Chromatographic ligands and chromatographic materials and uses thereof

Abstract

The present disclosure provides a chromatographic ligand defined by the following Formula I: # imgabs0 # wherein: X1 is selected from the group consisting of CO and SO2; each of R1-R5 is independently selected from the group consisting of H, F, Cl, O, N, S, C1-3 alkyl, and C1-3 alkyl-X2; any two adjacent moieties selected from R1-R5, together with the atom to which they are attached, can form a 5-or 6-membered heterocyclic or carbocyclic ring; and X2 is selected from the group consisting of O, S, NH (CO), (CO) NH, NH (SO2), and (SO2) NH; and 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 ring containing a 5-membered heterocyclic ring, which contains two oxygen atoms in the ring, X1 is SO2; and iii. When three of R1-R5 are CH3O, X1 is CO, or a pharmaceutically acceptable salt thereof. The present disclosure also relates to chromatography materials comprising a support and a chromatography ligand coupled to the support as disclosed herein. Further disclosed is the use of the chromatographic material for separating one or more target molecules from impurities, as well as a method for separating one or more target molecules from impurities.

Description

Technical Field

[0001] The present disclosure relates to chromatographic ligands and chromatographic 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 present disclosure relates to the separation of impurities of target molecules such as antibodies or antibody fragments from aggregates containing one or more target molecules. Background Art

[0002] Biomacromolecules, such as proteins, nucleic acids, and polysaccharides, often occur partially in the form of aggregates or polymers, such as dimers, trimers, or higher oligomers. In the field of biological production 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 that are completely different from those in its natural environment, the conditions may be favorable for 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 the bioprocess development and manufacture of biotherapeutic drugs. The aggregated form of the macromolecule may have lower biological activity than the non-aggregated form of the macromolecule; it may even completely lack the desired biological activity or may cause undesirable side effects. Therefore, it is essential for therapeutic safety that the therapeutic protein is in a non-aggregated state and that there are no molecular aggregates in the final product.

[0003] Preparative chromatography remains the main technology for purifying therapeutic proteins due to its benefits of resolution, scalability and robustness. Monoclonal antibodies are one of the most powerful therapeutic tools for curing more and more diseases. The continued development of upstream processing has led to increased purification complexity in terms of impurity types and contents, for example due to higher titers. In addition, the heterogeneity of monoclonal antibodies, in its life cycle from cell culture to purification, varies in its charge distribution and size due to various molecular modifications, which increases the challenges of the purification step. 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, etc., resulting in the formation of additional product-related impurities. Due to the increased molecular diversity of multispecific antibodies, the biopharmaceutical industry is also facing new purification challenges, in which new and difficult product-related impurities require more powerful purification steps to obtain the desired product quality. However, with the continuous increase of these challenges, key quality attributes remain high, which increases the requirements for high resolution and purification of chromatographic media.

[0004] Multimodal (or mixed-mode) chromatography, in which small molecule ligands provide more than one type of interaction, is an important tool for downstream processing of therapeutic proteins. A commercial example of multimodal chromatography is CaptoTM MMC ImpRes (Cytiva Sweden AB, Uppsala, Sweden). It is a weak cation exchange multimodal ligand that can achieve high selectivity over a wider pH and salt window compared to traditional ion exchangers. It effectively removes aggregates, viruses, and major contaminants during monoclonal antibody purification and is suitable for the purification of antibody fragments.

[0005] However, there continues to be a need in the art for alternative chromatography ligands and their use in methods to improve the separation of target molecules from impurities such as aggregates, such as high molecular weight aggregates of the target molecules. Summary of the invention

[0006] The above objectives of providing alternative chromatography ligands and methods to improve the separation of target molecules from impurities are achieved by the present disclosure, which relates to novel chromatography ligands and uses thereof.

[0007] More particularly, the presently disclosed chromatographic ligands are defined by the following formula I:

[0008]

[0009] in:

[0010] X1 is selected from CO and SO2;

[0011] Each of R1-R5 is independently selected from H, F, Cl, O, N, S, C 1-3 Alkyl and C 1-3 Alkyl-X2;

[0012] Any two adjacent moieties selected from R1 to R5 together with the atoms to which they are attached may form a 5- or 6-membered heterocyclic or carbocyclic ring; and

[0013] X2 is selected from O, S, NH(CO), (CO)NH, NH(SO2) and (SO2)NH; and

[0014] The conditions are:

[0015] i. When each of R1-R5 is H, X1 is SO2;

[0016] 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 two oxygen atoms in the ring, X1 is SO2; and

[0017] iii. When three of R1-R5 are CH3O, X1 is CO.

[0018] The present disclosure further provides a method for preparing a chromatography material, comprising immobilizing a plurality of the chromatography ligands defined above to a support.

[0019] There is also provided a chromatographic material comprising a chromatographic ligand as defined above coupled to a support.

[0020] Further provided is the use of a chromatography material disclosed herein for separating one or more target molecules from impurities.

[0021] The present disclosure also provides a method for separating one or more target molecules from impurities, comprising:

[0022] a) adding a liquid sample comprising one or more target molecules and impurities to a chromatography material as disclosed herein;

[0023] b) eluting the target molecule from the chromatography material;

[0024] c) optionally eluting impurities from the chromatography material.

[0025] Also provided is a method for separating one or more target molecules from impurities, comprising:

[0026] a) adding a liquid sample comprising one or more target molecules and impurities to a chromatography material as disclosed herein;

[0027] b) obtaining the target molecule in a flow-through mode, wherein the target molecule passes through the chromatographic material without substantially binding to the chromatographic material;

[0028] c) optionally eluting impurities from the chromatography material.

[0029] Preferred aspects of the disclosure are described below in the detailed description and dependent claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a flow chart of a first method for separating one or more target molecules from impurities according to the present disclosure.

[0031] Figure 2 is a flow chart of a second alternative method for separating one or more target molecules from impurities according to the present disclosure.

[0032] Figure 3 Shown is a comparison of the resolution between HMW aggregates of different ligand prototypes and a monoclonal antibody (mAb) for data obtained using Method 1 as described in Example 1 herein.

[0033] Figure 4 Shown is a comparison of the resolution between HMW aggregates of different ligand prototypes and mAb for data obtained using Method 2 as described in Example 1 herein.

[0034] Figure 5Retention data for three novel ligand prototypes as described in Example 1 herein are shown. DETAILED DESCRIPTION

[0035] The present disclosure relates to chromatography ligands for improving the separation of one or more target molecules from impurities, wherein the impurities may include aggregates and / or fragments of the target molecules.

[0036] As described in the Examples herein, the previously known multimodal Capto MMC ImpRes ligand (Cytiva Sweden AB, Uppsala, Sweden) was selected as the starting point for creating a large chemically diverse virtual library of 100 Capto MMC-like ligand structures. Based on these structures, bioinformatic predictions of physicochemical properties were used to generate a matrix of digital descriptors (e.g., pKa, cLogP, etc.). Subsequent principal component analysis (PCA) of these ligand descriptors resulted in a large chemical diversity map for selecting ligands for synthesis and coupling to an agarose base matrix. High-throughput plate-based screening and analysis of column retention and resolution then generated digital descriptors of chromatographic separation performance that were linked together with the chemical descriptors to guide further cycles of synthesis and column resolution evaluation.

[0037] The present 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. The embodiments herein show that when using a linear salt gradient elution, compared with the separation achieved by the reference ligand, the novel ligand that is more hydrophobic than the reference ligand achieves improved separation between monoclonal antibodies and product-related impurities (i.e., aggregates and the Fab fragments of the antibody). More specifically, by using novel ligands, monoclonal antibodies are obtained with higher purity and higher yield. The secondary hydrophobic and hydrogen bonding interactions provided by the ligand chemical structure of the more hydrophobic ligand are combined with electrostatic interactions, and their prominent effect leads to their better performance in removing Fab fragments and aggregate impurities.

