Chromatographic ligands and chromatographic materials and use thereof

Abstract

This disclosure provides chromatographic ligands defined by Formula I, wherein: X1 is selected from CO and SO2; R1 is selected from (C1-C7)alkyl, cycloalkyl, aryl, heterocyclic, (C1-C7)alkyl-X2, cycloalkyl-X2 and aryl-X2; X2 is selected from CO2H, SO3H, PO3H2 and heterocyclic compounds with pKa < 6; R2 is selected from H, (C1-C7)alkyl and (C1-C7)alkyl-X3; X3 is selected from CO2H, SO3H and PO3H2; L is selected from (C1-C7)alkyl and... (C1-C2)alkyl-X4-(C1-C2)alkyl, wherein X4 is selected from O, S, NH(CO), (CO)NH, NH(SO2), and (SO2)NH; optionally, L is substituted with a straight-chain or branched (C1-C7)alkyl group, and optionally, the straight-chain or branched (C1-C7)alkyl group is substituted with X5, wherein X5 is selected from CO2H, SO3H, PO3H2, O-(C1-C2)alkyl, S-(C1-C2)alkyl, OH, and (CO)NH2. This disclosure also relates to chromatographic materials comprising a support and a chromatographic ligand disclosed herein coupled to the support, and chromatographic apparatuses comprising said chromatographic materials. Further disclosure includes the use of said chromatographic materials or apparatuses for separating one or more target entities from impurities, and methods for separating one or more target entities from impurities.

Description

Technical Field

[0001] This disclosure relates to a chromatographic ligand, chromatographic material, and chromatographic apparatus, and its use for separating one or more target entities from impurities, as well as a method for separating one or more target entities from impurities. In particular, this disclosure relates to the separation of a target entity (such as an antibody) from impurities comprising aggregates of one or more target entities. Background Technology

[0002] Biological macromolecules (such as proteins, nucleic acids, and polysaccharides) can often exist partially in the form of aggregates or polymers, such as dimers, trimers, or higher oligomers. In the bioproduction of recombinant proteins, when the 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, these conditions may favor the formation of such aggregates via intermolecular disulfide bonds or other covalent bonds or via non-covalent interactions. The presence of such aggregates of target macromolecules is undesirable in many cases. Therefore, protein aggregation is a common problem encountered in the development of bioprocesses and the manufacture of biotherapeutic agents. The aggregated form of a macromolecule can have lower biological activity than its non-aggregated form; it may even lack the desired biological activity entirely or may cause undesirable side effects. Therefore, it is crucial for therapeutic safety that therapeutic proteins are in a non-aggregated state and that low levels of therapeutic molecular aggregates are present in the final product.

[0003] Preparative chromatography remains a primary technique for purifying therapeutic proteins due to its benefits in resolution, scalability, and robustness. Monoclonal antibodies are among the most powerful therapeutic tools for treating a growing number of diseases. Continued advancements in upstream processing have led to increased purification complexity in terms of the type and amount of impurities, for example, due to higher titers. Furthermore, the heterogeneity of monoclonal antibodies adds challenges to purification steps, as they exhibit different charge distributions and sizes due to various molecular modifications throughout their lifecycle from cell culture to purification. Potential molecular modifications include charged acidic and basic variants, fragments and high molecular weight (HMW) aggregates, as well as glycosylation, deamidation, incomplete disulfide bond formation, oxidation, and isomerization, resulting in additional product-related impurities. The biopharmaceutical industry also faces new purification challenges due to the increasing molecular diversity of multispecific antibodies, where novel and difficult product-related impurities require more robust purification steps to achieve the desired product quality. Additionally, regarding biological particles (such as viral particles packaged with pharmaceutically relevant genetic material), there is a need to separate product-related impurities (e.g., improperly packaged viral particles) from properly packaged viral particles. However, with these increasing challenges, key quality attributes remain high, thus increasing the demand for chromatographic media for high-resolution purification.

[0004] Multimodal (or mixed-mode) chromatography, where small molecule ligands provide more than one type of interaction, is an important tool in the downstream processing of therapeutic proteins. A commercial example of multimodal chromatography is Capto. TM MMC ImpRes (Cytiva Sweden AB, Uppsala, Sweden) is a weak cation exchange multimode ligand that achieves high selectivity over a wide pH and salt window compared to traditional ion exchangers. It enables efficient removal of aggregates, viruses, and major contaminants during the purification of monoclonal antibodies and is suitable for the purification of antibody fragments. Multimode Capto... TM MMC ligand analogs (see Kanwar Shekhawat et al.).

[0005] However, there has been a long-standing need in the art for alternative chromatographic ligands and their application in methods to improve the separation of target entities from impurities such as aggregates, including high molecular weight aggregates of the target entity. Summary of the Invention

[0006] This disclosure achieves the aforementioned objective of providing alternative chromatographic ligands and methods to improve the separation of target entities from impurities, and relates to novel chromatographic ligands and their uses.

[0007] More specifically, the currently disclosed chromatographic ligands are defined by Formula I: (I) in: X1 is selected from CO and SO2; R1 is selected from (C1-C7)alkyl, cycloalkyl, aryl, heterocyclic, (C1-C7)alkyl-X2, cycloalkyl-X2 and aryl-X2; X2 is selected from CO2H, SO3H, PO3H2 and heterocycles with pKa < 6; R2 is selected from H, (C1-C7)alkyl, and (C1-C7)alkyl-X3; X3 is selected from CO2H, SO3H and PO3H2; L is selected from (C1-C7) alkyl and (C1-C2) alkyl-X4-(C1-C2) alkyl, and X4 is selected from O, S, NH(CO), (CO)NH, NH(SO2) and (SO2)NH; Optionally, L is replaced by a straight-chain or branched (C1-C7) alkyl group. Optionally, the straight-chain or branched (C1-C7) alkyl group is replaced by X5, wherein X5 is selected from CO2H, SO3H, PO3H2, O-(C1-C2)alkyl, S-(C1-C2)alkyl, OH and (CO)NH2.

[0008] This disclosure also provides a method for preparing chromatographic materials, comprising immobilizing a plurality of chromatographic ligands as defined above onto a carrier.

[0009] A chromatographic material is also provided, comprising a currently disclosed chromatographic ligand coupled to a carrier.

[0010] The use of the chromatographic materials disclosed herein for separating one or more target entities from impurities is also provided.

[0011] This disclosure also provides a method for separating one or more target entities from impurities, comprising: A liquid sample containing one or more target entities and impurities is added to chromatographic materials as disclosed herein; Elution of the target entity from the chromatographic material; Impurities can be eluted from chromatographic materials.

[0012] A method for separating one or more target entities from impurities is also provided, comprising: A liquid sample containing one or more target entities and impurities is added to chromatographic materials as disclosed herein; Target entities are obtained through a flow-through process, and these target entities have passed through the chromatographic material but are essentially not bound to the chromatographic material. Impurities can be eluted from chromatographic materials.

[0013] Another chromatographic apparatus is provided, which contains currently disclosed chromatographic materials.

[0014] Preferred aspects of this disclosure are described below in the detailed description and dependent claims. Attached Figure Description

[0015] Figure 1 This is a flowchart of a first method for separating one or more target entities from impurities according to the present disclosure.

[0016] Figure 2 This is a flowchart of a second alternative method for separating one or more target entities from impurities according to this disclosure.

[0017] Figure 3 The cumulative yield of target entity monomers relative to aggregates for different novel ligand prototypes and reference ligands as described in Example 1 of this document is shown. Detailed Implementation

[0018] This disclosure relates to chromatographic ligands for improving the separation of one or more target entities from impurities, wherein the impurities may include aggregates and / or fragments of the target entity.

[0019] This disclosure describes the synthesis and chromatographic evaluation of novel multimodal ligands for a reference ligand (i.e., Capto MMC ImpRes (Cytiva™, Sweden)). Examples herein demonstrate that, when eluted using a linear salt gradient, improved separation resolution between monoclonal antibodies and product-related impurities in the form of aggregates of said antibodies is achieved by novel ligands, differing from the reference ligand in that of the reference ligand, through variations in cation exchanger groups and secondary interactions (e.g., hydrophobic interactions). More specifically, monoclonal antibodies are obtained with higher purity and higher yields by using the novel ligands. The combined effect of the novel cation exchange function and the secondary hydrophobic and hydrogen-bonding interactions provided by the ligand chemistry results in superior performance in removing aggregated impurities.

[0020] This disclosure provides a chromatographic ligand defined by Formula I: (I) in: X1 is selected from CO and SO2; R1 is selected from (C1-C7)alkyl, cycloalkyl, aryl, heterocyclic, (C1-C7)alkyl-X2, cycloalkyl-X2 and aryl-X2; X2 is selected from CO2H, SO3H, PO3H2 and heterocycles with pKa < 6; R2 is selected from H, (C1-C7)alkyl, and (C1-C7)alkyl-X3; X3 is selected from CO2H, SO3H and PO3H2; L is selected from (C1-C7) alkyl and (C1-C2) alkyl-X4-(C1-C2) alkyl, and X4 is selected from O, S, NH(CO), (CO)NH, NH(SO2) and (SO2)NH; Optionally, L is replaced by a straight-chain or branched (C1-C7) alkyl group. Optionally, the straight-chain or branched (C1-C7) alkyl group is replaced by X5, wherein X5 is selected from CO2H, SO3H, PO3H2, O-(C1-C2)alkyl, S-(C1-C2)alkyl, OH and (CO)NH2.

