Chromatography material, and uses thereof

Abstract

Disclosed herein is a chromatography material comprising a support and a plurality of ligands attached to the support, wherein at least a portion of the plurality of ligands have the structure given by Formula (I) wherein L is a linking group covalently attached to the support; and R1 is aryl, substituted aryl, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl. Also disclosed herein is a method of preparing said chromatography material, uses and methods for separating target entities from impurities, and a chromatography device comprising said chromatography material.

Description

[0001] Cytiva ref. P2023-0219-SE01

[0002] CHROMATOGRAPHY MATERIAL, AND USES THEREOF

[0003] TECHNICAL FIELD

[0004] The present disclosure relates to a chromatography materials comprising multimodal ligands, and a method for preparing said materials. The present disclosure also relates to use of the chromatography materials for separation of target entities, such as biomolecules, from impurities.

[0005] BACKGROUND

[0006] Preparative chromatography remains the primary technique for purification of biomolecules, such as therapeutic proteins, due to its benefits of resolution, scalability, and robustness. Multimodal (or mixed mode) chromatography is an important tool in downstream processing of biomolecules including therapeutic proteins. Multimodal chromatography materials typically comprise ligands attached to a support. The structure of the ligands provide more than one type of interaction with target entities or impurities, such as combinations of ionic, hydrophobic and hydrogen bonding interactions. In multimodal chromatography, samples comprising target entities and impurities are passed through the material and separated based on their interaction with the ligands of the chromatography material.

[0007] A commercialised example of a multimodal chromatography material is Capto™ MMC ImpRes (Cytiva Sweden AB, Uppsala, Sweden). This material comprises a weak cation exchange multimodal ligand that enables high selectivity in a broad pH and salt window compared with traditional ion exchangers. It achieves efficient removal of aggregates, viruses, and main contaminants in processes for the purification of monoclonal antibodies and is suitable for polishing of antibody fragments. However, there is a continuous need in the art for alternative chromatography materials.

[0008] WO03024588A1 discloses a multistep method of preparing chromatography materials such as Capto™ MMC. The disclosed method comprises: (i) preparing the ligand (a thiol compound); (ii) activating the ligand; (iii) coupling a reactive group to the support material; (iv) activating the reactive group; (v) coupling the activated ligand to the activated support material.

[0009] SUMMARY OF THE INVENTION

[0010] In a first aspect of the present invention, there is provided a chromatography material comprising a support and a plurality of ligands attached to the support, wherein at least a portion of the plurality of ligands have the structure given by Formula (I): Cytiva ref. P2023-0219-SE01

[0011] Formula (I) wherein:

[0012] L is a linking group covalently attached to the support;

[0013] Ri is aryl, substituted aryl, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

[0014] Compared with known chromatography materials, such as those disclosed in WO03024588A1, materials according to the first aspect can be prepared by a much simpler synthetic procedure. The inventors have surprisingly found that the base matrix does not need to be activated to achieve a successful coupling of the ligand to the base matrix, and many examples of ligands of Formula (I) are readily available commercially or can be easily synthesised. In contrast, the method disclosed in WO03024588A1 involves a multistep ligand synthesis and activation of the base matrix and ligand is required prior to coupling. The reduction in the number and complexity of the process steps may lead to a reduction in energy and material usage associated with the preparation of chromatography materials according to the present invention compared with known materials.

[0015] In a second aspect of the present invention, there is provided a method of preparing a chromatography material according to the first aspect. The method of the second aspect is an example of a simple method that can be used to couple the ligand to the support, which may involve direct coupling of the ligand to the base matrix without the need for functionalisation and / or activation of the base matrix prior to coupling.

[0016] In a third aspect of the present invention, there is provided a use of the chromatography material according to the first aspect for separating one or more target entities from one or more impurities.

[0017] In a fourth aspect of the present invention, there is a provided a method for separating one or more target entities from one or more impurities, the method comprising adding a liquid sample comprising one or more target entities and one or more impurities to a chromatography material according to the first aspect.