[0038] The present disclosure provides a chromatographic ligand defined by the following Formula I:

[0039]

[0040] in:

[0041] X1 is selected from CO and SO2;

[0042] Each of R1-R5 is independently selected from H, F, Cl, O, N, S, C 1-3 Alkyl and C 1-3 Alkyl-X2;

[0043] Any two adjacent moieties selected from R1 to R5 together with the atoms to which they are attached may form a 5- or 6-membered heterocyclic or carbocyclic ring; and

[0044] X2 is selected from O, S, NH(CO), (CO)NH, NH(SO2) and (SO2)NH; and

[0045] The conditions are:

[0046] i. When each of R1-R5 is H, X1 is SO2;

[0047] 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 two oxygen atoms in the ring, X1 is SO2; and

[0048] iii. When three of R1-R5 are CH3O, X1 is CO.

[0049] The 5- or 6-membered heterocyclic or carbocyclic ring may be unsaturated or saturated. Further, the 5- or 6-membered heterocyclic or carbocyclic ring may be non-polar, aromatic and / or aliphatic. The heterocyclic ring may contain up to three heteroatoms. When there are three heteroatoms, they are all N. When there are up to two heteroatoms, each of them may be independently selected from N, O and S.

[0050] In one embodiment, any two adjacent moieties selected from R1 to R5 together with the atoms to which they are attached may form a 5-membered heterocyclic ring containing at most one oxygen atom in the ring.

[0051] When the chromatographic ligand is coupled to a support, this can be illustrated according to the following (Formula Ia):

[0052]

[0053] The "support" portion of Formula Ia represents a support to which the ligand can be coupled, such as a chromatography bead. The ligand is linked to the support via a covalent thioether bond formed at the thiol.

[0054] Formula Ia (chromatographic ligand coupled to a support) may alternatively be described as

[0055]

[0056] The wavy part indicates the coupling to the support. Hereinafter, the term "support" is used in Formula Ia to indicate the position where the ligand can be bound to the support.

[0057] The term "chromatography ligand" means a molecule that has a known or unknown affinity for a given analyte and that can be coupled to the support of a chromatographic material, while "analyte" includes any specific binding partner for the ligand.

[0058] The target analytes to be separated according to the present disclosure are so-called target molecules and impurities present in a liquid sample.

[0059] In this context, the term "target molecule" is intended to include a macromolecule that is isolated from a liquid sample and purified from impurities prior to its intended application, e.g. as a therapeutic substance.

[0060] The term "macromolecule" has its conventional meaning in the field of bioprocessing, wherein the macromolecule is produced by cells in cell culture (often recombinant) and purified from the cell culture by any separation and purification means. Alternatively, the macromolecule is present in a biological solution, which 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 and antibody fragments, as well as carbohydrates and nucleic acid sequences, such as DNA and RNA. The macromolecules purified by using a chromatographic ligand according to the present disclosure are generally proteins or polypeptides, particularly therapeutic proteins or polypeptides, such as antibodies. Alternatively, the macromolecule may be a nucleic acid sequence, which may be used, for example, as a carrier, such as in therapeutic applications. A macromolecule or biomacromolecule may, for example, be a biopharmaceutical, i.e., a biomolecule, including but not limited to biomacromolecules intended to be used as pharmaceutical compounds. It should be understood that "macromolecule" is intended to mean a type of macromolecule, and the singular form of the term may include a large number of single macromolecules or samples of the same type.

[0061] It should be understood that the term "liquid sample" (or simply "sample") as used herein includes any type of sample obtainable from a cell culture, or from a fluid derived from a cell culture, which fluid is at least partially purified by any separation and purification means.

[0062] As used herein, the term "cell culture" refers to a culture of cells or a group of cells in culture, wherein the cells may be any type of cells, such as bacterial cells, viral cells, fungal cells, insect cells or mammalian cells. The cell culture may be unclarified, i.e., containing cells, or may be cell-depleted, i.e., a culture that contains no or only a small amount of cells before the cells are removed but contains biomolecules released from the cells. Further, the unclarified cell culture as used in the presently disclosed method may contain intact cells, disrupted cells, cell homogenates and / or cell lysates.

[0063] The term "antibody" as used herein means an immunoglobulin which may be natural or partially or completely synthetically produced. The term includes, but is not limited to, whole (intact) antibodies, such as monospecific and multispecific antibodies. The term also includes active antibody fragments, including Fab antigen-binding fragments, monovalent fragments, and bivalent fragments. The term also includes any protein having a binding domain homologous to an immunoglobulin binding domain. Such proteins may be derived from natural sources or may be partially or completely synthetically produced. The term further includes fusion proteins, which include antibodies or antibody fragments, such as monoclonal antibodies or monoclonal antibody fragments covalently linked to other proteins. Exemplary antibodies are immunoglobulin isotypes and fragments of 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) and scFv (so-called single-chain variable fragments, which are fusion proteins of immunoglobulin heavy chain and light chain variable regions), tandem scFv, BiTE (bispecific T cell engagement molecules), DART (dual affinity retargeting molecules) and diabodies (single chain and tandem diabodies). Bispecific monoclonal antibodies are an example of multispecific antibodies and are artificial proteins that can bind to two different types of antigens or two different epitopes on the same antigen at the same time. Chromatographic 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 antibodies to obtain high-quality final products. The presence of aggregates in therapeutic antibody preparations usually has a negative impact on patient safety and must be effectively removed during process manufacturing.

[0064] The term "vector" is used herein to refer to a viral particle, typically a recombinant viral particle, which is intended to be used to achieve gene transfer to modify a specific cell type or tissue. The viral particle can, for example, be engineered to provide a vector for expressing a therapeutic gene. Several viral types are currently being studied for delivering genetic material (e.g., genes) to cells to provide transient or permanent transgenic expression. These include adenovirus, retrovirus (gamma-retrovirus and lentivirus), poxvirus, adeno-associated virus (AAV), baculovirus, and herpes simplex virus.

[0065] "Viral particles" are used herein to refer to complete infectious viral particles. It includes a core, containing the genome of the virus (i.e., viral genome), in the form of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and the core is 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 nucleocapsids of some viruses are surrounded by a lipoprotein double-layer envelope. In the field of bioprocessing, in order to produce viral vectors for various applications such as therapy, the genome of the viral particles is modified to include a genetic insert containing the target genetic material. The modified viral particles are allowed to infect host cells in cell culture, and the viral particles are propagated in the host cells, and then the viral particles are purified from the cell culture by any separation and purification means.

[0066] In this article, the term "impurity" is intended to mean any molecule or substance present in a liquid sample and not a desired target molecule. The term "impurity" includes aggregates, such as aggregates of target molecules, such as high molecular weight aggregates of target molecules. The term "impurity" further includes fragments of target molecules, such as when the target molecule is a complete antibody and there are undesirable fragments of the antibody in the liquid sample. The term "impurity" also includes host cell proteins (HCPs), and in the case of bispecific antibodies, also includes homodimers.

[0067] The target molecule according to the present disclosure is generally a non-aggregating macromolecule, and the impurities from which the target molecule is to be separated generally include aggregates and / or fragments of the macromolecule, generally a protein, such as an antibody.

[0068] In this article, the term "non-aggregating macromolecule" is intended to mean a non-degradable macromolecule. Non-aggregating macromolecules may be referred to herein as "non-degradable macromolecules" or "complete macromolecules". In a typical embodiment herein, where the macromolecule is a protein or polypeptide, the non-aggregating macromolecule may be described as having a substantially complete tertiary structure, which generally involves a substantially hydrophilic surface of the macromolecule, while the hydrophobic portion is located inside the macromolecule. Therefore, the non-aggregating macromolecule has substantially no hydrophobic portion or hydrophobic group exposed on the surface.