[0021] If present, X5 can be bonded to any carbon atom in a straight-chain or branched (C1-C7) alkyl group.

[0022] The term “(C1-C7)alkyl” is intended to refer to C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, or C7 alkyl.

[0023] When R1 is a (C1-C7) alkyl group, the (C1-C7) alkyl group can be straight-chain or branched. When R1 is a (C1-C7) alkyl group, it is preferably a (C3-C7) alkyl group.

[0024] When R2 is a (C1-C7) alkyl, the (C1-C7) alkyl can be straight-chain or branched. When R2 is a (C1-C7) alkyl, it is preferably a (C1-C5) alkyl or a (C1-C4) alkyl, even more preferably a (C1-C3) alkyl, and currently most preferably a C1 alkyl.

[0025] When L is a (C1-C7) alkyl, it is preferably a (C1-C5) alkyl or a (C1-C4) alkyl, even more preferably a (C1-C3) alkyl, and currently most preferably a (C1-C2) alkyl or a C1 alkyl.

[0026] X2 is a polarizable acidic group. When X2 is a heterocycle with pKa < 6, it is preferably pKa < 5. A non-limiting example of a heterocycle with pKa < 6 is a tetrazolium.

[0027] According to the presently preferred non-limiting embodiment of Formula I, L is optionally substituted with a straight-chain or branched (C1-C7) alkyl group substituted with X5, wherein the straight-chain or branched (C1-C7) alkyl group is bonded to a C1 carbon atom, which is bonded to N.

[0028] According to another currently preferred non-limiting embodiment of Formula I, X2 is SO3H, and L is an unsubstituted C2 alkyl group, i.e., a C2 alkyl group not substituted by a straight-chain or branched (C1-C7) alkyl group. R1 and R2 are as defined above for Formula I. Optionally, R2 is H.

[0029] According to another currently preferred non-limiting embodiment of Formula I, X2 is CO2H, R2 is H, and L is a C1 alkyl group substituted with a straight-chain or branched (C1-C7) alkyl group optionally replaced by X5, as shown in Formula III: (III) R8 represents a straight-chain or branched (C1-C7) alkyl group that may be optionally replaced by X5, where the C1 alkyl group is replaced by X5.

[0030] If present, X5 is selected from CO2H, SO3H, PO3H2, O-(C1-C2)alkyl, S-(C1-C2)alkyl, OH, and (CO)NH2. According to this embodiment, X5 may optionally be selected from CO2H, O-(C1-C2)alkyl, S-(C1-C2)alkyl, OH, and (CO)NH2. Non-limiting examples of ligands according to this embodiment derived from different α-amino acids are shown below: Examples derived from serine: Examples derived from threonine: Examples derived from asparagine: Examples derived from glutamine: Examples derived from aspartic acid: Examples derived from glutamic acid: Examples derived from alanine: Examples derived from methionine: Examples derived from valine: Examples derived from isoleucine: Examples derived from leucine: According to another currently preferred non-limiting embodiment of Formula I, when R1 is aryl, the chromatographic ligand can be defined by Formula II: (II) in: Each of R3-R7 is independently selected from H, F, Cl, NH2, (C1-C3)alkyl, CO2H, SO3H and PO3H2 and X6-(C1-C3)alkyl; Any two adjacent portions selected from R3-R7, together with the atoms they are attached to, can form a 5- or 6-membered heterocycle or a carbocyclic ring; and X6 is selected from O, S, NH, N-(C1-C3)alkyl, NH(CO), (CO)NH, NH(SO2) and (SO2)NH.

[0031] The 5- or 6-membered heterocyclic ring or carbocyclic ring can be unsaturated or saturated. Furthermore, the 5- or 6-membered heterocyclic ring or carbocyclic ring can be nonpolar, aromatic, and / or aliphatic. The heterocyclic ring can contain up to three heteroatoms. When three heteroatoms are present, they are all N. When up to two heteroatoms are present, each of them can be independently selected from N, O, and S.

[0032] According to a non-limiting embodiment of Formula II, X1 is SO2 when each of R3-R7 is H.

[0033] In the presently preferred non-limiting embodiment of Formula II, any two adjacent portions selected from R3-R7 together with the atoms to which they are attached form a 5-membered heterocycle containing at most two oxygen atoms in the ring (or at most one oxygen atom in the ring).

[0034] According to a non-limiting embodiment of Formula II, when any two adjacent portions from R3-R7, together with the atoms they are attached to, form a 5-membered heterocycle containing two oxygen atoms in the ring, according to Formula IV, X1 is SO2: (IV) Where L, X2, and R2 are as defined for Equation I. R2 may preferably be H.

[0035] According to another non-limiting embodiment of Formula II, when three of R3-R7 are CH3O, X1 is CO.

[0036] The non-limiting implementation of Formula I is: According to the currently preferred non-limiting embodiment of Formula I, the currently disclosed chromatographic ligand can be defined by a chemical structure selected from any of (a)-(e): and .

[0037] According to the presently preferred embodiment, the chromatographic ligand may be defined by a chemical structure selected from either (a) or (b): a) and In this paper, the term "chromatographic ligand" means a molecule that has a known or unknown affinity for a given analyte and can be coupled to a carrier of chromatographic material, while "analyte" includes any specific binding pair of the ligand.

[0038] When a chromatographic ligand is coupled to a carrier, the ligand is linked to the carrier via a covalent thioether bond formed from a thiol (i.e., the HS-ligand becomes the carrier-S-ligand in Formula I and Formula II, respectively).

[0039] The analytes of interest separated according to this disclosure are so-called target entities and impurities that are present in liquid samples.

[0040] In this context, the term "target entity" is intended to include macromolecules and biological particles that will be separated from liquid samples and purified from impurities before being used for their intended application, such as as a therapeutic substance.

[0041] The term "macromolecule" has its conventional meaning in the field of bioprocessing, where the macromolecule is produced (usually recombinantly) from cells in a cell culture and purified from the cell culture by any means of separation and purification. Alternatively, macromolecules may be present in a biological solution that is not necessarily derived from a cell culture. Non-limiting examples of macromolecules are biomacromolecules, which are large biopolymers composed of monomers linked together, such as peptides and proteins (which may be natural or recombinant), including but not limited to enzymes, antibodies, portions and fragments of antibodies, and carbohydrate and nucleic acid sequences such as DNA and RNA. Macromolecules purified using chromatographic ligands according to this disclosure are typically proteins or polypeptides, particularly therapeutic proteins or polypeptides, such as antibodies, as further defined in detail below. Alternatively, macromolecules may be nucleic acid sequences that may or may not be contained in biological particles (such as viral particles). Nucleic acid sequences and / or viral particles may be used for therapeutic applications. Biomacromolecules or biological particles may, for example, be biopharmaceuticals intended to be used as pharmaceutical compounds. It should be understood that "macromolecule" is intended to refer to one type of macromolecule, and the singular form of the term may include a large number of individual macromolecules or samples of the same type. Similarly, "bioparticle" is intended to refer to a type of bioparticle, and the singular form of the term can include a large number of individual bioparticles or samples of the same type.

[0042] It should be understood that, as used herein, the term "liquid sample" (or simply "sample") encompasses any type of sample that may be obtained from cell cultures or from fluids derived from cell cultures, which have been at least partially purified by any separation and purification means. An alternative term for "liquid sample" is "feed," which is intended to refer to a mobile phase containing both target entities and impurities. When feed is added to chromatographic material, target entities and impurities can be retained in the chromatographic material to varying degrees by binding to chromatographic ligands. The binding to chromatographic ligands and the separation of target entities and impurities will depend on the specific molecules and their properties, such as isoelectric point, hydrophobicity, exposed charge, and the ability to perform additional interactions (such as π-π stacking and hydrogen bonding). The intrinsic properties of the molecules, the ligand properties, and the buffer composition will be used to achieve the separation of target entities and impurities.

[0043] In this document, the term "cell culture" refers to a culture of cultured cells or a group of cells, wherein 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 unclarified, i.e., containing cells, or can be cell-depleted, i.e., containing few or no cells but containing biomolecules released from the cells prior to cell removal. Furthermore, unclarified cell cultures used in the currently disclosed methods can contain intact cells, broken cells, cell homogenates, and / or cell lysates.

