecules from impurities can further comprise a step (a2) preceding step (a), which comprises pre-purifying one or more target molecules by separating the target molecules from a cell culture harvest containing the target molecules, thereby obtaining a pre-purified liquid sample containing the target molecules, and then applying the pre-purified liquid sample containing the target molecules to a chromatography material disclosed herein. Optionally, the pre-purifying step can comprise subjecting the cell culture harvest containing the target molecules to chromatography, or clarification followed by chromatography.

[0131] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Also, the singular forms "a," "an," and "the" are meant to include plural references unless otherwise stated.

[0132] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. [Example]

[0133] Introduction This example presents experiments demonstrating the successful separation of monoclonal antibodies from Fab fragments and high molecular weight aggregates using the novel ligands disclosed herein. Briefly, the load contained 92% pure monomeric monoclonal antibodies (mAbs), in addition to 6% Fab fragments and 2% high molecular weight (HMW) aggregates. Separation of mAbs, Fab fragments, and HMW aggregates on the chromatographic material was performed using a bind-and-elute method. Binding was performed under favorable binding conditions of 25 mM phosphate, pH 7, and elution was performed by linearly increasing the NaCl concentration from low to 1 M NaCl with 50 mM phosphate buffer, pH 7.5, as the elution buffer. Linear salt gradient elution eluted Fab fragments (smallest size) first, followed by mAbs, and then HMW aggregates (largest size). Aggregates eluted later in the linear gradient elution, indicating a stronger binding affinity for the ligand.

[0134] Materials and Methods material A liquid sample containing a monoclonal antibody (designated mAb1) was previously purified on a Protein A chromatography column and used in the experiments. mAb1 has a pI value of 8.6, a molecular weight of 150 kDa, and an extension coefficient of 1.58. The concentration of the buffer-exchanged mAb1 sample (pH 7 and conductivity 2.94 mS / cm) used in the isocratic retention test was approximately 20 mg / mL, had a monomer purity of 98.5%, and contained 1.5% HMW aggregates. For the linear salt gradient elution test, the concentration was approximately 18 mg / mL, had a monomer purity of 92%, and contained 6% Fab fragments and 2% HMW aggregates. Fab fragments were generated from mAb1 using papain digestion and added to the mAb1 sample. For the high-throughput plate-based assay, five model proteins, namely, cytochrome C, α-lactalbumin, lysozyme, ovalbumin, and human serum albumin (HSA) (Sigma-Aldrich, St. Louis, MO, USA), and a monoclonal antibody (mAb1) were used. Capto MMC ImpRes multimodal resin and Capto ImpRes base matrix were obtained from Cytiva (Uppsala, Sweden). Chemicals, including L-homocysteine ​​thiolactone hydrochloride, were obtained from Acros Organics. Acyl and sulfonyl chlorides, bromine, dichloromethane, and ethyl acetate were obtained from Sigma-Aldrich. All other chemicals, including sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, sodium acetate, sodium acetate trihydrate, sodium hydroxide, glacial acetic acid, and Blue Dextran 2000 used, were analytical grade and purchased from Merck (Darmstadt, Germany).

[0135] Instrumentation and Analysis All chromatographic experiments were performed on an Äkta explorer 10 chromatography system from Cytiva (Uppsala, Sweden) using manually packed Tricon 5 / 100 glass columns of approximately 2 mL column volume (CV). Protein concentrations of eluted fractions were determined using a SpectraMax M2 column from Molecular Devices LLC (California, USA). e Analysis was performed using a spectrophotometer. A SpectraMax 96-well plate reader was used. All gradient-eluted fractions were analyzed for % Fab fragment, mAb1, and HMW aggregate content using size-exclusion high-performance liquid chromatography (SEC-HPLC) using an Agilent 1200 Series HPLC from Agilent Technologies (Santa Clara, CA, USA). A Superdex™ 200 resin column containing 13 μm particles packed in a 10 × 300 mm high-resolution column from Cytiva (Uppsala, Sweden) was used. The mobile phase buffer composition was 200 mM sodium phosphate at pH 6.8. The flow rate used was 0.8 mL / min, and the total analysis time for each analysis was 25 min. Data analysis of UV280 nm obtained by SEC-HPLC was performed using Agilent ChemStation software on an Äkta chromatography system running Unicorn 5.31. Titrations were performed using a Metrohm autotitrator 905 Titrando system from Metrohm AG (Herisau, Switzerland) and analyzed with Tiamo software.