[0018] In a fifth aspect, there is provided a chromatography device comprising a chromatography material according to the first aspect. Cytiva ref. P2023-0219-SE01

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figures 1 and 2 illustrate methods of separating one or more target entities from one or more impurities according to the fourth aspect.

[0021] Figure 3 shows the results of testing chromatography materials according to the first aspect in the separation of four different target entities: Ribonuclease A (RNAse), Cytochrome C (Cyt C), Lysozyme (Lys) and a-Chymotrypsinogen at pH 4.5.

[0022] Figure 4 shows the results of testing chromatography materials according to the first aspect in the separation of four different target entities: Ribonuclease A (RNAse), Cytochrome C (Cyt C), Lysozyme (Lys) and a-Chymotrypsinogen at pH 7.6.

[0023] DEFINITIONS

[0024] Herein, the term "ligand" means a molecule that has a known or unknown affinity for a given analyte and can be coupled to a support of a chromatography material, whereas "analyte" includes any specific binding partner to the ligand. The analytes of interest to separate according to the present disclosure are so-called target entities and impurities, which are present in a liquid sample.

[0025] The herein disclosed chromatography material comprises a support to which the ligand is coupled. The term "support" has its conventional meaning in the field of bioprocessing and may alternatively be called a "support material", "matrix material", or a "solid phase", which are other terms conventionally used in this field.

[0026] The term "target entity" is intended to include macromolecules and biological particles, which are to be separated from a liquid sample and purified from impurities before being put to use in their intended applications, for example as therapeutic substances.

[0027] The term "macromolecule" has its conventional meaning in the field of bioprocessing, in which macromolecules are produced (often recombinantly) by cells in a cell culture and purified from the cell culture by any means of separation and purification. Alternatively, the macromolecules are present in a biological solution which does not necessarily originate from a cell culture. Non-limiting examples of macromolecules are biomacromolecules, which are large biological polymers that are made up of monomers linked together, such as peptides and proteins (which can be native or recombinant), including but not limited to enzymes, antibodies, parts of antibodies, and antibody fragments, as well as carbohydrates, and nucleic acid sequences, such as DNA and RNA. The macromolecule to be purified by use of the chromatography ligand according to the present disclosure is typically a protein or polypeptide, particularly a therapeutic protein or polypeptide, such as an antibody, as defined in detail Cytiva ref. P2023-0219-SE01 further below. Alternatively, the macromolecule may be a nucleic acid sequence, which may or may not be comprised by a biological particle such as a virus particle. The nucleic acid sequence and / or the virus particle may be used in a therapeutic application. A biomacromolecule or a biological particle may for example be a biopharmaceutical, which is intended for use as a pharmaceutical compound. It is to be understood that "a macromolecule" is intended to mean a type of macromolecule and that the singular form of the term may encompass a large number of individual macromolecules, or specimens, of the same type. Likewise, "a biological particle" is intended to mean a type of biological particle and the singular form of the term may encompass a large number of individual biological particles, or specimens, of the same type.

[0028] It is to be understood that the term "liquid sample" (or simply "sample") as used herein encompasses any type of sample obtainable from a cell culture, or from a fluid originating from a cell culture which fluid is at least partly purified, by any means of separation and purification. An alternative term to "liquid sample" is "feed", which is intended to mean a mobile phase, comprising target entities and impurities. When adding a feed to a chromatography material, the target entities and impurities may be retained in the chromatography material by binding to varying degrees to the chromatography ligand. The binding to a chromatography ligand and separation of a target entity and impurities will depend on the specific molecule and its properties, such as isoelectric point, hydrophobicity, exposed charges, and ability to additional interactions such as pi-pi stacking and hydrogen bonding. The molecule's inherent properties, the ligand properties and the buffer composition will be utilized to achieve separation of the target entity and impurities.