[0044] As used herein, the term "antibody" means an immunoglobulin that can be natural or partially or completely synthetically produced. This term includes, but is not limited to, complete antibodies, such as monospecific and multispecific antibodies. The term also includes active antibody fragments, including Fab antigen-binding fragments, monovalent fragments, and multivalent fragments. The term also covers any protein having a binding domain homologous to an immunoglobulin binding domain. Such proteins can be derived from natural sources or are produced partially or completely synthetically. The term also includes fusion proteins, which comprise antibodies or antibody fragments covalently linked to other proteins, such as monoclonal antibodies or monoclonal antibody fragments. Exemplary antibodies are fragments of immunoglobulin isotypes and different types, such as Fab, Fab', F(ab')2, Fv, dAb (single-domain antibody), and Fd (fragments obtained by hydrolyzing immunoglobulin molecules with papain and then reducing disulfide bonds), as well as scFv (so-called single-chain variable fragments, which are fusion proteins of the variable regions of the heavy and light chains of immunoglobulins), tandem scFv, BiTE (bispecific T-cell adaptor molecule), DART (dual-affinity retargeting molecule), and biantibodies (single-chain and tandem biantibodies). Bispecific monoclonal antibodies are examples of multispecific antibodies and are engineered proteins that can simultaneously bind to two different types of antigens or two different epitopes on the same antigen. The term "antibody" also includes antibody conjugates. Additionally, the term "part of an antibody" may be used herein to describe the antibody fragment itself, or an antibody conjugate or fusion protein that contains an antibody fragment or part of an antibody. Chromatographic ligands according to this disclosure can be used to purify, for example, therapeutic antibodies from impurities such as aggregates or fragments of the described therapeutic antibodies to obtain a high-quality final product. The presence of aggregates in therapeutic antibody formulations often has a negative impact on patient safety and must be effectively removed during the manufacturing process.

[0045] Non-limiting examples of bioparticles are viral particles, typically recombinant viral particles, designed to facilitate gene transfer to modify specific cell types or tissues. Viral particles can, for example, be engineered to provide vectors for expressing therapeutic genes. Several types of viruses are currently under investigation for delivering genetic material (e.g., genes) into cells to provide transient or permanent transgenic expression. These include adenoviruses, retroviruses (gamma retroviruses and lentiviruses), poxviruses, adeno-associated viruses (AAVs), baculoviruses, and herpes simplex viruses.

[0046] The term "viral particle" is used herein to refer to a complete infectious viral particle. It comprises a core containing the viral genome (i.e., the viral genome) in the form of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and this core is surrounded by a morphologically defined capsid. This capsid is called the capsid. The capsid and the encapsulated viral genome together constitute what is known as the nucleocapsid. 。Some viruses have a nucleocapsid surrounded by a lipoprotein bilayer. In the field of bioprocessing, for the purpose of producing viral vectors for various applications (such as therapies), the genome of viral particles is modified to include genetic inserts containing the genetic material of interest. The modified viral particles are then allowed to infect host cells in a cell culture, and the viral particles multiply in said host cells, after which the viral particles are purified from the cell culture by any isolation and purification method.

[0047] In this document, the term "impurity" is intended to refer to any molecule or substance present in a liquid sample that is not the desired target entity. The term "impurity" includes aggregates, such as aggregates of the target entity, such as dimers, and higher molecular weight aggregates of the target entity. The term "impurity" further includes fragments of the target entity, for example, when the target entity is a complete antibody and an undesirable fragment of said antibody is present in the liquid sample. In the case of a bispecific antibody, the term "impurity" can refer to several different types of product-related impurities that may need to be separated from the target entity. Since there are typically four chains (two heavy chains and two light chains) to combine, the theoretical mismatch number is high. Homodimers are a possible variant in which two identical heavy chains and two identical light chains match the "normal" mAb construct. Fragments of variant combinations of heavy and light chains are also common, such as half-antibodies consisting of only one heavy chain and one light chain. Furthermore, all free chains can occur. Therefore, the impurity profile of a bispecific antibody depends on the expression and assembly of the heavy and light chains.

[0048] The term "impurities" also includes host cell proteins (HCPs). HCPs may include, for example, enzymes such as proteases and / or lipases. Lipases are known in the art to be removed by using multimode chromatography ligands (see, for example, WO2020023566A1).

[0049] The term "impurity" also includes empty viral particles that do not contain genetic content. Removing such empty viral particles from viral particles that contain genetic content is of interest in the field of producing viral particles that can be used as vectors for delivering genetic material.

[0050] The target entities according to this disclosure are typically non-aggregated macromolecules, and the impurities from which the target entities are to be isolated typically include aggregates and / or fragments of the macromolecules, typically proteins such as antibodies.

[0051] In this document, the term "non-aggregated macromolecule" is intended to refer to a non-degradable macromolecule. Non-aggregated macromolecules may also be referred to herein as "non-degradable macromolecules" or "intact macromolecules." In a typical embodiment of this document, where the macromolecule is a protein or polypeptide, a non-aggregated macromolecule can be described as having a substantially intact tertiary structure, typically involving a substantially hydrophilic surface of the macromolecule, while hydrophobic portions are located within the macromolecule. Therefore, a non-aggregated macromolecule essentially does not have hydrophobic portions or hydrophobic groups exposed on its surface.

[0052] Conversely, in proteins or peptides that have begun to degrade, the tertiary structure is gradually disrupted, exposing hydrophobic portions to the environment surrounding the protein or peptide. Degrading or already degraded protein or peptide macromolecules can form aggregates. Non-aggregated macromolecules exist in a monomeric state. Aggregates of macromolecules can contain multimeric forms of the macromolecules, such as dimers, trimers, etc. Individual macromolecules undergoing degradation can form aggregates with other individual degradation samples of the same type of macromolecule, and / or can form aggregates with individual degradation samples of other types of degrading macromolecules, or combinations thereof. Because macromolecule aggregates contain degrading macromolecules, they possess hydrophobic portions exposed on their surfaces.

[0053] The term "high molecular weight (HMW) aggregate" is familiar to those skilled in the art. Such aggregates are formed by the self-association of target entities (e.g., monoclonal antibodies with a molecular weight of approximately 150 kDa) via covalent and non-covalent bonds. This results in the formation of dimers (e.g., approximately 300 kDa for monoclonal antibody dimers) or even higher-order aggregates, such as trimers (approximately 450 kDa for monoclonal antibody trimers). Depending on the nature of the target entity, these aggregates can be soluble or insoluble. Therefore, the term "high molecular weight aggregate" as used herein may refer to an aggregate of target entities whose molecular weight is approximately twice, or greater than twice, the molecular weight of the target entity.

[0054] In this paper, the term "hydrophobic portion" is intended to refer to the hydrophobic portion of a macromolecule or the hydrophobic group present in a macromolecule.

[0055] As used herein, the term "hydrophobic group" is defined as log P Molecular groups with a value > 0. Distribution coefficient (abbreviated as...) P The ratio is defined as a specific ratio of solute concentration between two solvents (two phases in a liquid phase), particularly for unionized solutes, and therefore the logarithm of this ratio is log[…]. P When one solvent is water and the other is a nonpolar solvent, then log PThe value is a measure of lipophilicity or hydrophobicity. A precedent for this definition is that the lipophilic and hydrophobic phase types are always represented in the numerator and denominator, respectively; for example, in... just In a two-phase system of octanol (hereinafter referred to as "octanol") and water: log P A value <0 indicates a higher percentage of solute in the hydrophilic phase. Conversely, a log value <0 indicates a higher percentage of solute in the hydrophilic phase. P A value >0 indicates a higher percentage of solute in the lipophilic phase (i.e., the hydrophobic phase).

[0056] As mentioned above, denatured macromolecules and their aggregates are generally more hydrophobic than intact, non-denatured, non-aggregated macromolecules. Therefore, aggregates bind to the hydrophobic groups of chromatographic ligands to a greater extent than non-aggregated macromolecules. Target entity aggregates also have a larger size than the target entity itself, and therefore a larger surface area for interaction with chromatographic ligands. Thus, target entity aggregates will exhibit stronger binding to ligands compared to the binding of the target entity to currently disclosed multimode ligands. Target entity aggregates also typically have more positively charged sites than monomeric target entities. Therefore, aggregates will generally elute later from chromatographic devices including multimode ligands than the target entity.

[0057] This disclosure also provides a method for preparing chromatographic materials, comprising immobilizing a plurality of chromatographic ligands as defined above onto a carrier.

[0058] The term "separation matrix" is used herein to refer to a material containing a support to which one or more ligands containing functional groups are coupled. The functional groups of the ligands bind to compounds (also referred to herein as analytes) to be separated from a liquid sample and / or from other compounds present in the liquid sample. The separation matrix may also contain compounds that couple the ligands to the support. The terms "connector," "extensionist," and "surface extensionist" may be used to describe such compounds, as further described below. The terms "chromatographic material" and "chromatographic matrix" are used herein to refer to one type of separation matrix.

[0059] The term “surface” in this document means all external surfaces, and in the case of porous carriers, includes both external surfaces and pore surfaces.

[0060] The separation matrix can be contained in any type of separation apparatus, as further defined elsewhere herein. As a non-limiting example, the chromatographic material may be packed into the column before the liquid sample is added to it.