[0069] In contrast, in proteins or polypeptides that begin to degrade, the tertiary structure is gradually destroyed, exposing the hydrophobic portion to the environment surrounding the protein or polypeptide. Protein or polypeptide macromolecules that are being degraded or have been degraded can form aggregates. The non-aggregated form of the macromolecule is in a monomeric state. The aggregates of macromolecules can contain polymeric forms of the macromolecules, such as dimers, trimers, etc. of the macromolecules. A single macromolecule that is being degraded can form aggregates with other single degradation samples of the same type of macromolecule, and / or can form aggregates with single degradation samples of other types of degraded macromolecules or combinations thereof. Since the aggregates of macromolecules contain degraded macromolecules, the aggregates of macromolecules have hydrophobic portions exposed on their surfaces.

[0070] So-called "high molecular weight (HMW) aggregates" are well-known terms to the skilled person. Such aggregates are formed by the self-association of a target molecule (e.g., a monoclonal antibody with a molecular weight of about 150 kDa) with each other through covalent and non-covalent bonding. This results in the formation of dimers (e.g., monoclonal antibody dimers are about 300 kDa) or even higher-order aggregates, such as trimers (monoclonal antibody trimers are about 450 kDa). These aggregates may be soluble or insoluble, depending on the properties of the target molecule. Therefore, the term "high molecular weight aggregate" may refer to an aggregate of a target molecule in this article, the molecular weight of which is about twice the molecular weight of the target molecule, or more than twice the molecular weight of the target molecule.

[0071] As used herein, the term "hydrophobic moiety" is intended to mean a hydrophobic portion of a macromolecule or a hydrophobic group present in a macromolecule.

[0072] The term "hydrophobic group" as used herein is defined as a group of molecules whose log P value>0. The partition coefficient, abbreviated as P, is defined as the specific ratio of the concentration of a solute between two solvents (two phases of the liquid phase), especially for unionized solutes, and the logarithm of this ratio is therefore log P. When one solvent is water and the other is a non-polar solvent, the log P value is a measure of lipophilicity or hydrophobicity. The precedent for the definition is that the lipophilic phase and the hydrophilic phase types are always located in the numerator and denominator, respectively; for example, in the two-phase system of n-octanol (hereinafter referred to as "octanol") and water:

[0073]

[0074] A log P value < 0 indicates that a higher percentage of the solute is in the hydrophilic phase. Conversely, a log P value > 0 indicates that a higher percentage of the solute is in the lipophilic phase, i.e., the hydrophobic phase.

[0075] As mentioned above, denatured macromolecules and macromolecular aggregates are generally more hydrophobic than complete, non-denatured, non-aggregating macromolecules. Therefore, the degree of binding of aggregates to the hydrophobic groups of chromatographic ligands is higher than that of non-aggregating macromolecules. The aggregates of target molecules also have a larger size than the target molecule itself, and therefore the surface area interacting with the chromatographic ligands is larger. Therefore, compared with the combination of the target molecule with the currently disclosed multi-modal ligands, the aggregates of the target molecule will show a stronger combination with the ligand. Therefore, usually the aggregates will be eluted from the chromatographic device later than the target molecule.

[0076] On the other hand, the size of the target molecule fragment is smaller than the complete target molecule, and therefore the surface for interaction with the ligand is smaller. Further, for example, the Fab fragment of a monoclonal antibody usually interacts with the multimodal ligand mainly via electrostatic interactions, while the complete monoclonal antibody usually interacts with the ligand via hydrophobic and electrostatic interactions. Therefore, the complete monoclonal antibody usually exhibits stronger binding to the currently disclosed multimodal ligand than the Fab fragment of the monoclonal antibody. Therefore, the complete antibody will usually elute from the chromatographic device later than the fragment of the complete antibody.

[0077] In summary, the use of the presently disclosed chromatographic ligands is based on exploiting the differences in binding of the ligands to non-aggregating macromolecules and aggregates and / or fragments of macromolecules, respectively.

[0078] As further described above, Formula I is:

[0079]

[0080] Formula I can be further defined by the following criteria:

[0081] X1 is selected from CO and SO2;

[0082] Each of R1-R5 is independently selected from H, F, Cl, O, N, S, C 1-3 Alkyl and C 1-3 Alkyl-X2;

[0083] Any two adjacent moieties selected from R1 to R5 together with the atoms to which they are attached may form a 5- or 6-membered heterocyclic or carbocyclic ring; and

[0084] X2 is selected from O, S, NH(CO), (CO)NH, NH(SO2) and (SO2)NH.

[0085] Formula I can be further defined by the following additional criteria:

[0086] i. When each of R1-R5 is H, X1 is SO2;

[0087] 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 two oxygen atoms in the ring, X1 is SO2; and

[0088] iii. When three of R1-R5 are CH3O, X1 is CO.

[0089] Further, the 5- or 6-membered heterocyclic or carbocyclic ring may be unsaturated or saturated. Further, the 5- or 6-membered heterocyclic or carbocyclic ring may be non-polar, aromatic and / or aliphatic. The heterocyclic ring may contain up to three heteroatoms. When there are three heteroatoms, they are all N. When there are up to two heteroatoms, each of them may be independently selected from N, O and S.

[0090] In the present context, the expression "lysozyme bulk binding capacity" is intended to mean the binding capacity of a chromatography material in batch mode for lysozyme, wherein the chromatography material comprises a chromatography ligand coupled to a support as explained elsewhere herein.

[0091] Lysozyme was one of five model proteins used in high-throughput plate-based studies to test the presently disclosed chromatography ligands when present in said material, as described in detail below in Example 1. More specifically, the binding capacity was studied by loading 60 μg of lysozyme onto 6 μL of chromatography material (i.e., support coupled to the chromatography ligand) in batch mode under binding conditions pH 7.5 and 480 mM NaCl.

[0092] 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 specific temperature and solution conditions such as pH and ionic strength.

[0093] The reason for being in the "linear part of the adsorption isotherm" is to understand the selectivity between protein and ligand under different binding conditions of pH and ionic strength. It will also make the measurement of the amount of bound protein robust to changes in the concentration of protein loaded on the chromatography material.

[0094] Protein-ligand selectivity studies for screening pH and ionic strength windows were never performed in the saturation part of the isotherm or under overload conditions, because the selectivity between protein-ligand is disturbed by competitive binding of other impurities (because it is impossible to obtain 100% pure monoclonal antibodies for studies), and protein-protein interactions occur under overload conditions, which is a characteristic of nonlinear isotherms. In this study, the maximum limit of the equilibrium binding capacity of lysozyme was 10 μg / μL chromatography material (60 μg lysozyme divided by 6 μL chromatography material). The experimentally observed percentage equilibrium binding capacity relative to the maximum equilibrium binding capacity (10 μg / μL resin) can be calculated for each resin for each binding condition. Here, those ligands that show at least 50%, such as 60%, of the maximum equilibrium binding capacity are selected, i.e., ligands that reach the amount of bound lysozyme at equilibrium with at least 50%, such as 60%, of the added amount of lysozyme.

[0095] The term "logS" has its conventional meaning in the art. LogS is directly related to the water solubility of a compound and is defined as a common solubility unit corresponding to the 10-based logarithm of the solubility of a molecule measured in mol / L. logS is a measure of hydrophobicity; the more negative the log S value, the more hydrophobic the chromatographic ligand.

[0096] The presently disclosed chromatographic ligands may also be further defined by having a logS from about -2.5 to about -5. The Log S values ​​of the chromatographic ligand structures are calculated with the support modeled as a methyl group. In other words, they are calculated for a methyl-S-ligand structure. More particularly, a chromatographic ligand is provided herein wherein when -SH of Formula I has been replaced with methyl sulfide (-S-CH3), the ligand has a logS from about -2.5 to about -5.

[0097] More particularly, the presently disclosed chromatographic ligands may be defined by a chemical structure selected from any one of (a) to (h):

[0098] a.