[0029] The term "cell culture" refers to a culture of cells or a group of cells being cultivated, wherein the cells may be any type of cells, such as bacterial cells, viral cells, fungal cells, insect cells, or mammalian cells. A cell culture may be unclarified, i.e., comprising cells, or may be cell-depleted, i.e., a culture comprising no or few cells but comprising biomolecules released from the cells before removing the cells. Further, an unclarified cell culture as used in the presently disclosed method may comprise intact cells, disrupted cells, a cell homogenate, and / or a cell lysate.

[0030] The term "antibody" as used herein means an immunoglobulin which may be natural or partly or wholly synthetically produced. The term includes, but is not limited to, whole (complete) antibodies, such as monospecific and multispecific antibodies. The term also includes active antibody fragments, including Fab antigen-binding fragments, univalent fragments, and multi-valent fragments. The term also covers any protein having a binding domain which is homologous to an immunoglobulin binding domain. Such proteins can be derived from natural sources or be partly or wholly synthetically produced. The term further includes fusion proteins including an antibody or antibody fragment, e.g, Cytiva ref. P2023-0219-SE01 monoclonal antibody or monoclonal antibody fragment covalently linked to other proteins. Exemplary, antibodies are the immunoglobulin isotypes and different types of fragments, such as Fab, Fab', F (ab1) 2, Fv, dAb (single domain antibody), and Fd (fragment obtained by papain hydrolysis of an immunoglobulin molecule followed by reduction of the disulfide bonds), as well as scFv (so-called single-chain variable fragment, which is a fusion protein of the variable regions of the heavy and light chains of immunoglobulins), tandem scFvs, BiTEs (bispecific T-cell engager molecules), DARTs (dualaffinity retargeting molecules), and diabodies (single chain and tandem diabodies). A bispecific monoclonal antibody is an example of a multispecific antibody and is an engineered protein that can simultaneously bind to two different types of antigen 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 an antibody fragment as such, or an antibody conjugate or a fusion protein, which comprises an antibody fragment or part of an antibody. The chromatography ligand according to the present disclosure may be used to purify for example a therapeutic antibody from impurities such as aggregates or fragments of said therapeutic antibody, to attain a high-quality end product. The presence of aggregates in therapeutic antibody preparations generally have a negative impact on patient safety and must be effectively removed during the manufacturing process.

[0031] A non-limiting example of a biological particle is a virus particle, normally a recombinant virus particle, which is intended for use to achieve gene transfer to modify specific cell type or tissue. A virus particle can for example be engineered to provide a vector expressing therapeutic genes. Several virus types are currently being investigated for use to deliver genetic material (e.g., genes) to cells to provide either transient or permanent transgene expression. These include adenoviruses, retroviruses (y- retroviruses and lentiviruses), poxviruses, adeno-associated viruses (AAV), baculoviruses, and herpes simplex viruses.

[0032] A "virus particle" is herein used to denote a complete infectious virus particle. It includes a core, comprising the genome of the virus (i.e., the viral genome), either 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 nucleocapsid of some viruses is surrounded by a lipoprotein bilayer envelope. In the field of bioprocessing, for the purpose of producing viral vectors for various applications such as therapy, the genome of a virus particle is modified to include a genetic insert, comprising genetic material of interest. Modified virus particles are allowed to infect host cells in a cell culture and the virus particles are propagated in said host cells, after which the virus particles are purified from the cell culture by any means of separation and purification. Cytiva ref. P2023-0219-SE01

[0033] Herein, the term "impurities" is intended to mean any molecule or substance which is present in the liquid sample, and which is not a desired target entity. The term "impurities" includes aggregates, such as aggregates of a target entity, such as high molecular weight aggregates of a target entity. The term "impurities" further includes fragments of a target entity, for example when the target entity is an intact antibody and undesired fragments of said antibody are present in the liquid sample. In cases where the target entity is a bispecific antibody, the term "impurities" may relate to several different types of product-related impurities that may need to be separated from the target entity. As there are typically four chains to combine, the theoretical number of mismatches is high. The homodimer is one likely variant where two identical heavy chains and two identical light chains have matched up to a "normal" mAb construct. Also fragments of variant combinations of heavy and light chains are common, e.g. half an antibody consisting of only one heavy and one light chain. Also, all free chains can occur. Thus, the impurity profile for bispecific antibodies depends on the expression and assembly of the heavy and light chains.