[0136] method Synthesis of prototype Capto MMC ligands Multimodal Capto MMC thioether ligands are derived from thiolactone proligands. The Capto MMC ligand prototype library was synthesized via acylation or sulfonylation of commercially available D,L-homocysteine ​​thiolactone hydrochloride with an equimolar amount of the appropriate acyl or sulfonyl chloride in dichloromethane (DCM) in the presence of diisopropylethylamine (DIPEA). After the reaction was complete, typically after 16 h, the solvent was removed under vacuum. The residue, typically a pale yellow oil, was dissolved in ethyl acetate (EtOAc) and washed sequentially in a separatory funnel with an aqueous solution of citric acid (10% w / v), an aqueous solution of KCO 10% (w / w), water, and brine. The organic phase was dried over MgSO 4 , filtered, and evaporated under reduced pressure to yield the product as an overall white or pale yellow solid.

[0137] More specifically, the eight multimodal ligands of the present disclosure were synthesized as follows.

[0138] N-Toluoyl-D,L-homocysteine ​​thiolactone (L01) - D,L-homocysteine ​​thiolactone hydrochloride (18.75 mmol, 2.87 g) and diisopropylethylamine (37.5 mmol, 6.53 mL) are dissolved in DCM (30 mL) and the mixture is cooled in an ice bath. Toluoyl chloride (18.75 mmol) is added slowly to the stirred solution as described in the general procedure to give the product as a white solid. 1 H NMR (CDCl3, 300 MHz): δ = 7.70 (d 2H, Ar-H), 7.24 (d, 2H, Ar-H), 6.61 (s, 1H, amide-H), 4.68 (m, 1H, thiolactone-H), 3.49–3.25 (m, 2H, thiolactone-H), 3.10 (m, 1H, thiolactone-H), 2.40 (s, 3H, Ar-CH3), 2.03 (s, 3H, thiolactone-H).

[0139] N-Pentafluorobenzoyl-D,L-homocysteine ​​thiolactone (L02) - D,L-homocysteine ​​thiolactone hydrochloride (18 mmol, 2.787 g) and diisopropylethylamine (36.9 mmol, 6.492 mL) are dissolved in DCM (40 mL) and the mixture is cooled in an ice bath. Pentafluorobenzoyl chloride (18 mmol, 2.62 mL) is added slowly to the stirred solution as described in the general procedure to give the product as a white solid. 1 H NMR (CDCl3, 300 MHz): δ = 6.58 (s, 1H, amide-H), 4.63 (m, 1H, thiolactone-H), 3.40 (m, 2H, thiolactone-H), 3.13 (m, 1H, thiolactone-H), 2.07 (m, 1H, thiolactone-H).

[0140] N-Benzenesulfonyl-D,L-homocysteine ​​thiolactone (L05) - D,L-homocysteine ​​thiolactone hydrochloride (16 mmol, 2.478 g) and diisopropylethylamine (32.8 mmol, 5.771 mL) are dissolved in DCM (40 mL) and the mixture is cooled in an ice bath. Benzenesulfonyl chloride (16 mmol, 2.063 mL) is added slowly to the stirred solution as described in the general procedure to give the product as a white solid. 1 H NMR (CDCl3, 300 MHz): δ = 7.90 (m, 2H, Ar-H), 7.70-7.52 (d, 2H, Ar-H), 5.23 (s, 1H, amide-H), 3.78 (m, 1H, thiolactone-H), 3.32-3.23 (m, 2H, thiolactone-H), 2.86 (m, 1H, thiolactone-H), 2.07 (m, 1H, thiolactone-H).