[0061] The chromatographic materials disclosed herein comprise a carrier coupled with a ligand. The term “carrier” has its conventional meaning in the field of bioprocessing and may alternatively be referred to as “carrier material,” “matrix material,” or “solid phase,” which are other terms commonly used in the art.

[0062] As further mentioned above, when a chromatographic ligand is coupled to a carrier, the ligand is linked to the carrier via a covalent thioether bond formed at the thiol. Such a linker or extender may also be included in the term "carrier" when the ligand is coupled to the carrier using a linker or extender (as described in further detail below).

[0063] The carrier can be made of different types of materials and can have different shapes or forms, as described in more detail below.

[0064] The carrier of chromatographic materials can contain porous particles, non-porous particles, or expanded bed media, or it can be a convective flow material.

[0065] Porous particles can be alternatively referred to as beads, and chromatographic materials containing such particles or beads can be referred to as resins.

[0066] Porous particles can be made, for example, from agarose (such as cross-linked agarose). A non-limiting example of porous agarose particles is Capto ImpRes resin (Cytiva, Sweden), which contains substantially spherical particles.

[0067] Suitable particle sizes for currently disclosed chromatographic materials can be in the diameter range of 5 μm to 200 μm, such as 10 μm to 100 μm, for example 20 μm to 70 μm. In one specific embodiment, the average particle size is in the range of about 25 µm to about 60 µm, such as about 25 µm, 30 µm, 35 µm, 40 µm, 45 µm, 50 µm or 60 µm.

[0068] The suitable average pore size of the currently disclosed chromatographic materials in particulate form can be 2 to 3 times that of the target entity and impurities to be separated, including but not limited to an average pore size of about 20 nm (e.g., suitable for separating monoclonal antibodies) to about 80 nm, such as about 20 nm, 30 nm, 50 nm, 75 nm or 80 nm, preferably about 30 nm to about 80 nm.

[0069] Those skilled in the art can easily select appropriate particle size and porosity according to the method to be used.

[0070] Other non-limiting examples of porous particles are magnetic beads and polystyrene beads. A non-limiting example of magnetic particles is Mag Sepharose™ (Cytiva, Sweden).

[0071] A non-limiting example of non-porous particles is a solid glass bead.

[0072] A non-limiting example of an expanded bed medium is STREAMLINE™ resin (Cytiva, Sweden).

[0073] The matrix material can be a convective flow matrix material. Such materials can be, for example, adsorbed membranes, where the flow through such materials is convective rather than diffuse. Convective matrix materials include any matrix in which a hydraulic pressure differential is applied between the inflow and outflow of the matrix, forcing the matrix to infuse, thereby essentially achieving convective transport of matter into or out of the matrix, which can be achieved very rapidly at high flow rates.

[0074] Examples of convection matrix materials include porous adsorption membranes, fibrous materials, and monolithic materials.

[0075] The adsorption membrane can be, for example, a polymer membrane, such as a polyethersulfone membrane, such as the Mustang™ membrane chromatography capsule (Cytiva, Sweden). Another example of an adsorption membrane is a polymer nanofiber membrane, such as, for example, cellulose, cellulose acetate, and cellulose fibers, which have been treated to be used as an adsorbent. Treatment may include one or more of crosslinking, derivatization, and ligand coupling. The matrix material can be a nonwoven material containing fibers, such as those made from cellulose. Such fibrous substrates can be based on electrospun polymer fibers or cellulose fibers and can have, for example, a cross-sectional diameter of 10 nm–1000 nm, such as 200 nm–800 nm, 200 nm–400 nm, or 300 nm–400 nm. For example, convection-based chromatography and membrane adsorbents are described in US20140296464A1, US20160288089A1, US2019308169A1 and US2019234914A1, which are hereby incorporated in their entirety by reference.

[0076] The polymer used for polymer membranes can be a natural or synthetic polymer, including derived polymers. For example, hydroxylated polymers can be used to provide hydroxylated polymer membranes. Hydroxylated polymers can include sugar polymers. In addition to cellulose and polyethersulfone as mentioned above, polymers that can be used for adsorption membranes include polymers based on polytetrafluoroethylene (PTFE), polypropylene, polyamide, or polyacrylamide.

[0077] Alternatively, the adsorption membrane can be a monolithic material or a conventional membrane made through emulsification. Another alternative is 3D printed materials.

[0078] The mean flow porosity (MFP) is an indicator of a material's flow properties and is measured using capillary flow porosity determination, which is based on displacing a wetting liquid with a known surface tension from the pores of a sample by applying gas at gradually increasing pressure. A higher MFP indicates greater liquid flow through the material at a given pressure. The mean flow porosity is calculated based on the point where 50% of the flow passes through the sample. Therefore, the mean flow porosity corresponds to the porosity calculated at the pressure where the wet and semi-dry profiles intersect.

[0079] A non-limiting example of a monolithic material is CIMmultus® (Sartorius, Germany).

[0080] Chromatographic materials may contain compounds that couple ligands to a support. The terms “connector,” “extendon,” and “surface extender” can be used to describe such compounds, as further described below.

[0081] Linkers can comprise a portion resulting from a reaction that couples a ligand to a support. Well-known coupling chemistry is known to those skilled in the art and can involve reactions with, for example, allyl glycidyl ether (AGE), divinyl sulfone (DVS), or epichlorohydrin (ECH). Thus, linkers can contain a portion obtained through the reaction of a support with allyl glycidyl ether, through the reaction of an optionally activated or derived support with divinyl sulfone, or through a reaction with epichlorohydrin, and connected to the support via this portion. For example, a linker can comprise a vinyl sulfone moiety. Accepted methods include, for example, introducing an epoxy group using epichlorohydrin or 1,4-butanediol diglycidyl ether. Another example is introducing a double bond onto the support using a reagent such as divinyl sulfone. Linkers formed from these reagents can be further extended by allowing new functions for ligand coupling.

[0082] Spacers, such as straight-chain or branched 2-12 carbon alkyl groups or 2-12 carbon ethers, can be used between hydrophobic ligands and linkers (e.g., vinyl sulfones). Linkers can also react with extensor groups and connect them to ligands or spacers. A spacer is the carbon chain length between the linker and ligand, used to improve the basic stability of the linker-ligand bond.

[0083] The ligand can be coupled to the carrier via an "extension group," or simply "extension." The extension can be selected from polysaccharide and polymer structures. The extension can be... For example Dextran, acrylamide, or polyglycerol. If dextran is used as the extensor, its molecular weight can range from 5,000 Daltons to 2,000,000 Daltons. The extensor group can be synthesized from a support by polymerization (length not determined) or coupled to the surface of a support.

[0084] In some implementations, no extension subgroup is used, and the ligand is directly immobilized / attached to the support. The ligand can be attached to the matrix material via oxygen.

[0085] The term "eluting agent" is used in its conventional sense in the art, that is, a buffer solution with suitable pH and / or ionic strength to release one or more compounds from a separation matrix.

[0086] The term "eluting agent" is used in its conventional sense in the art, that is, the portion of a liquid sample that is eluted from a chromatographic column after the liquid sample has been loaded onto the column.

[0087] In the method for preparing chromatographic materials disclosed herein, the density of the plurality of ligands immobilized on the support can be from about 15 µmol / mL to about 100 µmol / mL, such as about 15 µmol / mL, 20 µmol / mL, 25 µmol / mL, 30 µmol / mL, 35 µmol / mL, 40 µmol / mL, 45 µmol / mL, 50 µmol / mL, 55 µmol / mL, 60 µmol / mL, 65 µmol / mL, 70 µmol / mL, 75 µmol / mL, 80 µmol / mL, 85 µmol / mL, 90 µmol / mL, 95 µmol / mL or 100 µmol / mL, preferably from about 20 µmol / mL to about 50 µmol / mL.

[0088] The ligand density (the amount of ligand per volume of resin) is determined by pH electrode titration, as is well known in the art (see, for example, Kanwar Shekhawat L. et al.). Titration can be performed with NaOH (aqueous solution) from an acidic sample of the resin slurry, or with HCl (aqueous solution) from an alkaline sample of the resin slurry. More specifically, in the case of strong cation exchange ligands, ion exchange is first performed with a high concentration of HCl (aqueous solution), followed by a low concentration of HCl (aqueous solution). The hydrated hydrogen counterion is then quantified by titration with NaOH (aqueous solution). For ligands with one cation exchange site, the ion capacity is equal to the ligand density.

[0089] This disclosure also provides a chromatographic material comprising a chromatographic ligand as defined above, coupled to a carrier. The chromatographic material and carrier are as defined and illustrated above.

[0090] In the currently preferred embodiment, the carrier comprises beads with a volume-weighted median diameter (d50v) of about 20 µm to about 60 µm, such as about 20 µm, 25 µm, 30 µm, 35 µm, 40 µm, 45 µm, 50 µm, 55 µm or 60 µm, preferably about 30 µm to about 55 µm, and currently more preferably about 40 µm to 50 µm.