[0034] The term "impurities" also includes host cell proteins (HCP). HCP may for example include enzymes, such as protease(s), and / or lipase(s). It is known in the art to remove lipases by using multimodal chromatography ligands (see e.g., W02020023566A1). The term "impurities" also includes empty virus particles with no genetic content. The removal of such empty virus particles from virus particles with genetic content is of interest in the field of generating virus particles to be used as vectors for delivering genetic material.

[0035] Herein, the term "non-aggregated macromolecule" is intended to mean a non-degraded macromolecule. A non-aggregated macromolecule may herein alternatively be called "non-degraded macromolecule" or "intact macromolecule". In a typical embodiment herein, in which the macromolecule is a protein or a polypeptide, the non-aggregated macromolecule may be described as having an essentially intact tertiary structure, which usually involves an essentially hydrophilic surface of the macromolecule, while hydrophobic moieties are located in the interior of the macromolecule. Hence, a non-aggregated macromolecule essentially does not have hydrophobic moieties or hydrophobic groups exposed on the surface.

[0036] In contrast, in a protein or polypeptide which starts to degrade, the tertiary structure is gradually destroyed, which exposes hydrophobic moieties to the environment surrounding the protein or polypeptide. A protein or polypeptide macromolecule which is being degraded, or has been degraded, may form aggregates. A non-aggregated form of a macromolecule is in a monomeric state. Aggregates of a macromolecule may contain multimeric forms of the macromolecule, such as dimers, trimers etc. of the macromolecule. An individual macromolecule which is degrading may form aggregates with Cytiva ref. P2023-0219-SE01 other individual, degrading, specimens of the same type of macromolecule, and / or may form aggregates with individual, degrading, specimens of other types of degrading macromolecules, or a combination thereof. Since aggregates of macromolecules contain degrading macromolecules, it follows that aggregates of macromolecules have hydrophobic moieties exposed on their surfaces.

[0037] So-called "high molecular weight (HMW) aggregates" is a term well-known to the skilled person. Such aggregates are formed by self-association of the target entities (e.g., a monoclonal antibody having a molecular weight of approx. 150kDa) with each other via covalent and non-covalent bonding. This results in the formation of dimers (e.g., approx. 300kDa for monoclonal antibody dimers) or even higher order of aggregates, e.g., trimers (approx. 450kDa for monoclonal antibody trimers). These aggregates can be either soluble or non-soluble based on the nature of the target entity. Hence, the term "high molecular weight aggregate" may refer herein to an aggregate of a target entity, which aggregate has a molecular weight which is approximately twice the molecular weight of the target entity, or larger than twice the molecular weight of the target entity.

[0038] Herein, the term "hydrophobic moiety" is intended to mean a hydrophobic part of the macromolecule or a hydrophobic group present in the macromolecule.

[0039] The term "hydrophobic group" as used herein is defined as a group of molecules which has a log P value > 0. The partition coefficient, abbreviated P, is defined as a particular ratio of the concentrations of a solute between the two solvents (a biphase of liquid phases), specifically for un-ionized solutes, and the logarithm of the ratio is thus log P. When one of the solvents is water and the other is a nonpolar solvent, then the log P value is a measure of lipophilicity or hydrophobicity. The defined precedent is for the lipophilic and hydrophilic phase types to always be in the numerator and denominator respectively; for example, in a biphasic system of n-octanol (hereafter simply "octanol") and water: concentration of solute in octanol P = - - - concentration of solute in water

[0040] 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 a higher percentage of the solute in the lipophilic phase, i.e., the hydrophobic phase.