[0141] N-(4-ethylbenzoyl)-D,L-homocysteine ​​thiolactone (L08) - D,L-homocysteine ​​thiolactone hydrochloride (18.75 mmol, 2.87 g) and diisopropylethylamine (37.5 mmol, 6.53 mL) are dissolved in DCM and the mixture is cooled in an ice bath. 4-Ethylbenzoyl chloride (18.75 mmol) is added slowly to the stirred solution as described in the general procedure to give the product as a white solid (>90%). 1 H NMR (CDCl3, 300 MHz): δ = 8.35 (s, 1H, Ar), 8.00-7.80 (m, 4H, Ar), 7.68-7.49 (m, 2H, Ar), 6.74 (s, 1H, amide-H), 4.78-4.64 (m, 1H), 3.55-3.27 (m, 2H), 3.23-3.13 (m, 1H), 2.20-1.95 (m, 1H).

[0142] N-Benzodioxanesulfonyl-D,L-homocysteine ​​thiolactone (L09) - D,L-homocysteine ​​thiolactone hydrochloride (15 mmol, 2.323 g) and diisopropylethylamine (30 mmol, 5.28 mL) are dissolved in DCM (25 mL) and the mixture is cooled in an ice bath. 1,4-Benzodioxane-6-sulfonyl chloride (15 mmol, 3.705 g) is added slowly to the stirred solution as described in the general procedure to give the product as a white solid. 1 H NMR (CDCl3, 300 MHz): δ = 7.45–7.30 (m, 2H, Ar), 6.94 (d, 2H, Ar), 5.11 (s, 1H, amide-H), 4.30 (m, 4H, alpha), 3.69 (m, 1H, thiolactone-H), 3.25 (m, 2H, thiolactone-H), 2.86 (m, 1H, thiolactone-H), 2.05 (m, 1H, thiolactone-H).

[0143] N-(2-Naphthoyl)-D,L-homocysteine ​​thiolactone (L11) - D,L-homocysteine ​​thiolactone hydrochloride (18.75 mmol, 2.87 g) and diisopropylethylamine (37.5 mmol, 6.53 mL) were dissolved in DCM (30 mL) and the mixture was cooled in an ice bath. 2-Naphthoyl chloride (18.75 mmol) was added slowly to the stirred solution as described in the general procedure to give the product as a white solid (92%, 4.67 g). 1 H NMR (CDCl3, 300 MHz): δ = 8.35 (s, 1H, Ar), 8.00-7.80 (m, 4H, Ar), 7.68-7.49 (m, 2H, Ar), 6.74 (s, 1H, amide-H), 4.78-4.64 (m, 1H), 3.55-3.27 (m, 2H), 3.23-3.13 (m, 1H), 2.20-1.95 (m, 1H).

[0144] N-Pentafluorobenzenesulfonyl homocysteine ​​thiolactone (L17)—D,L-Homocysteine ​​thiolactone HCl (10.02 mmol, 1.02 equiv., 1.567 g), dichloromethane (20 mL), and diisopropylethylamine (2.04 equiv., 20.04 mmol, 3.553 mL) are added to a Schlenk flask, flushed with N2, and stirred until dissolved. The solution is cooled in an ice bath, and pentafluorobenzenesulfonyl chloride (1 equiv., 10 mmol, 2.666 g, 1.484 mL) is added dropwise. The reaction mixture is allowed to warm to room temperature with stirring under N2 overnight. The reaction is complete by TLC (DCM with a drop of EtOH or 1:1 EtOAc / cyclohexane). The reaction mixture is concentrated on a rotovap and then diluted with EtOAc (100 mL). This was washed with citric acid (10 wt%, 3 x 50 mL), followed by K2CO3 (10 wt%, 3 x 50 mL), and brine (2 x 50 mL). The organic phase was concentrated on a rotovap, and the brown solid residue (1.7 g, theoretical yield 3.75 g) was dried under house vacuum. The crude product was recrystallized from boiling EtOH / HO (approximately 30:10 mL) to give a light brown solid (0.896 g, 24% yield). 1 H NMR (300 MHz, CDCl3) δ 5.72 (br, 1H, NH), 4.20 (m, 1H, CH), 3.36 (m, 2H), 2.88 (m, 1H), 2.18 (m, 1H).