[0091] The chromatographic materials disclosed herein can have densities of multiple ligands immobilized on a support ranging from about 15 µmol / mL to about 100 µmol / mL, such as about 15 µmol / mL, 20 µmol / mL, 25 µmol / mL, 30 µmol / mL, 35 µmol / mL, 40 µmol / mL, 45 µmol / mL, 50 µmol / mL, 55 µmol / mL, 60 µmol / mL, 65 µmol / mL, 70 µmol / mL, 75 µmol / mL, 80 µmol / mL, 85 µmol / mL, 90 µmol / mL, 95 µmol / mL, or 100 µmol / mL, preferably about 20 µmol / mL to about 50 µmol / mL. The methods for determining the ligand density are as described above.

[0092] The ligands of the chromatographic material are defined by Formula I as described in further detail above.

[0093] The use of the chromatographic materials disclosed herein for separating one or more target entities from impurities is also provided.

[0094] In the context of the described uses, one or more target entities may be one or more antibodies and / or portions of antibodies, as described in detail elsewhere herein. Preferably, the antibody is a monoclonal antibody. Alternatively, the monoclonal antibody is a multispecific monoclonal antibody, such as a bispecific monoclonal antibody. Alternatively, one or more target entities may be one or more antibody fragments. Optionally, one or more antibody fragments may be selected from antigen-binding fragments described and exemplified in detail elsewhere herein, such as Fab, Fab', F(ab')2, scFv, Fv, dAb, or Fd.

[0095] Furthermore, in the context of the stated uses, impurities may comprise aggregates of one or more target entities, such as high-molecular-weight aggregates of target entities, as described in detail elsewhere herein. For example, when the target entity is an antibody (such as a monoclonal antibody), the impurity may comprise aggregates of said antibody, such as high-molecular-weight aggregates of one or more antibody fragments. Alternatively, when the target entity is an antibody fragment, the impurity may comprise aggregates of one or more antibody fragments, such as high-molecular-weight aggregates of one or more antibody fragments. Alternatively, when the target entity is a bispecific antibody, the impurity may comprise product-related impurities, such as various mismatch entities of said bispecific antibody, such as homodimers of said bispecific antibody. Regardless of the target entity, the impurity may comprise host cell proteins.

[0096] This disclosure provides, for example, Figure 1 The method 100 shown for separating one or more target entities from impurities further addresses or at least mitigates problems associated with existing methods for separating one or more target entities from impurities, the method comprising: A liquid sample containing one or more target entities and impurities is added (110) to a chromatographic material as disclosed herein; Elute (120) target entities from chromatographic material; (130) impurities can be eluted from the chromatographic material.

[0097] According to method 100, after adding (110) a liquid sample to the chromatographic material and selecting appropriate chromatographic conditions, the target entity present in the liquid sample will bind to the ligands of the chromatographic material. Impurities may optionally bind to the ligands of the chromatographic material, depending on what impurities are present in the liquid sample and what binding conditions are applied. Impurities that do not bind to the ligands will ultimately remain in the flow fraction during the binding step.

[0098] Target entities and, optionally, impurities (if bound to ligands) can be eluted from the chromatographic material by applying an elution buffer containing salt (120). Elution can be performed isotropically, i.e., by maintaining a constant composition of the elution buffer throughout the elution process, or by i) a salt gradient, (ii) a pH gradient, or a combination of (i) and (ii). In other words, elution can be performed by applying (i) a constant pH and / or salt concentration, (ii) a salt gradient, (iii) a pH gradient, or a combination of (ii) and (iii).

[0099] Buffer systems (including binding buffers and elution buffers suitable for separating various types of target entities (e.g., monoclonal antibodies) are well known in the art and can be readily selected by those skilled in the art. According to non-limiting examples applicable to the chromatographic materials currently disclosed, binding of the target entity for monoclonal antibodies and optionally, binding of impurities can occur at a salt concentration of 0 mM to 200 mM NaCl within a pH range of about 4.5–6.5. During the binding step, the pH and salt concentration are generally kept constant. Furthermore, elution of the target entity and optionally, elution of impurities can be carried out at a salt concentration of 200 mM–750 mM NaCl within a pH range of about 5.5–7.5, more particularly by applying a constant pH or a pH gradient within a pH range of about 5.5–7.5, and by applying a constant salt concentration or a salt gradient within a salt concentration range of 200 mM–750 mM NaCl.

[0100] It should be understood that the term "gradient" used in the context of elution conditions includes both continuous gradients and step gradients. Continuous gradients can be linear or nonlinear, or a combination thereof.

[0101] This disclosure also provides an alternative method for separating one or more target entities from impurities, such as Figure 2 As shown, the method 200 includes: A liquid sample containing one or more target entities and impurities is added (210) to a chromatographic material as disclosed herein; (220) Target entities were obtained by circulation mode, which had passed through the chromatographic material and were essentially not bound to the chromatographic material; (230) Impurities can be eluted from the chromatographic material.

[0102] According to method 200, the target entity is substantially not bound to the chromatographic material, while at least one or more impurities are substantially bound to the chromatographic material.

[0103] The above-described flow method 200 may be particularly suitable when using chromatographic materials containing a carrier in membrane form (e.g., Fibro), as described in further detail above.

[0104] like Figure 1 and Figure 2 As shown, the above-mentioned method for separating target entities from impurities is based on multi-mode interactions between chromatographic ligands and molecules or particles present in liquid samples, namely electrostatic interactions, hydrophobic interactions, hydrogen bonds, etc.

[0105] In the context of the method, one or more target entities can be one or more antibodies, as described in detail elsewhere herein. Preferably, the antibody is a monoclonal antibody. Alternatively, the monoclonal antibody is a multispecific monoclonal antibody, such as a bispecific monoclonal antibody. Alternatively, one or more target entities can be one or more antibody fragments. Optionally, one or more antibody fragments can be selected from antigen-binding fragments described and illustrated in detail elsewhere herein, such as Fab, Fab', F(ab')2, scFv, Fv, dAb, or Fd.

[0106] Furthermore, in the context of the method, the impurity may comprise aggregates of one or more target entities, such as high-molecular-weight aggregates of target entities, as described in detail elsewhere herein. For example, when the target entity is an antibody, the impurity may comprise aggregates of said antibody, such as high-molecular-weight aggregates of one or more antibody fragments. Alternatively, when the target entity is an antibody fragment, the impurity may comprise aggregates of one or more antibody fragments, such as high-molecular-weight aggregates of one or more antibody fragments. Additionally, when the target entity is a bispecific antibody, the impurity may comprise one or more mismatched entities of said bispecific antibody.

[0107] The method disclosed above for separating the target entity from impurities may include step 102 before step 110 or step 202 before step 210, wherein step 102 / 202 includes pretreatment of the liquid sample. Optionally, the pretreatment may include cell lysis, clarification, and / or filtration of the cell culture harvest containing the target entity.

[0108] The methods disclosed above for separating target entities from impurities may include step 104 before step 110 or step 204 before step 210, wherein step 104 / 204 includes prepurifying one or more target entities by separating the target entity from a cell culture harvest containing the target entity, thereby obtaining a prepurified liquid sample containing the target entity, and then adding the prepurified liquid sample containing the target entity to the chromatographic material disclosed herein. Optionally, the prepurification may include subjecting the cell culture harvest containing the target entity to chromatography, or subjecting it to clarification and then to chromatography. According to a non-limiting example, the prepurification step 104 / 204 may include applying affinity chromatography. For example, a protein A-based affinity resin may be used to purify monoclonal antibodies. Alternatively, the prepurification step may include using magnetic beads, bulk materials, or membranes. Affinity chromatography may be performed using periodic countercurrent (PCC) chromatography.

[0109] The methods disclosed above for separating a target entity from impurities may include one or more purification steps 140 performed after the step of eluting (120) the target entity, or one or more purification steps 240 performed after the step of obtaining (220) the target entity from the currently disclosed multimode chromatographic material. Non-limiting examples of such one or more purification steps 140 may include hydrophobic interaction chromatography, chromatography using Capto Core beads (Cytiva, Sweden), additional orthogonal ion exchange steps, such as anion exchange chromatography steps and / or multimode anion exchange chromatography.

[0110] This disclosure also provides a chromatographic apparatus comprising chromatographic materials as described in detail elsewhere herein. Definitions of the terms, examples, and general descriptions of target entities, support materials, and multimodal ligands provided elsewhere herein also apply to this aspect of the disclosure.

[0111] Chromatographic apparatus may be a chromatographic column. A chromatographic column may contain chromatographic material with a support, which may include porous particles, solid media, expanded bed media, or non-porous particles, such as polystyrene beads or glass beads.

[0112] Alternatively, the chromatographic apparatus can be a membrane adsorbent unit, such as a capsule or cartridge. The membrane adsorbent unit may contain chromatographic material with a support comprising a convective flow material, such as a porous adsorption membrane or a fibrous material.