[0041] As mentioned above, denatured macromolecules, as well as aggregates of a macromolecule are typically more hydrophobic than an intact, non-denatured, non-aggregated macromolecule. Aggregates therefore bind to a hydrophobic group of a chromatography ligand to a higher extent than a non-aggregated macromolecule. Aggregates of a target entity also have a larger size than the target entity as such, and thus have a larger surface area interacting with a chromatography ligand. Cytiva ref. P2023-0219-SE01

[0042] Consequently, compared to a target entity's binding to the presently disclosed multimodal ligand, aggregates of the target entity will exhibit a stronger binding to the ligand. Aggregates of target entities generally also have more positively charged sites than a monomeric target entity. Thereby, in general an aggregate will elute later from a chromatography device including a multimodal ligand than the target entity.

[0043] The term "separation matrix" is used herein to denote a material comprising a support to which one or more ligands comprising functional groups have been coupled. The functional groups of the ligand(s) bind compounds herein also called analytes, which are to be separated from a liquid sample and / or which are to be separated from other compounds present in the liquid sample. A separation matrix may further comprise a compound which couples the ligand(s) to the support. The terms "linker", "extender", and "surface extender" may be used to describe such a compound, as further described below. The terms "chromatography material" and "chromatography matrix" are used herein to denote a type of separation matrix.

[0044] The term "surface" herein means all external surfaces and includes in the case of a porous support outer surfaces as well as pore surfaces.

[0045] The separation matrix may be contained in any type of separation device, as further defined elsewhere herein. As a non-limiting example, a chromatography material may be packed in a chromatography column, before adding a liquid sample to the chromatography material being contained in the chromatography column.

[0046] The term "eluent" is used in its conventional meaning in this field, i.e., a buffer of suitable pH and / or ionic strength to release one or more compounds from a separation matrix.

[0047] The term "eluate" is used in its conventional meaning in this field, i.e., the part(s) of a liquid sample which are eluted from a chromatography column after having loaded the liquid sample onto the chromatography column.

[0048] It is to be understood that the term "gradient" as used in the context of elution conditions encompasses both continuous gradients and step gradients. A continuous gradient may be linear or non-linear, or a combination thereof.

[0049] DETAILED DESCRIPTION

[0050] The present disclosure is directed to chromatography materials comprising multimodal ligands that perform comparably or better than known materials, and can be prepared by a much simpler synthetic procedure. Cytiva ref. P2023-0219-SE01

[0051] The first aspect provides a chromatography material comprising a support and a plurality of ligands attached to the support, wherein at least a portion of the plurality of ligands have the structure given by Formula (I):

[0052] Formula (I) wherein:

[0053] L is a linking group covalently attached to the support;

[0054] Ri is aryl, substituted aryl, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

[0055] Multimodal chromatography makes use of more than one type of interaction between the chromatography material and the entities in a sample (e.g. target entities, impurities). Possible types of interaction include ionic, hydrophobic and hydrogen bonding. Ligands of Formula (I) are suitable for multimodal chromatography. The carboxylate group acts as a cation exchange group. The Ri group, being alkyl or aryl, provides a hydrophobic interaction. It should be noted that the ligand of Formula (I) is illustrated as the conjugate base (i.e. deprotonated), which is the form of the ligand at a pH above the pKa of the carboxylate group and is the active form for chromatography. Chromatography will be carried out at a pH above the pKa of the carboxylate group, to make use of the cation exchange interaction of the carboxylate group with an analyte. However, when not under chromatography conditions, the pH may be below the pKa of the carboxylate group and so the ligand can also exist in the protonated form, for example when the chromatography material is in storage. Additionally, as will be described in more detail below, chromatography materials of the first aspect may be prepared from the reaction of carboxylate esters with a support material. As such, materials of the first aspect may comprise a ligand in the protonated form, carboxylate ester form, or in the form according to Formula (I). Thus, in some examples, the ligand may be in the form given by Formula (la) below prior to forming the active chromatography material (given by Formula (I) above). The active chromatography can be formed by deprotonation (when R2 is H) or hydrolysis of the ester (when R2 is alkyl). As will be described in more detail herein, the method according to the second aspect may be carried out under alkaline conditions, and therefore the chromatography material may be obtained directly in the form given by Formula (I), as deprotonation or ester hydrolysis of the starting ligand material may occur under the alkaline reaction conditions. Cytiva ref. P2023-0219-SE01