[0145] N-(3,5-Diethoxybenzoyl)homocysteine ​​thiolactone (L18)—D,L-Homocysteine ​​thiolactone HCl (10.2 mmol, 1.02 equiv., 1.567 g), dichloromethane (20 mL), and diisopropylethylamine (2.04 equiv., 20.4 mmol, 2.636 g, 3.55 mL) were added to a Schlenk flask, flushed with N2, and stirred until dissolved. The solution was cooled in an ice bath, and acid chloride (1 equiv., 10 mmol, 2.287 g) in DCM (5 mL) was added dropwise. After 30 min, the ice bath was removed, and the reaction mixture was allowed to warm to room temperature with stirring under N2 overnight. TLC (DCM with a drop of EtOH or 1:1 EtOAc / cyclohexane) showed one spot plus baseline. The reaction mixture is concentrated on a rotovap and then diluted with EtOAc (100 mL). It is washed with citric acid (10 wt%, 3 x 50 mL), followed by K2CO3 (10 wt%, 3 x 50 mL), and brine (2 x 50 mL). The organic phase is concentrated on a rotovap, and the white solid is dried under house vacuum. Yield 2.78 g (90% yield). 1 H NMR (300 MHz, CDCl3) δ 6.89 (d, 2H, ArH), 6.58 (t, 1H, ArH), 6.50 (br, 1H, NH), 4.64 (m, 1H, CH), 4.06 (q, 4H, O-CH2-CH3), 3.42 (m, 1H), 3.34 (m, 1H), 3.10 (m, 1H), 2.01 (m, 1H), 1.41 (t, 6H, -CH3).

[0146] Following aqueous workup and, where appropriate, crystallization, the resulting N-acylated or sulfonylated thiolactones are hydrolyzed with aqueous NaOH to provide carboxylic acid cation-exchange groups, and then with thiols used to nucleophilically couple to functionalized agarose-based matrices (Capto ImpRes, Cytiva). Briefly, the agarose-based matrix is ​​first functionalized with terminal allyl groups using allyl glycidyl ether (AGE), followed by bromination with elemental bromine. The brominated gel forms epoxy ends that are good electrophiles under basic conditions, allowing for the formation of SN React with MMC ligand prototypes via 2-nucleophilic substitution. The loading of the multimodal ligand is 25-30 μmol / mL, determined by titration. 樹脂 is.

[0147] High-throughput plate-based screening assay Reference Capto MMC ImpRes resin (approximately 25 μmol / mL 樹脂 Binding capacity (BC) data of the linear portion of the isotherms of six different proteins, namely, cytochrome C, α-lactalbumin, lysozyme, ovalbumin, HSA, and one monoclonal antibody (mAb1), on a novel multimodal cation-exchange resin containing 10 μg / μL were obtained through high-throughput, plate-based rapid screening. 樹脂 BC data at low protein loads were determined in 6 μL of PreDictor™ 96-well filter plates for four different binding pHs (4.5, 5.5, 6.5, 7.5) and eight different NaCl salt concentrations (0, 55, 133, 257, 480, 750, 1250, 1750 mM) using a Tecan robotic workstation, an automated liquid handling system from Tecan Group Ltd. (Mannedorf, Switzerland). Thus, ligand prototypes were tested for 32 different binding pH and salt conditions. Binding pHs of 4.5 and 5.5 were based on 25 mM acetate buffer, and pHs of 6.5 and 7.5 were based on 50 mM phosphate buffer. Reservoir volumes for binding pH were calculated using a proprietary Excel application (Cytiva, Uppsala, Sweden). All protein solutions were prepared using 5 mM pH 7 phosphate buffer, but mAb1 loading was buffer exchanged in 5 mM pH 7 phosphate to avoid buffer effects on the target pH and salt concentration. Some experiments were repeated twice.