[0113] Alternatively, the chromatographic apparatus may be a container containing non-porous particles such as solid glass beads or porous particles such as magnetic beads.

[0114] This disclosure also provides the use of the above-described chromatographic apparatus for separating target entities from impurities. Similarly, the definitions of terms, examples, and general descriptions of target entities, support materials, and multimode ligands as provided elsewhere herein also apply to this aspect of the disclosure.

[0115] As is well known in the art, a chromatographic cycle typically includes the following steps: equilibration of the chromatographic material, loading of a sample containing the target entity and impurities, washing of the chromatographic material, elution of the target entity (unless obtained in flow during the loading step), in-situ cleaning (CIP) of the chromatographic material, and reequilibration of the chromatographic material. It should be understood that the chromatographic material is typically present in the chromatographic apparatus throughout all steps of the chromatographic cycle.

[0116] 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 relating to this invention. Furthermore, unless otherwise stated, the singular forms “a,” “an,” and “the” are intended to include plural references.

[0117] All publications, patent applications, patents and other references mentioned in this article are incorporated herein by reference in their entirety.

[0118] Experimental Section Example 1 introduce This embodiment demonstrates experimental evidence of successful separation of monoclonal antibodies from high molecular weight (HMW) aggregates using the novel ligands disclosed herein. In short, the loading contained 92% pure monomeric monoclonal antibody (mAb) and 2% high molecular weight (HMW) aggregates. Separation of the mAb and HMW aggregates on the chromatographic material was performed using a binding and elution method. Binding was carried out under favorable binding conditions of 50 mM pH 5.5 acetate, and elution was performed by linearly increasing the NaCl concentration from zero to 400 ml NaCl, using 50 mM pH 5.5 acetate buffer as the elution buffer. In linear salt gradient elution, the mAb was eluted first, followed by the HMW aggregates (largest in size). The later the aggregates eluted in the linear gradient elution, the stronger their binding affinity to the ligand.

[0119] Materials and methods Liquid samples containing a pre-purified monoclonal antibody (called mAb1) on a protein A column were used in the experiments. Capto MMC ImpRes multimode resin and Capto ImpRes base matrix were obtained from Cytiva (Uppsala, Sweden). Chemicals including D,L-homocysteine ​​thiolactone hydrochloride were obtained from Acros Organics, and acyl and sulfonyl chlorides, bromine, dichloromethane, and ethyl acetate were obtained from Sigma Aldrich. Sodium chloride and sodium dihydrogen phosphate were obtained from VWR (Randor, USA). All other chemicals used (including acetic acid, sodium acetate trihydrate, and disodium hydrogen phosphate) were of analytical grade and purchased from Merck (Darmstadt, Germany).

[0120] Synthesis of Capto MMC ligand prototype The currently disclosed multimode ligand prototypes are synthesized as follows: L25 (Taurine-L07): First, N-cyclopropionyl-D,L-homocysteine ​​thiolactone (L07-t) was prepared as follows. D,L-homocysteine ​​thiolactone hydrochloride (20 mmol, 3.097 g) and diisopropylethylamine (41 mmol, 7.213 mL) were dissolved in dichloromethane (DCM) (40 mL), and the mixture was cooled in an ice bath. Cyclopropane carboxyl chloride (20 mmol, 1.852 mL) was slowly added to the stirred solution, and the mixture was warmed to room temperature overnight. Aqueous post-treatment yielded a product as a white solid.1 ¹H NMR (CDCl₃, 300 MHz): δ = 6.12 (s, ¹H, amide- H ), 4.52(m, 1H, thiolactone- H ), 3.45-3.20 (m, 2H, thiolactone- H ), 2.96 (m, 1H, thiolactone- H ), 1.94 (m, 1H, thiolactone- H ), 1.45 (m, 1H, alph- H ), 0.98 (m, 2H, alph- H ), 0.78(m, 2H, alph- H ).

[0121] In a 50 mL pear-shaped flask, with the aid of a warm water bath, N Cyclopropionyl-D,L-homocysteine ​​thiolactone (L07-t, 5 mmol, 0.926 g) was dissolved in a mixture of acetonitrile (10 mL) and triethylamine (2.5 mL). Taurine (1.250 g, 10 mmol, 2 equivalents) was dissolved in a mixture of deionized water (10 mL) and triethylamine (2.5 mL) using a cold water bath. The taurine solution was added dropwise to the thiolactone solution with stirring. The homogeneous reaction mixture was bubbled with N2 and stirred at room temperature. After 3.5 hours, the reaction was completed by thin-layer chromatography (TLC, with a 1:1 EtOAc / cyclohexane silica plate). The reaction mixture was concentrated on a rotary evaporator (rotovap) to a turbid white viscous oil. The residue was washed with acetonitrile (5 × 10 mL), the solution was decanted from the colorless solid, and the combined washes were filtered. A solution of KPF6 in MeCN (approximately 1.2 g in 15 mL) was added with stirring, immediately yielding a white precipitate (ppt). The ppt was collected by filtration, washed with acetonitrile (MeCN, methyl cyanide), and rapidly transferred to a round-bottom flask for drying under vacuum. A white solid potassium salt (1.338 g, 76.8%) was obtained and purified by NMR. 1 H NMR (300 MHz, D2O) δ 4.39 (m, 1H, C H ), 3.52 (t,2H, NH-C H 2-CH2-SO3), 3.01 (t, 2H, NH-CH2-C H 2-SO3), 2.63-2.45 (m, 2H), 1.97 (m, 2H), 1.62 (m, 1H, cyclopropyl C)H ), 0.79 (m, 4H, cyclopropyl C) H 2). 13 C NMR (75 MHz, D2O) δ 177.75, 173.85, 52.71, 49.50, 35.10, 34.89, 20.15, 13.82, 7.16, 7.04.

[0122] L26 (Taurine-L05): First, prepare as follows... N -Benzenesulfonyl-D,L-homocysteine ​​thiolactone (L05-t). D,L-homocysteine ​​thiolactone hydrochloride (16 mmol, 2.478 g) and diisopropylethylamine (32.8 mmol, 5.771 mL) were dissolved in DCM (40 mL), and the mixture was cooled in an ice bath. Benzenesulfonyl chloride (16 mmol, 2.063 mL) was slowly added to the stirred solution. The reaction was allowed to warm to room temperature. Aqueous post-treatment gave the product as a white solid. 1 HNMR (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 ).

[0123] Will NBenzenesulfonyl homocysteine ​​thiolactone (L05-t, 0.515 g, 2 mmol) was dissolved in MeCN (4 mL) and triethylamine (1.5 mL). A solution of taurine (0.501 g, 4 mmol, 2 equivalents) in deionized water (DIW) (4 mL) and triethylamine (0.5 mL) was added to this solution. The homogeneous reaction mixture was bubbled with N2 and stirred at room temperature. After 3.5 hours, the reaction was completed by TLC (with a silicon plate having a 1:1 EtOAc / cyclohexane ratio). The reaction mixture was concentrated in a pear-shaped flask on a rotary evaporator to a cloudy white viscous oil. The residue was washed with acetonitrile (5 × 10 mL), the solution was decanted from the colorless solid (excess taurine), and the combined washes were filtered. A solution of potassium hexafluorophosphate (KPF6) in MeCN (approximately 1.2 g in 15 mL) was added with stirring, immediately yielding a turbid mixture from which a viscous oil precipitated. This was washed with MeCN and dried under vacuum to give a beige hygroscopic solid (0.425 g, 51%). The purity of the product was confirmed by NMR. 1 H NMR (300 MHz, D2O) δ 7.73 (m, 2H), 7.61 (m, 1H), 7.50 (m, 2H), 3.84 (m,1H), 3.16 (t, 2H), 2.67 (t, 2H), 2.40 (m, 1H), 2.32 (m, 1H), 1.75 (m, 2H). 13 CNMR (75 MHz, D2O) δ 172.50, 137.97, 133.84, 129.52, 126.88, 54.92, 49.23, 35.88, 34.82, 19.74.

[0124] L32 (Taurine-L09): First, prepare as follows... N 1,4-Benzodioxanesulfonyl-D,L-homocysteine ​​thiolactone (L09-t). D,L-homocysteine ​​thiolactone hydrochloride (15 mmol, 2.323 g) and diisopropylethylamine (30 mmol, 5.28 mL) were dissolved in DCM (25 mL), and the mixture was cooled in an ice bath. 1,4-Benzodioxane-6-sulfonyl chloride (15 mmol, 3.705 g) was slowly added to the stirred solution, and the reaction mixture was warmed to room temperature overnight. Aqueous post-treatment gave the product as a white solid. 1H NMR (CDCl3, 300 MHz): δ = 7.45-7.30 (m, 2H), 6.94 (d, 2H), 5.11 (s, 1H), 4.30 (m, 4H), 3.69 (m, 1H), 3.25 (m, 2H), 2.86(m, 1H), 2.05 (m, 1H).