[0099]

[0100] b.

[0101]

[0102] c.

[0103]

[0104] d.

[0105]

[0106] e.

[0107]

[0108] f.

[0109]

[0110] g.

[0111]

[0112] and

[0113]

[0114] When the above chromatographic ligands are coupled to a support, they can be as follows:

[0115] a.

[0116]

[0117] b.

[0118]

[0119] c.

[0120]

[0121] d.

[0122]

[0123] e.

[0124]

[0125] f.

[0126]

[0127] g.

[0128]

[0129] and

[0130] h.

[0131]

[0132] According to currently preferred embodiments, the presently disclosed chromatographic ligands may be defined by a chemical structure selected from (a)-(f):

[0133] a.

[0134]

[0135] b.

[0136]

[0137] c.

[0138]

[0139] d.

[0140]

[0141] e.

[0142]

[0143] and

[0144] f.

[0145]

[0146] When the above chromatographic ligands are coupled to a support, they can be as follows:

[0147] a)

[0148] b)

[0149] c)

[0150] d)

[0151] e)

[0152] and

[0153] f)

[0154] Table 1 describes the chemical structures, predicted logS values ​​and lysozyme bulk binding capacities of the ligands (a)-(h) listed above as well as reference ligands.

[0155] Table 1. Chemical structures, predicted Log S values ​​and lysozyme bulk binding capacities of resins based on target and reference ligands. The predicted Log S values ​​are for structures with supports modeled as methyl groups. In other words, they are calculated for methyl-S-ligand structures.

[0156]

[0157] *The amount of lysozyme bound to the ligand is expressed as % of the amount of lysozyme added to the resin.

[0158] The present disclosure further provides a method for preparing a chromatography material, comprising immobilizing a plurality of the chromatography ligands defined above to a support.

[0159] The term "separation matrix" is used herein to refer to a material comprising a support to which one or more ligands comprising functional groups are coupled. The functional group of the ligand is bound to a compound to be separated from a liquid sample and / or to be separated from other compounds present in the liquid sample, also referred to herein as an analyte. The separation matrix may further comprise a compound coupling the ligand to the support. The terms "linker", "extender" and "surface extension agent" may be used to describe such compounds, as further described below. The term "resin" is sometimes used for separation matrices in the art. The terms "chromatographic material" and "chromatographic matrix" are used herein to refer to a type of separation matrix.

[0160] The term "surface" is intended herein to mean all external surfaces and, in the case of porous supports, includes external surfaces as well as pore surfaces.

[0161] The separation matrix may be comprised in any type of separation device, as further defined elsewhere herein.As a non-limiting example, the chromatography material may be packed in a chromatography column prior to adding the liquid sample to the chromatography column material contained in the chromatography column.

[0162] The chromatographic materials disclosed herein comprise a support to which a ligand is coupled. The term "support" has its conventional meaning in the bioprocessing arts, and may alternatively be referred to as "support material" or "solid phase", which are other terms conventionally used in the art.

[0163] In Formula Ia, the portion of the ligand intended to be coupled to the support is illustrated.

[0164]

[0165] In this regard, the term "support" at the upper left of Formula Ia indicates the position at which the ligand can be coupled to the support. If a linker or extender is used to couple the ligand to the support (as described in further detail below), such a linker or extender may also be included in the term "support".

[0166] Also disclosed herein are compounds defined by Formula I, and as further defined when referring to a chromatographic ligand.

[0167] The disclosed chromatographic materials comprise a support to which a ligand is coupled. The support can be made of different types of materials and can have different shapes or forms, as described in more detail below.

[0168] The support can be made of 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 materials, for example, agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan gum, alginate, pectin, starch, etc. Natural polymer supports are readily prepared and optionally cross-linked according to standard methods such as reverse suspension gelation (S Hjerten: Biochim Biophys Acta 79 (2), 393-398 (1964). In a particularly advantageous embodiment, the support is a relatively rigid but porous agarose prepared by methods that enhance its flow properties, see, for example, US 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, for example styrene or styrene derivatives, divinylbenzene, acrylamide, acrylate, methacrylate, vinyl ester, vinyl amide, 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), 70-75(1988)). Natural or synthetic polymer supports can also be obtained from commercial sources such as Cytiva of Sweden, for example in the form of porous particles. In still an 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.

[0169] The support for the chromatographic material may be in the form of particles, such as substantially spherical, elongated or irregularly shaped particles.

[0170] Capto ImpRes base chromatography matrix (Cytiva, Uppsala, Sweden) comprises a support in the form of substantially spherical particles or beads having a diameter of about 40 μm. This is a non-limiting example of a particle suitable for comprising the presently disclosed ligands by coupling the ligands to the support.

[0171] Suitable particle sizes for the presently disclosed chromatography materials may be in the range of 5-500 μm in diameter, such as 10-200 μm, for example 20-100 μm. In a particular embodiment, the average particle size is in the range of about 20 μm to about 50 μm, such as 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.

[0172] Suitable average pore sizes for the presently disclosed chromatographic materials in particulate form may have any size larger than the target molecules and impurities to be separated, including but not limited to average pore diameters from about 9 nm (e.g., suitable for separation of 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.

[0173] Those skilled in the art can easily select the appropriate particle size and porosity depending on the process to be used.

[0174] The chromatographic material may be dried, such as dried particles, which are soaked in a liquid to maintain their original form when used. For example, such a dried chromatographic material may comprise dried agarose particles.

[0175] The support of the chromatographic material may alternatively be in the form of magnetic particles. The term "magnetic particles" is defined herein as particles that can be attracted by a magnetic field. At the same time, the magnetic particles used in the currently disclosed method should not aggregate in the absence of a magnetic field. In other words, the behavior of the magnetic particles should be similar to that of superparamagnetic particles. The particles may have any symmetrical shape, such as a sphere or a cube, or any asymmetric 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", "mag particles" and "mag beads" are used interchangeably herein without limiting the scope to magnetic particles having a spherical shape. Magnetic particles suitable for the currently disclosed method have been described in WO2018122089, which is hereby incorporated by reference in its entirety.

[0176] The support for the chromatographic material may alternatively take any other shape conventionally used in separations, such as a monolith, a filter or membrane, a capillary, a chip, a nanofiber, a surface, and the like.

[0177] When the support of the chromatographic material comprises a monolith, suitable average pore diameters in the monolith for separating target molecules from impurities range from a minimum average pore diameter of about 9-12 nm (e.g. suitable for separation of monoclonal antibodies), and up to a maximum pore diameter of about 5 μm, such as about 0.5, 1.0, 2.0, 3.0, 4.0 or 5.0 μm.

[0178] When the support of the chromatographic material comprises nanofibers, such nanofibers may, for example, comprise electrospun polymer nanofibers. When used, such nanofibers will form a stationary phase comprising a plurality of pores which are permeable to the mobile phase.

[0179] The support of chromatographic material can comprise a membranous structure, such as a single membrane, a membrane stack or a filter. The membrane can be an adsorptive membrane. When the support of chromatographic material comprises a membranous structure, the suitable pore diameter range for separating target molecules from impurities in the membranous structure is a minimum average pore diameter of about 9-12 nm (e.g., suitable for the separation of monoclonal antibodies), and a maximum pore diameter of about 5 μm at most, such as about 0.5, 1.0, 2.0, 3.0, 4.0 or 5.0 μm. When chromatographic material comprises a membranous structure, this membranous structure can, for example, comprise a nonwoven web of polymer nanofibers.

[0180] In the context of a membranous structure, non-limiting examples of suitable polymers may be selected from polysulfones, polyamides, nylons, polyacrylic acids, polymethacrylic acids, polyacrylonitrile, polystyrene and polyethylene oxide, and mixtures thereof.