[0148] Estimation of system dead volume, column porosity, and resin particle porosity The external dead volume of the Äkta system was estimated by pulse injection of 200 μL of 1 M NaCl in 10 mM phosphate buffer, pH 6.5, in the absence of a column; i.e., a zero-dead-volume connector was used instead of the column. Column porosity ε of manually packed columns with Capto MMC ImpRes resin bound to different synthetic ligand prototypes was calculated. C was estimated by pulse injection of 200 μL of 3 mg / mL Blue Dextran 2000 (molecular weight 2000 kDa) into the column in 10 mM phosphate buffer at pH 6.5. C is the equation:

[0149]

number

[0150] The calculation was carried out using the formula: Dex is the column retention volume of Blue Dextran, and V systemdead is the dead volume of the Akta system, and V C is the geometric column volume. Resin particle porosity ε P is the equation: ε T =ε C +ε P (1-ε C ) (2) The calculation was performed using the formula: T is the total column porosity estimated by pulse injection of 200 μL of protein solution (concentration ≈ 8.23 ​​mg / mL) under non-binding conditions of pH 12 100 mM Na3PO4 onto the column. T is the equation:

[0151]

number

[0152] where t is the retention time of the protein under non-binding conditions, Q is the flow rate, and V C is the column volume.

[0153] Isocratic retention test Each isocratic retention experiment was carried out in five different steps, including: step 1: equilibration (5 CV; pH 7 25 mM phosphate buffer + X (200 mM - 1800 mM) NaCl); step 2: sample load (approximately 2 mg protein per mL of resin; 200 μL sample load volume); step 3: isocratic elution (until UV was less than 2 mAU); step 4: strip (5 CV; pH 7.5 50 mM phosphate buffer + 1000 mM NaCl); step 5: clean-in-place (CIP, 3 CV) step with 1 N NaOH for column regeneration and sanitization. The flow rate was set at 0.5 mL / min. The fluid outflow was monitored at 280 nm for peak detection. The retention factor k was calculated using the equation:

[0154]

number

[0155] V R is the retention volume corrected for the dead volume of the protein system under isocratic conditions, V0 is the column void volume, and is expressed by the equation:

[0156]

number

[0157] The isocratic retention factor k was calculated using the formula: The total number of countersalt ions and water molecules released during mAb1 adsorption, as well as the hydrophobic contact area (HCA), were estimated by fitting the characteristic "U"-shaped curve obtained for multimodal chromatography to the selective interaction model developed by Perkins et al. [28, 29]. The equation relating the total number of released countersalt ions and released water molecules to the retention factor k is shown below:

[0158]

number

[0159] In the formula, -(Δν + +Δν - ) is the total number of counterions released during mAb1 adsorption, which is governed by electrostatic interactions at low salt concentrations, (-Δν1) is the total number of water molecules released during the adsorption of one mAb1, which is governed by hydrophobic interactions at high salt concentrations; c is an integration constant; C S is the salt concentration of the mobile phase; m is the molar concentration of water; n is the valence of the salt ion;

[0160]

number

[0161] is the thermodynamic property of the salt ion and a is the activity of the salt ion. The isocratic retention factor data was fitted to the simplified form of equation (6) shown below. lnk=α+βlnC S -γC S (7)

[0162] The model parameters β and γ are calculated using the following formula: (Δν + +Δν - )=gβ (8)

[0163]

number

[0164] By (Δν + +Δν - ) and (Δν1).

[0165] The hydrophobic contact area (HCA) during mAb1 adsorption on the resin was calculated by fitting the isocratic retention factor data to Melander's three parameter equation

[30] , given by: logk=A-BlogC S +CC S (10) where B and C are the electrostatic and hydrophobic interaction parameters, respectively. From the parameter C, HCA is calculated using equation

[31] :

[0166]

number

[0167] where R is the universal gas constant, T is the absolute temperature, and σ S is the molar surface tension increment of the salt.