[0125] Taurine (504 mg, 4 mmol) was dissolved in a DIW (4 mL) containing triethylamine (0.5 mL). This was then added to... N Benzadioxanesulfonyl-homocysteine ​​thiolactone (L09-t, 633 mg, 2 mmol) was dissolved in a solution of MeCN (4 mL) containing triethylamine (1.5 mL). It was bubbled with N2, and an additional 1 mL of MeCN was added. The mixture was maintained in a warm bath (35°C) for approximately 10 minutes to ensure homogeneity. It was then capped and stirred at room temperature under N2. After 3.5 hours, no SM was observed by TLC (1:1 CyH / EtOAc). The mixture was concentrated on a rotary evaporator, and the residue was washed with MeCN (5 × 10 mL) and decanted from a white solid. The combined washes were filtered through a P4 glass frit. The addition of excess KPF6 (0.8 g, in 12 mL MeCN) immediately formed a white precipitate. This was collected on the glass frit and dried under a stream of dry air. 0.54 g of white solid (56% yield) was given, which was purified by NMR. 1 H NMR (300 MHz, D2O) δ 7.23-7.21 (m, 2H), 6.95 (m, 1H), 4.24 (m, 4H, OC H 2- ), 3.82 (m, 1H), 3.23 (t, 2H), 2.72 (t, 2H), 2.50-2.42 (m, 1H), 2.35-2.26 (m, 1H), 1.74 (m, 2H). 13 C NMR (75MHz, D2O) δ 172.64, 147.82, 143.30, 130.40, 120.93, 118.09, 116.36, 64.73,64.36, 54.86, 49.32, 35.92, 34.90, 19.79.

[0126] L34 (Taurine-L06): First, prepare as follows ND,L-Butyrylhomocysteine ​​Thiolactone (L06-t). D,L-homocysteine ​​thiolactone hydrochloride (20 mmol, 3.097 g) and diisopropylethylamine (41 mmol, 7.213 mL) were dissolved in DCM (40 mL), and the mixture was cooled in an ice bath. Butyryl chloride (20 mmol, 2.10 mL) was slowly added to the stirred solution, and the reaction mixture was warmed to room temperature overnight. Aqueous post-treatment yielded a product as a white solid. 1 HNMR (CDCl3, 300 MHz): δ = 5.96 (s, 1H), 4.52 (m, 1H), 3.45-3.20 (m, 2H), 2.96(m, 1H), 2.23 (t, 2H), 1.94 (m, 1H), 1.66 (m, 2H), 0.97 (t, 3H).

[0127] Will N Butyrylhomocysteine ​​thiolactone (L06-t, 0.946 g, 5 mmol) was dissolved in 10 mL MeCN containing 4 mL triethylamine. A solution of taurine (1.261 g, 10 mmol, 2 equivalents) in 10 mL DIW and 1 mL triethylamine was added. The mixture was bubbled with N2 and then stirred at room temperature under N2 for 3.5 hours. The starting material was consumed by TLC. The reaction mixture was then... In a vacuum Concentrate the solution and wash the residue with MeCN (6 × 20 mL). Filter the combined MeCN washes through a P.4 glass frit filter and add excess KPF6 to the MeCN (20 mL). Filter the emulsion suspension through a P.3 glass frit filter and wash with 3 × 2 mL MeCN. Dry the solid under a stream of dry air for approximately 15 minutes to give 1.17 g of white solid. The solid was present in the supernatant and stored overnight in a refrigerator. Reduce the volume from approximately 100 mL to 60 mL under N2 and filter to give an additional 0.121 g of white solid. 74% yield. 1 H NMR (300 MHz, D2O) δ 4.40 (m, 1H), 3.54(m, 2H), 3.02 (t, 2H), 2.64-2.56 (m, 1H), 2.52-2.43 (m, 1H), 2.23 (t, 2H),1.97 (m, 2H), 1.56 (m, 2H), 0.85 (t, 3H). 13C NMR (75 MHz, DO) δ 177.53, 177.41, 173.66, 52.45, 49.51, 37.35, 35.12, 34.76, 20.18, 18.86, 12.78.

[0128] L33 (Asp-L09): L09 thiolactone (L09-t, 0.787 g, 2.5 mmol, prepared as described above) was dissolved in MeCN (5 mL) containing triethylamine (TEA) (1 mL). A solution of aspartic acid (1.005 g, 7.5 mmol, 3 equivalents) in DIW (5 mL) and TEA (1.5 mL) was added. The mixture was bubbled with N2. Additional MeCN was added while gently heating in a warm water bath until all ppt dissolved. The reaction mixture (RM) was stirred at 45°C under N2. After 20 hours, the RM was concentrated on a rotary evaporator, and the residue was then suspended in an aqueous NaOH solution (30 mL) (pH > 11). It was washed with DCM (3 × 20 mL). The combined DCM washes were washed with an aqueous NaOH solution. The combined aqueous phases were acidified with an aqueous HCl solution to pH < 2–3, yielding a turbid suspension. The aqueous mixture was then washed with EtOAc (5 × 30 mL). The combined EtOAc wash solution was dried with brine, then dried with MgSO4, and then... In a vacuum The solvent was removed. A white solid (1.106 g, 98% yield) was obtained as a mixture of diastereomers. 1 ¹H NMR (300 MHz, D₂O in the presence of Na₂CO₃) δ 7.24–7.16 (m, 2H), 6.91 (m, 1H), 4.22 (br, 4H), 4.03 (m, 1H), 3.66 (m, 1H), 3.47 (m, 1H, minor diastereomer), 2.33–2.19 (m, 3H, overlap), 2.16–2.09 (m, 1H), 1.68 (m, 2H). 13C NMR (75 MHz, D2O in the presence of Na2CO3) δ 178.54, 178.49, 177.81, 177.72, 174.66, 173.76, 173.63, 164.32, 147.49, 147.05, 146.93, 143.13, 143.11, 132.46, 132.22, 130.74, 120.76, 120.58, 120.43, 117.88, 116.22, 116.02, 115.92, 64.66, 64.34, 64.30, 61.65, 57.91, 57.34, 56.86, 56.73, 52.61, 52.44, 39.77, 39.68, 38.76, 23.20, 20.50, 20.45, 20.35, 16.71, 13.16.

[0129] The ligands are coupled to a functionalized agarose matrix (Capto ImpRes, Cytiva). In short, the agarose matrix is ​​first terminally functionalized with allyl glycidyl ether (AGE). The allyl-functionalized resin is then brominated with elemental bromine and subsequently treated with the ligand solution at high pH. As determined by titration, the multimode ligand density is typically 20 µmol / mL–35 µmol / mL. 树脂 .

[0130] linear salt gradient elution study The pre-purified mAb sample buffer on the protein A column was exchanged for binding buffer 50 mM acetate pH 5.5. The purification step was performed using a Tricorn 5 / 200 column with a 6-minute residence time. Column runs were performed on an ÄKTA pure 25 system. mAb samples were loaded onto the column, packed with resin containing ligand L33 to achieve 50% dynamic binding capacity, and then washed with binding buffer. Elution was performed from 0–100% elution buffer in a 30-column-volume salt gradient. The elution buffer consisted of 50 mM acetate pH 5.5 and 400 mM sodium chloride. The eluted fractions were collected and the protein concentration and mAb1 and HMW aggregate content (%) were analyzed.

[0131] Combined salt and pH gradient studies The pre-purified mAb sample buffer on the protein A column was exchanged for binding buffer 50 mM acetate at pH 5.5 and diluted to approximately 2 mg / mL. The purification step was performed using a Tricorn 5 / 200 column with a residence time of 6 minutes. Column runs were performed on an ÄKTA pure 25 system. The mAb sample was loaded onto the column, packed with resin containing ligand L33 to a 50% dynamic binding capacity, and then washed with binding buffer. Elution was performed from 0–100% in elution buffer B at 20 column volumes of combined salts and pH gradients. Elution buffer A consisted of 25 mM sodium acetate, 25 mM sodium dihydrogen phosphate (pH 5.5), and 50 mM sodium chloride, while elution buffer B consisted of 25 mM sodium acetate, 25 mM sodium dihydrogen phosphate (pH 7.5), and 400 mM sodium chloride. The fractions from the elution were collected and the protein concentration and the percentage of mAb1 and HMW aggregates were analyzed.

[0132] Each chromatographic experiment is performed in five different steps, including equilibration, sample loading, column washing, elution, in-situ cleaning (CIP), and reequilibration.

[0133] Results and discussion By using a resin with the same properties as the reference Capto MMC ImpRes resin (25 µmol / mL-39 µmol / mL) 树脂 Similar ion capacities (20 µmol / mL - 70 µmol / mL) 树脂 All prototypes of new ligands were prepared to minimize the effect of ligand density, as determined by ion capacity measurements, on chromatographic performance.