[0181] Alternatively, the polymer may be a cellulose polymer, such as selected from cellulose and partial derivatives of cellulose, in particular cellulose esters, cross-linked cellulose, grafted cellulose or ligand-coupled cellulose. Cellulose fiber chromatography (known as Fibro chromatography; Cytiva, Sweden) is an ultrafast chromatographic purification using short process times and high productivity with high flow rates and high capacity of cellulose fibers. When the support of the chromatographic material comprises cellulose fibers such as Fibro, the range of suitable pore diameters in the cellulose fibers for separating target molecules from impurities is a minimum average pore diameter of about 9-12 nm (e.g., suitable for separating monoclonal antibodies), and a maximum pore diameter of about 5 μm at most, such as about 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0 or 5.0 μm.

[0182] The term "membrane chromatography" has its conventional meaning in the field of bioprocessing. In membrane chromatography, there is a combination of components of a fluid (e.g., single molecules, associates, or particles) with a solid surface in contact with the fluid. Molecules can approach the active surface of the solid phase by convective transport. The advantage of a membrane adsorber over a packed chromatographic column is that it is suitable for operation at a much higher flow rate. This is also referred to as convection-based chromatography. Convection-based chromatographic matrices include any matrix in which the application of a hydraulic pressure difference between the inflow and outflow of the matrix forces the matrix to be perfused, thereby achieving essentially convective transport of substances into or out of the matrix, which is achieved very quickly at high flow rates. Convection-based chromatography and membrane adsorbers are described, for example, in US20140296464A1, US20160288089A1, WO2018011600A1, WO2018037244A1, WO2013068741A1, WO2015052465A1, US7867784B2, which are hereby incorporated by reference in their entirety.

[0183] The coupling of the ligand to the chromatographic material support as disclosed herein can be provided by introducing a joint between the support and the ligand. The coupling can be carried out according to any conventional covalent coupling method, such as by using epichlorohydrin; epibromohydrin; allyl-glycidyl ether; diepoxides, such as butanediol diglycidyl ether; halogen-substituted aliphatic substances, such as dichloropropanol; and divinyl sulfone. Other non-limiting examples of suitable joints are: polyethylene glycol (PEG) with 2-6 carbon atoms, carbohydrates with 3-6 carbon atoms, and polyols with 3-6 carbon atoms. These methods are all well known in the art and are easily carried out by technicians.

[0184] The ligand can be coupled to the support via a longer linker molecule, also known as a "surface extender", or simply "extender". Extenders are well known in the art and are typically used to spatially increase the distance between the ligand and the support. Extenders sometimes represent tentacles or flexible arms. For a more detailed description of possible chemical structures, see, for example, US 6,428,707, which is hereby included herein by reference. In short, the extender can be in the form of a polymer, such as a homo- or copolymer. The hydrophilic polymer extender can be of synthetic origin, i.e., having a synthetic backbone, or of biological origin, i.e., a biopolymer with a naturally occurring backbone. Typical synthetic polymers are polyvinyl alcohol, polypropylene- and polymethacrylamide, polyvinyl ethers, etc. Typical biopolymers are polysaccharides, such as starch, cellulose, dextran, agarose.

[0185] The term "eluent" is used in the art in its conventional sense, ie, a buffer having a suitable pH and / or ionic strength to release one or more compounds from a separation matrix.

[0186] The term "eluate" is used in the art in its conventional sense, ie, the portion of a liquid sample that elutes from a chromatography column after the liquid sample has been loaded onto the chromatography column.

[0187] In the methods for preparing chromatographic materials disclosed herein, the density of the plurality of ligands immobilized on the support may be about 15 to about 50 μmol / mL, such as about 15, 20, 25, 30, 35, 40, 45 or 50 μmol / mL, preferably about 20 to about 35 μmol / mL.

[0188] The present disclosure also provides a chromatography material comprising the chromatography ligand defined above coupled to a support. The chromatography material and the support are as defined and exemplified above.

[0189] In a presently preferred embodiment, the support comprises beads having a diameter of about 25 μm to about 50 μm, preferably about 30 μm to about 45 μm.

[0190] The chromatographic materials disclosed herein can have a density of the plurality of ligands immobilized on the support of about 15 to about 50 μmol / mL, such as about 15, 20, 25, 30, 35, 40, 45 or 50 μmol / mL, preferably about 20 to about 35 μmol / mL.

[0191] The chromatographic material comprising the chromatographic ligand as disclosed herein can be further defined by having a lysozyme bulk binding capacity in the linear part of the adsorption isotherm under binding conditions pH 7.5 and 480 mM NaCl, and further defined by the amount of lysozyme bound to the chromatographic material at equilibrium being at least 50%, such as 60%, of the amount of lysozyme added to the chromatographic material. The meanings of the terms "lysozyme bulk binding capacity" and "linear part of the adsorption isotherm" are as further defined above.

[0192] As described elsewhere herein, a chromatographic ligand can be defined by having a log S from about -2.5 to about -5.

[0193] The ligands of the chromatographic material are defined by formula I, as described in further detail above.

[0194] Further provided is the use of a chromatography material disclosed herein for separating one or more target molecules from impurities.

[0195] In the context of the use, 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, such as 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 described and exemplified in detail elsewhere herein.

[0196] Further, in the context of the use, the impurities may comprise aggregates of one or more target molecules, such as high molecular weight aggregates of the target molecules, as described in detail elsewhere herein. For example, when the target molecule is an antibody, the impurities may comprise aggregates of the antibody, such as high molecular weight aggregates of one or more antibody fragments. Alternatively, when the target molecule is an antibody fragment, the impurities may comprise aggregates of one or more antibody fragments, such as high molecular weight aggregates of one or more antibody fragments.

[0197] The present disclosure also provides Figure 1 The method for separating one or more target molecules from impurities is shown to solve or at least alleviate the problems associated with existing methods for separating one or more target molecules from impurities, including:

[0198] a) adding a liquid sample comprising one or more target molecules and impurities to a chromatography material as disclosed herein;

[0199] b) eluting the target molecule from the chromatography material;

[0200] c) optionally eluting impurities from the chromatography material.

[0201] 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 separating various types of target molecules (e.g., monoclonal antibodies) are well known in the art and can be easily selected by a skilled person.

[0202] It should be understood that the term "gradient" as used in the context of elution conditions includes both continuous and step gradients. A continuous gradient may be linear or non-linear, or a combination thereof.

[0203] The present disclosure further provides an alternative method for separating one or more target molecules from impurities, such as Figure 2 As shown, the method includes:

[0204] a) adding a liquid sample comprising one or more target molecules and impurities to a chromatography material as disclosed herein;

[0205] b) obtaining the target molecule in a flow-through mode, wherein the target molecule passes through the chromatographic material without substantially binding to the chromatographic material;

[0206] c) optionally eluting impurities from the chromatography material.

[0207] The above-described flow-through method may be particularly suitable when using a chromatography material comprising a support in the form of cellulose fibers (eg Fibro) or nanofibers, as described in further detail above.

[0208] like Figure 1 and 2 As shown, the above method for separating target molecules from impurities is based on multimodal interactions between chromatographic ligands and molecules present in the liquid sample, namely electrostatic interactions, hydrophobic interactions, hydrogen bonds, etc.

[0209] 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, such as 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 described and exemplified in detail elsewhere herein.

[0210] Further in the context of the method, the impurities may comprise aggregates of one or more target molecules, such as high molecular weight aggregates of the target molecules, as described in detail elsewhere herein. For example, when the target molecule is an antibody, the impurities may comprise aggregates of the antibody, such as high molecular weight aggregates of one or more antibody fragments. Alternatively, when the target molecule is an antibody fragment, the impurities may comprise aggregates of one or more antibody fragments, such as high molecular weight aggregates of one or more antibody fragments.

[0211] The method disclosed above for separating a target molecule from impurities may further include a step (a1) before step (a), wherein step (a1) includes pre-treating the liquid sample. Optionally, the pre-treatment may include subjecting the cell culture harvest containing the target molecule to cell lysis, clarification and / or filtration.