[0168] Linear salt gradient elution test Each linear NaCl salt gradient experiment was performed in five different steps, including:

[0169] (i) Method 1: Bind and Elute

[0170] [Table 2]

[0171] (ii) Method 2: Bind and Elute

[0172] [Table 3]

[0173] The elution was collected in fractions and analyzed for protein concentration and % content of mAb1, Fab fragments, and HMW aggregates using SE-HPLC as described above. Using the SE-HPLC data, the linear gradient elution peak was deconvoluted into its component mAb1 and Fab, according to the following equation:

[0174]

number

[0175] Resolution R between mAb1 and Fab using swas used to calculate where t R is the retention time of the peak, and w 0.5h is the full width at half height of the maximum peak. The retention times of the mAb1 and Fab fragment peaks are t R2 and t R1 and the full width at half height of the maximum peak of Fab and mAb1 are

[0176]

number

[0177] It is shown as follows.

[0178] Separation resolution for HMW aggregates was compared by plotting cumulative mAb1 yield (%) against cumulative HMW aggregates (%). The cumulative mAb1 yield (%) and HMW aggregates (%) were calculated using the following formula:

[0179]

number

[0180] was calculated using SE-HPLC data.

[0181] Results and Discussion The effect of ligand density, as determined by ion mass measurements, on chromatographic performance was measured using the reference Capto MMC ImpRes resin (25 μmol / mL 樹脂 ) and similar ion content (20-31 μmol / mL 樹脂 The cost of the chromatographic assay was minimized by generating all prototypes containing the new ligands. The synthesized prototypes were tested for chromatographic performance in a high-throughput plate-based assay.

[0182] Isocratic retention of mAb1 in the original Capto MMC analogue To understand the behavior of mAb1 adsorption on the new Capto MMC ligand prototypes and begin to qualitatively connect ligand structure to binding, mAb1 retention was analyzed as a function of different isocratic chromatographic conditions for the new prototypes and reference ligands. mAb1 vs. NaCl concentration (C S The dimensionless isocratic retention factor ln(k) versus pH was varied from 200 mM to 1800 mM at pH 7 and plotted (not shown).

[0183] The prototype exhibits a "U"-shaped dependence of protein retention as a function of salt concentration, with minimal retention at approximately 800 mM NaCl. The "U"-shaped behavior is characteristic of multimodal resins and is the result of contributions from ion exchange and other secondary interactions, such as hydrophobic interactions. For binding pH values ​​below the pI value of mAb1 (approximately 8.6) and low NaCl concentrations, protein retention decreases with increasing salt concentration. Similar to classical CIEX chromatography, this is because buffer cations compete with the positively charged mAb for negatively charged sites in multimodal chromatography. However, at high NaCl concentrations (≥1000 mM), protein retention increases with salt concentration, behaving similarly to HIC resins.

[0184] At intermediate regimes, approximately 800-1000 mM NaCl, there is a transition occurring in the mAb1 adsorption mechanism from primary electrostatic interactions to secondary hydrophobic interactions.

[0185] Figure 3 shows a comparison of the isocratic mAb1 retention factor ln(k) for L09, L02, and L00 at varying NaCl concentrations from 200 mM to 1800 mM at pH 7. More specifically, Figure 3 shows the ln(k) versus ln(C) for mAb1 adsorption for (closed squares) L00, (x) L09, and (open squares) L02 at pH 7. S ) is shown. The solid line (-) overlays the fits to equations (7) and (10).

[0186] Chromatographic performance using high-throughput binding capacity (BC) data Response surface analysis of high-throughput BC data We performed high-throughput plate-based experiments that provide information on protein binding to several targets under both varying pH and salt concentrations in parallel, making them extremely useful for evaluating the diverse binding and elution properties of multimodal resins. These plate experiments were performed under low loadings in the linear portion of the isotherm, providing information on binding behavior and selection opportunities rather than maximum binding capacity. Again, the multimodal prototypes exhibited a "U"-shaped dependence of binding with salt concentration, attributable to multimodal electrostatic and other secondary interactions.