[0134] linear salt gradient elution study In a linear salt gradient, the novel multimode ligands L32 and L33 outperformed the reference MMC ligand in aggregate removal. Compared to sample loading, L32 and L33 provided 90% and 95% aggregate reductions, respectively, while MMC provided 75% aggregate reduction. The experimental conditions for L32 and L33 were as described above. The experimental conditions for MMC were the same except for the binding and elution buffers. The binding buffer was 50 mM acetate with 50 mM sodium chloride at pH 5.5, and the elution buffer was 50 mM phosphate with 750 mM sodium chloride at pH 6.

[0135] Figure 3 The cumulative monomer yields of L32, L33, and MMC relative to the aggregate yields are shown respectively.

[0136] in conclusion These results indicate that, compared to the reference MMC ligand, the new multimodal ligand prototype can achieve efficient removal of aggregate material from challenging monoclonal antibodies and provide improved and different selectivity for product-related impurities.

[0137] Example 2 The experimental design was performed using the same antibody as in Example 1, with the following changes: a) In ligand chemistry: i. Formula I, where NL-X2 = Asp, X1 = CO, and R1 = naphthalene or benzodioxane; ii. Formula I, where NL-X2 = Gly-Gly, X1 = SO2, and R1 = benzodioxane; iii. Formula I, where X2 = tetrazolium, X1 = SO2, and R1 = benzodioxane.

[0138] b) Regarding the carrier: i. Ligands coupled to synthetic matrix material particles, such as Source TM 30 (Cytiva, Sweden); ii. Ligands coupled to porous adsorption membranes, such as Fibro or Mustang; iii. Ligands coupled to magnetic beads.

[0139] c) Regarding impurities: i. Determine the affinity of different ligands for different polymeric forms and fragments of antibodies.

[0140] d) Regarding chromatographic conditions: i. Combined salt and pH gradient.

[0141] Example 3 Experiments were designed as in Example 1 using other target entities (e.g., bispecific antibodies) different from those in Example 1.

[0142] It should be understood that this disclosure is not limited to the exemplary embodiments described above, and several conceivable modifications may be made to this disclosure within the scope of the appended claims. Any reference numerals enclosed in parentheses in the claims should not be construed as limiting the claims.

[0143] References Kanwar Shekhawat L. et al., Journal of Chromatography A, 1699 (2023)464018 WO2020023566A1 US20140296464A1 US20160288089A1 US2019308169A1 US2019234914A1

Claims

1. A chromatographic ligand defined by Formula I: in: X1 is selected from CO and SO2; R1 is selected from (C1-C7)alkyl, cycloalkyl, aryl, heterocyclic, (C1-C7)alkyl-X2, cycloalkyl-X2 and aryl-X2; X2 is selected from CO2H, SO3H, PO3H2 and heterocycles with pKa < 6; R2 is selected from H, (C1-C7)alkyl, and (C1-C7)alkyl-X3; X3 is selected from CO2H, SO3H and PO3H2; L is selected from (C1-C7) alkyl and (C1-C2) alkyl-X4-(C1-C2) alkyl, and X4 is selected from O, S, NH(CO), (CO)NH, NH(SO2) and (SO2)NH; Optionally, L is replaced by a straight-chain or branched (C1-C7) alkyl group. Optionally, the straight-chain or branched (C1-C7) alkyl group is replaced by X5, wherein X5 is selected from CO2H, SO3H, PO3H2, O-(C1-C2)alkyl, S-(C1-C2)alkyl, OH and (CO)NH2.

2. The chromatographic ligand according to claim 1, wherein X2 is SO3H and L is a C2 alkyl group. Optionally, R2 is H.

3. The chromatographic ligand according to claim 1, wherein X2 is CO2H, R2 is H, and L is a C1 alkyl group that is optionally substituted with a straight-chain or branched (C1-C7) alkyl group that is replaced by X5.

4. The chromatographic ligand according to claim 1, when R1 is an aryl group, is defined by formula II: (II) in: Each of R3-R7 is independently selected from H, F, Cl, NH2, (C1-C3)alkyl, CO2H, SO3H and PO3H2 and X6-(C1-C3)alkyl; Any two adjacent portions selected from R3-R7, together with the atoms they are attached to, can form a 5- or 6-membered heterocycle or a carbocyclic ring; and X6 is selected from O, S, NH, N-(C1-C3)alkyl, NH(CO), (CO)NH, NH(SO2) and (SO2)NH.

5. The chromatographic ligand according to claim 1, wherein the formula is defined as consisting of the group selected from (a)-(e): and 。 6. A method for preparing chromatographic materials, comprising immobilizing a plurality of ligands according to any one of claims 1 to 5 to a support, wherein the support comprises porous particles, non-porous particles or an expanded bed medium, or a convective flow material, such as a porous adsorption membrane, a fibrous material or a monolithic material.

7. The method of claim 6, wherein the carrier comprises porous particles having a diameter of about 10 µm to about 100 µm, preferably about 25 µm to about 60 µm.

8. The method according to claim 6 or 7, wherein the density of the plurality of ligands immobilized on the carrier is from about 15 µmol / mL to about 100 µmol / mL, preferably from about 20 µmol / mL to about 50 µmol / mL.

9. A chromatographic material comprising a chromatographic ligand coupled to a carrier according to any one of claims 1 to 5, wherein the carrier comprises porous particles, non-porous particles or an expanded bed medium, or a convective flow material, such as a porous adsorption membrane, a fibrous material or a monolithic material. Optionally, the carrier comprises porous particles with a diameter of about 10 µm to about 100 µm, preferably about 25 µm to about 60 µm.

10. The chromatographic material according to claim 9, wherein the density of the plurality of ligands coupled to the carrier is from about 15 µmol / mL to about 100 µmol / mL, preferably from about 20 µmol / mL to about 50 µmol / mL.

11. Use of the chromatographic material according to claim 9 or 10 for separating one or more target entities from impurities.

12. The use according to claim 11, wherein the one or more target entities comprise one or more antibodies and / or portions of antibodies, preferably wherein the antibody is a monoclonal antibody, optionally wherein the monoclonal antibody is a multispecific monoclonal antibody, optionally wherein the multispecific antibody is a bispecific antibody, and the impurity comprises a mismatch entity of the bispecific antibody, such as a homodimer of the bispecific antibody.

13. The use according to claim 12, wherein the impurity comprises an aggregate of one or more antibodies.

14. The use according to claim 11 or 12, wherein the one or more target entities are one or more antibody fragments, optionally wherein the one or more antibody fragments are selected from antigen-binding fragments, such as Fab, Fab', F(ab')2, scFv, Fv, dAb or Fd.

15. The use according to claim 14, wherein the impurity comprises an aggregate of the one or more antibody fragments.

16. The use according to any one of claims 11 to 15, wherein the impurity comprises a host cell protein.

17. A method (100) for separating one or more target entities from impurities, comprising: A liquid sample containing one or more target entities and impurities is added (110) to the chromatographic material according to any one of claims 9 to 10; The target entity is eluted from the chromatographic material (120); The impurities can be eluted (130) from the chromatographic material.

18. The method (100) of claim 17, wherein the target entity and optionally the impurities are eluted from the chromatographic material by using an elution buffer containing salt and by applying (i) a constant pH and / or salt concentration, (ii) a salt gradient, (iii) a pH gradient, or a combination of (ii) and (iii) during the elution step.

19. A method (200) for separating one or more target entities from impurities, comprising: a. Adding (210) a liquid sample containing one or more target entities and impurities to the chromatographic material according to any one of claims 9 to 10; b. Obtain the target entity (220) in a flow-through mode, wherein the target entity has passed through the chromatographic material and is substantially not bound to the chromatographic material; c. Optionally, the impurities (230) can be eluted from the chromatographic material.

20. The method of any one of claims 17 to 19, wherein the one or more target entities comprise one or more antibodies and / or portions of antibodies, preferably wherein the antibody is a monoclonal antibody, optionally wherein the monoclonal antibody is a multispecific monoclonal antibody, optionally wherein the multispecific antibody is a bispecific antibody, and the impurity comprises a mismatch entity of the bispecific antibody, such as a homodimer of the bispecific antibody.

21. The method of claim 20, wherein the impurity comprises an aggregate of one or more antibodies.

22. The method according to any one of claims 17 to 20, wherein the one or more target entities are one or more antibody fragments, optionally wherein the one or more antibody fragments are selected from antigen-binding fragments, such as Fab, Fab', F(ab')2, scFv, Fv, dAb, or Fd.

23. The method of claim 22, wherein the impurity comprises an aggregate of the one or more antibody fragments.

24. The method according to any one of claims 17 to 23, wherein the impurity comprises a host cell protein.

25. A chromatographic apparatus comprising the chromatographic material according to claim 9 or 10, optionally wherein the chromatographic apparatus is disposable. Optionally, the chromatographic apparatus is selected from: a. A chromatographic column, optionally comprising chromatographic material with a support, said support comprising porous particles, a bulk medium, an expanded bed medium, or non-porous particles; b. A membrane adsorbent unit, optionally comprising a chromatographic material having a support, said support comprising a convective flow material, such as a porous adsorption membrane or a fibrous material; and c. A container containing non-porous particles such as solid glass beads or porous particles such as magnetic beads.

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