[0212] The method for separating a target molecule from impurities disclosed above may further include a step (a2) before step (a), wherein step (a2) includes 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 adding the pre-purified liquid sample containing the target molecules to the chromatographic material disclosed herein. Optionally, the pre-purification may include subjecting the cell culture harvest containing the target molecules to chromatography, or clarifying and then performing chromatography.

[0213] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention relates. In addition, unless otherwise specified, the singular forms "a", "an" and "the" are intended to include plural references.

[0214] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0215] Example 1

[0216] introduction

[0217] This example shows an experiment to confirm the successful separation of monoclonal antibodies from Fab fragments and high molecular weight aggregates by using novel ligands disclosed herein. In short, the load contains a monomer monoclonal antibody (mAb) with a purity of 92% and an additional 6% Fab fragment and 2% high molecular weight (HMW) aggregates. The separation of mAb, Fab fragments and HMW aggregates on the chromatographic material was carried out with a binding and elution method. Binding was carried out under the favorable binding conditions of 25mM pH 7 phosphate, and elution was carried out by increasing the NaCl concentration linearly from a low concentration to 1M NaCl, 50mM pH 7.5 phosphate buffer as an elution buffer. In a linear salt gradient elution, the Fab fragment (smallest size) was eluted first, followed by the mAb, and then the HMW aggregates (largest size). The elution of the aggregates in the linear gradient elution was much later, indicating that the binding affinity to the ligand was stronger.

[0218] Materials and methods

[0219] Material

[0220] Liquid samples containing a monoclonal antibody (called mAb1) pre-purified on a protein A column were used in the experiment. mAb1 has a pI value of 8.6, a molecular weight of 150kDa, and an elongation coefficient of 1.58. The concentration of the buffer exchange mAb1 sample (pH 7 and conductivity of 2.94mS / cm) used for isocratic retention studies was ~20mg / mL, and the monomer purity was 98.5% and contained 1.5% HMW aggregates, and the concentration used for linear salt gradient elution studies was ~18mg / mL, and the monomer purity was 92% and contained 6% Fab fragments and 2% HMW aggregates. Fab fragments were generated from mAb1 using a papain digestion method and spiked into mAb1 samples. For high-throughput plate-based studies, 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, and acyl chlorides and sulfonyl chlorides, bromine, dichloromethane, and ethyl acetate were obtained from Sigma Aldrich. All other chemicals used, including sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, sodium acetate, sodium acetate trihydrate, sodium hydroxide, glacial acetic acid, blue dextran 2000, were of analytical grade and purchased from Merck (Darmstadt, Germany).

[0221] Equipment and analysis

[0222] All chromatographic experiments were performed on a Cytiva (Uppsala, Sweden) The chromatography was performed on a SpectraMax M2 HPLC-MS / MS system from Molecular Devices LLC (California, USA) using a manually packed Tricon 5 / 100 glass column with a column volume (CV) of ~2 mL. e The protein concentration of the eluted fractions was analyzed spectrophotometrically. A SpectraMax 96-well plate reader was used. The Fab fragments, mAb1, and HMW aggregate content (%) of all gradient elution fractions were analyzed using size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) using an Agilent 1200 series HPLC from Agilent technologies (Santa Clara, CA, USA). A Superdex HPLC containing 13 μm particles packed in a 10×300 mm high resolution column from Cytiva (Uppsala, Sweden) was used. TM 200 resin column. The mobile phase buffer consisted of 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. The software "Agilent ChemStation" and the software "Unicorn 5.31" were used. The chromatography system performed analysis of the data obtained by SEC-HPLC at UV 280 nm. Titrations were performed using a Metrohm Autotitrator 905 Titrando system from Metrohm AG (Herisau, Switzerland) and analyzed with Tiamo software.

[0223] method

[0224] Synthesis of Capto MMC ligand prototype

[0225] The multimodal Capto MMC thioether ligands are derived from thiolactone proligands. The Capto MMC ligand prototype library was synthesized by acylation or sulfonylation of commercially available D,L-homocysteine ​​thiolactone hydrochloride with an equimolar amount of the appropriate acyl chloride or sulfonyl chloride in dichloromethane (DCM) in the presence of diisopropylethylamine (DIPEA). After the reaction is complete, typically 16 h, the solvent is removed in vacuo and the residue (typically a pale yellow oil) is dissolved in ethyl acetate (EtOAc) and washed sequentially in a separatory funnel with aqueous citric acid (10% w / v), K2CO3 10% (w / w) aqueous solution, water, and brine. The organic phase is dried over MgSO4, filtered, and evaporated under reduced pressure to give the product, typically a white or pale yellow solid.

[0226] More specifically, the eight multimodal ligand syntheses currently disclosed are as follows:

[0227] N-Methylbenzoyl-D,L-homocysteine ​​thiolactone (L01) - 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. To the stirred solution was slowly added methylbenzoyl chloride (18.75 mmol) 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).

[0228] 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) were dissolved in DCM (40 mL) and the mixture was cooled in an ice bath. Pentafluorobenzoyl chloride (18 mmol, 2.62 mL) was slowly added 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).

[0229] 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) 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 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).

[0230] 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) were dissolved in DCM and the mixture was cooled in an ice bath. To the stirred solution was slowly added 4-ethylbenzoyl chloride (18.75 mmol) as described in the general procedure to give the product as a white solid (>90%). 1 H NMR (CDCl3, 300MHz): 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).

[0231] N-Benzodioxanesulfonyl-D,L-homocysteine ​​thiolactone (L09) - 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 as described in the general procedure to give the product as a white solid. 1H 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, alph), 3.69 (m, 1H, thiolactone-H), 3.25 (m, 2H, thiolactone-H), 2.86 (m, 1H, thiolactone-H), 2.05 (m, 1H, thiolactone-H).

[0232] 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. To the stirred solution was slowly added 2-naphthoyl chloride (18.75 mmol) as described in the general procedure to give the product as a white solid (92%, 4.67 g). 1 H NMR (CDCl3, 300MHz): 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).

[0233] N-Pentafluorobenzenesulfonyl homocysteine ​​thiolactone (L17) - D, L-homocysteine ​​thiolactone HCl (10.02mmol, 1.02eq, 1.567g), dichloromethane (20mL) and diisopropylethylamine (2.04eq, 20.04mmol, 3.553mL) were added to a Schlenk flask and purged with N2 and stirred until dissolved. The solution was cooled in an ice bath and pentafluorobenzenesulfonyl chloride (1eq, 10mmol, 2.666g, 1.484mL) was added dropwise. The reaction mixture was allowed to warm to rt while stirring overnight under N2. The reaction was complete by TLC (DCM containing a few drops of EtOH, or 1:1 EtOAc / cyclohexane). The reaction mixture was concentrated on a rotary evaporator (rotovap) and then diluted with EtOAc (100mL). It was washed with citric acid (10 wt%, 3 x 50 mL) then K2CO3 (10 wt%, 3 x 50 mL) and brine (2 x 50 mL). The organic phase was concentrated on a rotary evaporator and the brown solid residue (1.7 g, theoretical yield 3.75 g) was dried under house vac. The crude product was recrystallized from boiling EtOH / H2O (about 30:10 mL) to give a light brown solid (0.896 g, 24% yield).1 H NMR (300MHz, CDCl3) δ5.72(br,1H,NH), 4.20(m,1H,CH), 3.36(m,2H), 2.88(m,1H), 2.18(m,1H).