[0187] Principal component analysis of BC High-throughput plate-based binding data were collected for prototypes containing six model proteins each at 32 different pH / [NaCl] binding conditions. To gain further insight into this large amount of data, principal component analysis (PCA) was performed, yielding six principal components (PCs). From these six PCs, the first two PCs explained a total of 85.7% of the variance in the data. The trend of PC1 scores, including the binding capacity of mAb1 at pH 4.5 and 1750 mM NaCl, shown in the chromatographic diversity map (not shown), indicated that PC1 scores strongly correlated with high salt binding to mAb1. This indicated that the overall results from detailed testing of mAb1 appear to be generalizable and accessible for rapid, high-throughput plate-based testing.

[0188] High-throughput binding capacity of ligand prototypes Table 2 shows the high-throughput binding capacity data in terms of % bounds for specific proteins under given binding conditions.

[0189] [Table 4]

[0190] Linear salt gradient elution test Separation resolution R between Fab fragments and mAb s Table 3 shows the R for the resolution of separation between Fab fragments and monoclonal antibody (mAb1). s All new ligand prototypes showed better separation resolution than the reference ligand (L00), but L09 had the highest separation resolution, followed by L02, L05, L01 and L08.

[0191] [Table 5]

[0192] Resolution of separation between HMW aggregates and mAb (i) Method 1: Bind and Elute Figure 4 shows a comparison of separation resolution between HMW aggregates and monoclonal antibodies (mAbs) for different ligand prototypes for data obtained using Method 1, further described above. The load contained approximately 2% HMW aggregates. Comparison of the cumulative plots showed that the novel ligands outperformed the reference ligand (L00), with L09 being one of the most promising for aggregate removal.

[0193] (ii) Method 2: Bind and Elute Figure 5 shows a comparison of separation resolution between HMW aggregates and mAbs for different ligand prototypes for data obtained using Method 2, further described above. The load contained approximately 1.95% HMW aggregates. L09 outperformed L08 and L11 for HMW aggregate removal in dual pH-salt linear gradient elution.

[0194] conclusion These results demonstrate that the new multimodal ligand prototype achieves effective removal of aggregate species from problematic monoclonal antibodies, resulting in improved selectivity for product-related impurities. It can be concluded that more hydrophobic ligands outperform less hydrophobic ligands. Along with electrostatic interactions, the humble role of secondary hydrophobic and hydrogen-bonding interactions provided by the ligand chemistry of the more hydrophobic ligands results in better performance with respect to removal of Fab fragment and HMW aggregate impurities. [Example]

[0195] The experimental design is performed with the same antibodies as in Example 1, with the following modifications: (a) Testing different ligands attached to the support of the chromatographic material, i.e., ligands containing different hydrophobic groups. (b) Determination of the affinity of different ligands for different multimeric forms of the antibody. [Example]

[0196] The experimental design is carried out as in Example 1 with a target molecule other than that in Example 1, such as a bispecific antibody (approximately 200 kDa), or a protein that is not an antibody, such as a protein of 50 to 100 kDa.

[0197] It is to be understood that the present disclosure is not limited to its exemplary embodiments described above, and that several possible modifications of the present disclosure are possible within the scope of the following claims.

Claims

1. A chromatography ligand as defined by formula I. 【Chemistry 1】 [In the formula, X 1 CO and SO 2 is selected from R 1 ~R 5 Each of these is H, F, Cl, O, N, S, C 1～3 Alkyl, and C 1～3 Alkyl-X 2 are independently selected from R 1 ~R 5 any two adjacent moieties selected from can form, together with the atoms to which they are attached, a 5- or 6-membered heterocyclic or carbocyclic ring; X 2 is O, S, NH(CO), (CO)NH, NH(SO 2 ), and (SO 2 )NH, however, i.R 1 ~R 5 If each of is H, then X 1 SO 2 and ii. R 1 ~R 5 When any two adjacent moieties selected from the group consisting of: 1 SO 2 and iii. R 1 ~R 5 These three are CH 3 If O, then X 1 is CO]

2. R 1 ~R 5 10. The chromatography ligand of claim 1, wherein any two adjacent moieties selected from:

3. (a) to (f): 【Chemistry 2】 3. The chromatography ligand according to claim 1 or 2, which is defined by a chemical structure selected from the group consisting of:

4. The ligand is a thiol (-SH) of formula I, which is a methyl thioether (-S-CH 3 4. The chromatography ligand of claim 1, which when replaced with .alpha., has a logS of about -2.5 to about -5.