[0056] Formula (la) wherein:

[0057] L is a linking group covalently attached to the support;

[0058] Ri is aryl, substituted aryl, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; and R2 is hydrogen or C1-C5 alkyl.

[0059] In some embodiments, the ligand is attached to a N, S or O atom of the linking group via a covalent bond. In some embodiments, the ligand is attached to a O atom of the linking group via a covalent bond. In some embodiments, the linking group L may include a spacer between the support material and the ligand, such as a linear alkyl chain. In some embodiments, there may be no spacer between the support material and the ligand. In such embodiments, the linking group L is a group that is part of the support material, and the covalent bond may be formed by reaction of the ligand with said group. For example, the ligand may be attached to an oxygen atom of the support as a result of a reaction between the ligand and a surface hydroxyl group of the support material.

[0060] In some embodiments, Ri is aryl, substituted aryl, cycloalkyl or substituted cycloalkyl. Aryl, substituted aryl, cycloalkyl or substituted cycloalkyl may be monocyclic or bicyclic, preferably monocyclic. Substituted aryl and substituted cycloalkyl may be monosubstituted or may contain more than one substitution. For example, substituted aryl may be monosubstituted, disubstituted or trisubstituted.

[0061] In some embodiments, Ri is: wherein R3 is H, halo, or C1-C5 alkyl; and * indicates the point of attachment of Ri in Formula (I). Such aryl groups may be particularly suited to providing hydrophobic interactions in multimodal chromatography. The C1-C5 alkyl may be linear, branched or cyclic, and may be substituted or unsubstituted. In some embodiments, C1-C5 alkyl may be methyl, ethyl, propyl, isopropyl, butyl, Cytiva ref. P2023-0219-SE01 isobutyl, pentyl or isopentyl. In some embodiments, R3 is H, Cl or Br. In some embodiments, R3 is H. In some embodiments, Ri is:

[0062] In some embodiments, Ri is C2-C6 alkyl or C2-C6 halo-substituted alkyl. The term "C2-C6 alkyl" is intended to mean Ci alkyl, C2alkyl, C3 alkyl, C4alkyl, C5alkyl, or C6alkyl. C2-C6 alkyl and C2-C6 halo- substituted alkyl may be linear or branched. In some embodiments, C2-C6 alkyl is ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl or isohexyl. In some embodiments, C2-C6 alkyl is propyl, isopropyl, butyl, isobutyl, hexyl, or isohexyl, preferably propyl, isopropyl, butyl or hexyl. C2-C6 halo-substituted alkyl may be monosubstituted, or may contain more than one halo-substitution. For example, C2-C6 halo-substituted alkyl may be monosubstituted, disubstituted or trisubstituted. In some embodiments, C2-C6 halo-substituted alkyl is C2-C6 chloro-substituted alkyl or C2-C6 bromosubstituted alkyl. In some such embodiments, C2-C6 halo-substituted alkyl may be bromoethyl.

[0063] The quantity of ligands coupled to the support may be expressed as a ligand density, which is the amount of ligands present relative to the amount of the support. In some examples, ligand density may be expressed relative to the volume of the chromatography material, such as by a value in pmol mL1. In some examples, ligand density may be expressed relative to the molar amount of the support, such as by a value in pmoliiganmolsuppon1. Ligand density is determined by titration using a pH electrode, as is well known in the art [see Kanwar Shekhawat L. et al., Journal of Chromatography A, 1699 (2023) 464018 for example]. Titration can be performed with NaOH(aqj from an acidic sample of resin slurry, or by titration with HCI(aq; from a basic sample of resin slurry. More specifically in the case of the strong cation exchange ligands, ion exchange is performed first with high, then low concentration HCI(aq). The hydronium counterions are then quantified by titration with NaOH(aqj. For ligands bearing one cation exchange site the ionic capacity is taken to be equal to the ligand density.