[0234] N-(3,5-diethoxy-benzoyl) homocysteine ​​thiolactone (L18) - D, L-homocysteine ​​thiolactone HCl (10.2mmol, 1.02eq, 1.567g), dichloromethane (20mL) and diisopropylethylamine (2.04eq, 20.4mmol, 2.636g, 3.55mL) were added to a Schlenk flask and purged with N2 and stirred until dissolved. The solution was cooled in an ice bath and the acyl chloride (1eq, 10mmol, 2.287g) in DCM (5mL) was added dropwise. After 30min, the ice bath was removed and the reaction mixture was allowed to warm to rt while stirring overnight under N2. TLC (DCM containing a few drops of EtOH, or 1:1 EtOAc / cyclohexane) showed a spot + baseline. The reaction mixture was concentrated on a rotary evaporator and then diluted with EtOAc (100mL). It was washed with citric acid (10 wt%, 3 x 50 mL) then K2CO3 (10 wt%, 3 x 50 mL) and brine (2 x 50 mL). The organic phase was concentrated on a rotary evaporator and the white solid was dried under house vacuum. Yield 2.78 g (90% yield). 1 H NMR (300MHz, CDCl3) δ6.89(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).

[0235] After aqueous treatment and crystallization where appropriate, the resulting N-acylated or sulfonylated thiolactones are hydrolyzed in aqueous NaOH to provide carboxylate cation exchange groups and thiols, which are then used for nucleophilic coupling to a functionalized agarose base matrix (Capto ImpRes, Cytiva). Briefly, the agarose matrix is ​​first functionalized with terminal allyl groups using allyl glycidyl ether (AGE) and subsequently brominated with elemental bromine. Bromination of the gel under alkaline conditions forms epoxy termini, which are good electrophiles that can be catalyzed by S N 2 Nucleophilic substitution reaction with the MMC ligand prototype. The capacity of the multimodal ligand as determined by titration was 25-30 μmol / mL resin.

[0236] High-throughput plate-based screening studies

[0237] Binding capacity (BC) data for the linear portion of the isotherm for six different proteins, namely cytochrome C, α-lactalbumin, lysozyme, ovalbumin, HSA, and a monoclonal antibody (mAb1), were obtained on novel multimodal cation exchange resins including reference CaptoMMC ImpRes resin (~25 μmol / mL resin) by high throughput plate-based rapid screening. Switzerland) automated liquid handling system Tecan robotic workstation, in the 6μL PreDictor TM Four different binding pH (4.5, 5.5, 6.5, 7.5) and eight different NaCl salt concentrations (0, 55, 133, 257, 480, 750, 1250, 1750mM) low protein loadings were measured in 96-well filter plates for BC data of 10 μg / μL resin. Therefore, 32 different ligand prototypes were tested for binding pH and salt conditions. Based on 25mM acetate buffer, and pH 6.5 and 7.5 based on 50mM phosphate buffer, the volume of the stock solution in conjunction with pH was calculated using a proprietary Excel application (Cytiva, Uppsala, Sweden). All protein solutions were prepared using 5mM pH 7 phosphate buffers, and mAb1 was loaded in 5mM pH 7 phosphates for buffer exchange to avoid the effect of buffer on target pH and salt concentration. Several experiments were repeated in duplicate.

[0238] Evaluation of system dead volume, column porosity, and resin particle porosity

[0239] In the absence of a column, i.e., replacing the column with a zero dead volume connector, the ion exchange rate was evaluated by pulse injection of 200 μL of 1 M NaCl in 10 mM phosphate buffer, pH 6.5. The column porosity ε of manually packed columns with the resin Capto MMC ImpRes coupled to different synthetic ligand prototypes was evaluated by pulse injection of 200 μL of 3 mg / mL blue dextran 2000 (Mol. Wt. 2000 kDa) in pH 6.5 10 mM phosphate buffer into the column. C Column porosity ε C Use the following equation to calculate:

[0240]

[0241] Where V Dex is the column retention volume of blue dextran, V systemdead for The system dead volume, and VC is the geometric column volume. The porosity of the resin particles ε P Use the following equation to calculate:

[0242] ε T =ε C +ε P (1-ε C ) (2)

[0243] where ε T is the total column porosity evaluated by pulse injection of 200 μL of protein solution (concentration ≈ 8.23 ​​mg / mL) into the column under non-binding conditions of 100 mM Na3PO4 at pH 12. Total column porosity ε T Use the following equation to calculate:

[0244]

[0245] where t0 is the retention time of the protein under non-binding conditions, Q is the flow rate, and V C is the column volume.

[0246] Isocratic retention studies

[0247] Each isocratic retention experiment was performed in 5 different steps, including: Step 1: Equilibration (5CV; pH 7 25mM phosphate buffer + X (200mM-1800mM) NaCl); Step 2: Sample loading (~2mg protein per mL resin; sample loading volume is 200μL); Step 3: Isocratic elution (until UV is less than 2mAU); Step 4: Stripping (5CV; pH 7.5 50mM phosphate buffer + 1000mM NaCl); Step 5: Cleaning in place (CIP, 3CV) step with 1N NaOH for column regeneration and disinfection. The flow rate was set to 0.5mL / min. The fluid effluent was monitored at 280nm for peak detection. The retention factor k was calculated using the following equation:

[0248]

[0249] V R The retention volume was corrected for the system dead volume of the protein under isocratic conditions and V0 was the column void volume and calculated using the following equation:

[0250] V0=V C ε C (5)

[0251] Based on the isocratic retention factor k, the total number of counter salt ions and water molecules released during mAb1 adsorption, and the hydrophobic contact area (HCA) estimated by fitting the characteristic “U”-shaped curve obtained for multimodal chromatograms to the preferential interaction model developed by Perkins et al. [28,29], the equation relating the total number of counter salt ions released and water molecules released to the retention factor k is given by:

[0252]

[0253] Where -(Δv + +Δv - ) is the number of counterions released during mAb1 adsorption, which is controlled by electrostatic interactions at low salt concentrations, and (-Δv1) is the total number of water molecules released during the adsorption of one mAb1 molecule, which is controlled by hydrophobic interactions at higher salt concentrations; c is the 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; and a are the thermodynamic properties of the salt ion and the activity of the salt ion, respectively. The isocratic retention factor data were fitted to a simplified form of equation (6) given by:

[0254] ln k=α+βlnC S -γC S (7)

[0255] The model parameters β and γ are related to (Δv + +Δv - ) and (Δv1) are related to:

[0256] (Δv + +Δv - )=gβ (8)

[0257]

[0258] The hydrophobic contact area (HCA) during adsorption of mAb1 on the resin was calculated by fitting the isocratic retention factor data to Melander's 3-parameter equation

[30] given by:

[0259] logk=A-BlogC S +CC S (10)

[0260] Where B and C are the electrostatic and hydrophobic interaction parameters, respectively. Based on parameter C, HCA is calculated using equation

[31] :

[0261]

[0262] where R is the universal gas constant, T is the absolute temperature, and σ S is the molar surface tension increment of the salt.

[0263] Linear salt gradient elution studies

[0264] Each linear NaCl salt gradient experiment was performed in 5 different steps, including:

[0265] (i) Method 1: Binding and elution

[0266]

[0267] (ii) Method 2: Binding and elution

[0268]

[0269] The eluate was collected in fractions and analyzed for protein concentration and mAb1, Fab fragment and HMW aggregate content (%) using SE-HPLC as described above. Using SE-HPLC data, the linear gradient elution peak was deconvoluted into its components mAb1 and Fab and used to calculate the resolution R between mAb1 and Fab using the following expression s :

[0270]

[0271] where t R is the peak retention time and w 0，5h is the full width at half maximum peak height. The retention times of mAb1 and Fab fragment peaks are t R2 and t R1 and the full width at half maximum peak height of Fab and mAb1 are represented by and express.