5. 5. A method for preparing a chromatographic material, comprising the step of immobilizing a plurality of ligands according to any one of claims 1 to 4 on a support, optionally wherein the support comprises beads having a diameter of from about 25 μm to about 50 μm, preferably from about 30 μm to about 45 μm.

6. The method according to claim 5, wherein the density of the plurality of ligands immobilized on the support is about 15 to about 50 μmol / mL, preferably about 20 to about 35 μmol / mL.

7. 5. A chromatography material comprising the chromatography ligand of any one of claims 1 to 4 bound to a support, optionally wherein the support comprises beads having a diameter of from about 25 μm to about 50 μm, preferably from about 30 μm to about 45 μm.

8. 8. The chromatographic material of claim 7, wherein the density of the plurality of ligands bound to the support is from about 15 to about 50 μmol / mL, preferably from about 20 to about 35 μmol / mL.

9. 9. The chromatography material according to claim 7 or 8, wherein the material has a lysozyme batch binding capacity in the linear part of the adsorption isotherm at binding conditions of pH 7.5 and 480 mM NaCl, and the amount of lysozyme bound to the chromatography material at equilibrium is at least 50%, for example 60%, of the amount of lysozyme added to the chromatography material.

10. 10. Use of a chromatography material according to any one of claims 7 to 9 for separating one or more target molecules from impurities.

11. 11. The use according to claim 10, wherein the one or more target molecules are one or more antibodies, preferably the antibodies are monoclonal antibodies, optionally the monoclonal antibodies are multispecific monoclonal antibodies, such as bispecific monoclonal antibodies.

12. 12. The use of claim 11, wherein the impurities comprise aggregates of one or more antibodies.

13. The one or more target molecules are one or more antibody fragments, and optionally the one or more antibody fragments are antigen-binding fragments, e.g., Fab, Fab', F(ab') 2 , scFv, Fv, dAb, or Fd.

14. 14. The use of claim 13, wherein the impurities comprise aggregates of one or more antibody fragments.

15. 1. A method for separating one or more target molecules from impurities, comprising: a) applying a liquid sample containing one or more target molecules and impurities to a chromatographic material according to any one of claims 7 to 9, b) eluting the target molecule from the chromatographic material; c) optionally eluting the impurities from the chromatographic material A method comprising:

16. 16. The method of claim 15, wherein the target molecule, and optionally impurities, are eluted from the chromatographic material by applying an elution buffer comprising: (i) a salt gradient; (ii) a pH gradient; or a combination of (i) and (ii).

17. 1. A method for separating one or more target molecules from impurities, comprising: a) applying a liquid sample containing one or more target molecules and impurities to a chromatographic material according to any one of claims 7 to 9, b) obtaining the target molecules in a flow-through manner, wherein the target molecules pass through the chromatographic material essentially without binding to the chromatographic material; c) optionally eluting the impurities from the chromatographic material A method comprising:

18. 18. The method of any one of claims 15 to 17, wherein the one or more target molecules are one or more antibodies, preferably the antibodies are monoclonal antibodies, optionally the monoclonal antibodies are multispecific monoclonal antibodies, such as bispecific monoclonal antibodies.

19. 19. The method of claim 18, wherein the impurities comprise aggregates of one or more antibodies.

20. The one or more target molecules are one or more antibody fragments, and optionally the one or more antibody fragments are antigen-binding fragments, e.g., Fab, Fab', F(ab') 2 20. The method of any one of claims 15 to 19, wherein the antibody is selected from the group consisting of an Fv, an scFv, an Fv, a dAb, or an Fd.

21. 21. The method of claim 20, wherein the impurities comprise aggregates of one or more antibody fragments.

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