[0272] The resolution of HMW aggregates was compared by plotting the cumulative mAb1 yield (%) versus the cumulative HMW aggregate (%). Using the SE-HPLC data, the cumulative mAb1 yield (%) and HMW aggregate (%) were calculated using the following expressions:

[0273]

[0274] Results and discussion

[0275] By comparing the reference Capto MMC ImpRes resin (25 μmol / mL 树脂 ) have similar ionic capacities (20-31 μmol / mL 树脂) to prepare all prototypes while minimizing the effect of ligand density on chromatographic performance as determined by ion capacity measurements. The chromatographic performance of the synthesized prototypes was tested in a high-throughput plate-based study.

[0276] Isocratic retention of mAb1 on Capto MMC-like prototype

[0277] In order to understand the adsorption behavior of mAb1 on the new Capto MMC ligand prototypes and begin to qualitatively link ligand structure to binding, mAb1 retention was analyzed as a function of different isocratic chromatographic conditions for the new prototypes and reference ligands. The retention of mAb1 versus NaCl concentration (C S ) varies from 200 mM to 1800 mM at pH 7 (not shown).

[0278] The prototype showed a "U"-shaped dependence of protein retention as a function of salt concentration, with minimum retention at approximately 800 mM NaCl. The "U"-shaped behavior is a characteristic of multimodal resins and is also a result of contributions from ion exchange and other secondary interactions such as hydrophobic interactions. For binding pH values ​​below the pI value of mAb1 (~8.6) and lower NaCl concentrations, protein retention decreases with increasing salt concentration. As with traditional CIEX chromatography, this is because buffer cations compete with positively charged mAbs for negatively charged sites on multiple peaks. However, at high NaCl concentrations (≥1000 mM), protein retention increases with salt concentration, behaving like HIC resins.

[0279] At the intermediate stage of about 800–1000 mM NaCl, the mAb1 adsorption mechanism undergoes a transition from primary electrostatic interactions to secondary hydrophobic interactions.

[0280] Figure 3 A comparison of the isocratic mAbl retention factor ln(k) at pH 7 for L09, L02, and L00 versus NaCl concentrations varying from 200 mM to 1800 mM is shown. More specifically, Figure 3 The results show that at pH 7, the ln(k) of mAb1 adsorbed onto (■)L00, (x)L09, and (□)L02 is relative to ln(C S ). Solid line Overlapping fit with equation (7) and equation (10).

[0281] Chromatographic performance using high-throughput binding capacity (BC) data

[0282] Response surface analysis of high-throughput BC data

[0283] We set out to perform high-throughput plate-based studies that provide information about protein binding to many targets simultaneously at both various pH and salt concentrations, and this is very useful for evaluating the different binding and elution properties of multimodal resins. These plate experiments were performed at low loadings in the linear portion of the isotherm, providing information about binding behavior and selectivity opportunities rather than maximum binding capacity. Likewise, the multimodal prototype showed a "U"-shaped dependence of binding on salt concentration, which was attributed to multimodal electrostatic and other secondary interactions.

[0284] Principal component analysis of BC

[0285] High-throughput plate-based binding data were collected for the prototype, with each of the six model proteins under 32 different pH / [NaCl] binding conditions. In order to gain further insights from this large amount of data, a principal component analysis (PCA) was performed, resulting in six principal components (PCs). Of these six PCs, the first two PCs described a total of 85.7% of the data variance. As shown in the chromatographic diversity plot (not shown), at pH 4.5, 1750 mM NaCl, the PC1 score trended with mAb1 binding capacity, indicating that the PC1 score was strongly correlated with high salt binding to mAb1. This suggests that the overall conclusions from the detailed study of mAb1 appear to be general and can be obtained from rapid, high-throughput, plate-based studies.

[0286] High-throughput binding capacity of ligand prototypes

[0287] Table 2 shows high throughput binding capacity data for % binding of specific proteins under given binding conditions.

[0288] Table 2: High throughput binding capacity of different ligand prototypes for proteins (lysozyme and monoclonal antibody, respectively) under different binding conditions.

[0289]

[0290] Linear salt gradient elution studies

[0291] The resolution between Fab fragment and mAb is Rs

[0292] Table 3 shows the R separation between Fab fragments and monoclonal antibody (mAb1) s All new ligand prototypes showed better resolution than the reference ligand (L00), with L09 having the highest resolution, followed by L02, L05, L01, and L08.

[0293] Table 3: R s value

[0294] Ligand <![CDATA[R S ]]> L00 0.7 L01 0.9 L02 1.1 L05 1.0 L08 0.9 L09 1.3

[0295] Resolution between HMW aggregates and mAb

[0296] (i) Method 1: Binding and elution

[0297] Figure 4 Comparison of resolution between HMW aggregates and monoclonal antibodies (mAbs) for different ligand prototypes is shown, for data obtained using Method 1, as described further above. The load contained ~2% HMW aggregates. Comparison of the cumulative relationship plots shows that the new ligands perform better than the reference ligand (L00), with L09 being the most promising one for aggregate removal.

[0298] (ii) Method 2: Binding and elution

[0299] Figure 5 Comparison of resolution between HMW aggregates and mAbs for different ligand prototypes is shown, for data obtained using Method 2, as described further above. The load contained -1.95% HMW aggregates. In the dual pH-salt linear gradient elution, L09 performed better than L08 and L11 for HMW aggregate removal.

[0300] in conclusion

[0301] These results indicate that the new multimodal ligand prototype can achieve efficient removal of aggregate species from challenging monoclonal antibodies and provide improved selectivity for product-related impurities. It can be concluded that more hydrophobic ligands outperform less hydrophobic ligands. The prominent role of secondary hydrophobic and hydrogen bonding interactions provided by the ligand chemical structure of more hydrophobic ligands combined with electrostatic interactions leads to their better performance in removing Fab fragments and HMW aggregate impurities.

[0302] Example 2

[0303] The experimental design was performed using the same antibodies as in Example 1, but with the following changes:

[0304] (a) Testing different ligands coupled to the chromatographic material support, i.e. ligands containing different hydrophobic groups.

[0305] (b) Determination of the affinity of different ligands for different multimeric forms of the antibody.

[0306] Example 3

[0307] As in Example 1, the experimental design was performed with other target molecules than those in Example 1, such as a bispecific antibody (about 200 kDa), or a non-antibody protein, such as a 50-100 kDa protein.

[0308] It should be understood that the present disclosure is not limited to the exemplary embodiments thereof described above and that several conceivable modifications of the disclosure are possible within the scope of the appended claims.

Claims

1. A chromatographic ligand defined by the following formula I: in: X1 is selected from CO and SO2; Each of R1-R5 is independently selected from 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 together with the atoms to which they are attached may form 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; and The conditions are: 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 two oxygen atoms in the ring, X1 is SO2; and iii. When three of R1-R5 are CH3O, X1 is CO.

2. The chromatographic ligand according to claim 1, wherein any two adjacent moieties selected from R1 to R5 together with the atoms to which they are attached form a 5-membered heterocyclic ring containing at most one oxygen atom in the ring.

3. The chromatographic ligand according to any one of claims 1-2, which is defined by a chemical structure selected from the following (a)-(f): a) b) c) d) e) and f) 4. The chromatographic ligand of any one of claims 1 to 3, wherein when the thiol (-SH) of formula I is replaced with methyl sulfide (-S-CH3), the ligand has a logS of about -2.5 to about -5.

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

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

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

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

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

13. The use according to claim 12, wherein the one or more target molecules 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.

14. Use according to claim 13, wherein the impurities comprise aggregates of the one or more antibody fragments.

15. A method for separating one or more target molecules from impurities, comprising: a) adding a liquid sample comprising 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 said impurities from said chromatography material.

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

17. A method for separating one or more target molecules from impurities, comprising: a) adding a liquid sample comprising one or more target molecules and impurities to a chromatographic material according to any one of claims 7 to 9; b) obtaining the target molecule in a flow-through mode, wherein the target molecule passes through the chromatography material without substantially binding to the chromatography material; c) optionally eluting said impurities from said chromatography material.

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

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

20. The method according to any one of claims 15-19, wherein the one or more target molecules 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.

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

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