[0064] In some embodiments, the density of the plurality of ligands having the structure given by Formula (I) may be from about 15 to about 150 pmol mL1, such as about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 pmol mL1. In some embodiments, the density of the plurality of ligands having the structure given by Cytiva ref. P2023-0219-SE01

[0065] Formula (I) may be from about 15 to about 130 pmol mL1, or from about 15 to about 70 pmol mL1, or from about 20 to about 50 pmol mL1. The method used for determination of ligand density is the titration method as described above.

[0066] In some embodiments, the chromatography material may comprise more than one type ligand according to formula (I) with different Ri groups. Such materials may be described as mixed-ligand chromatography materials. In some embodiments, the chromatography material may comprise ligands according to formula (I) along with further ligands having a different structure, such as other chromatography ligands known in the art. In some embodiments, the total density of all of the ligands attached to the support (i.e. including ligands of formula (I) and further ligands) may be from about 15 to about 150 pmol mL1, such as about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 pmol mL1. In some embodiments, the total density of all of the ligands attached to the support may be from about 15 to about 130 pmol mL1, or from about 15 to about 70 pmol mL1, or from about 20 to about 50 pmol mL1.

[0067] The support may be made of different types of materials and may have different shapes or forms, as described in more detail below.

[0068] The support of the chromatography material may comprise porous particles, non-porous particles, or expanded bed media, or may be a convective flow material. Porous particles may alternatively be called beads, and a chromatography material comprising such particles or beads may be called a resin. Porous particles may for example be made of agarose, such as cross-linked agarose. A non-limiting example of porous agarose particles is Capto ImpRes resins (Cytiva, Sweden), comprising substantially spherical particles. 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).

[0069] Suitable particle sizes of the presently disclosed chromatography material may be in a diameter range of 5-200 pm, such as 10-100 pm, e.g., 20-70 pm. In a specific embodiment, the average particle size is in the range of from about 25 pm to about 60 pm, such as about 25, 30, 35, 40, 45, 50, or 60 pm. Suitable average pore sizes of the presently disclosed chromatography material in the form of particles may be of sizes 2-3 times larger than the target entities and impurities to be separated, including but not limited to an average pore diameter of from about 20 nm (e.g., suitable for separation of monoclonal antibodies) to about 80 nm, such as about 20 nm, 30 nm, 50 nm, 75 nm, or 80 nm, preferably from about 30 nm to about 80 nm. The skilled person in this field can easily choose the suitable particle size and porosity depending on the process to be used.

[0070] Non-limiting examples of non-porous particles are solid glass beads. A non-limiting example of expanded bed media is STREAMLINE™ resins (Cytiva, Sweden). Cytiva ref. P2023-0219-SE01

[0071] The matrix material may be a convective flow matrix material. Such a material may for example be an adsorptive membrane where a flow through such materials is convective rather than diffusional. A convective matrix material includes any matrix in which application of a hydraulic pressure difference between the inflow and outflow of the matrix forces perfusion of the matrix, achieving substantially convective transport of the substance(s) into the matrix or out of the matrix, which can be effected very rapidly at a high flow rate. Examples of convective matrix materials include porous adsorptive membranes, fibrous materials, and monolithic materials.

[0072] The adsorptive membrane can for example be a polymeric membrane, such as a polyether sulfone membrane, e.g., Mustang™ membrane chromatography capsules (Cytiva, Sweden). Another example of an adsorptive membrane is a polymer nanofiber membrane, such as for example cellulose, cellulose acetate and cellulose fibres, which have been treated for use as an adsorbent. Treatment may include one or more of cross-linking, derivatization, and coupling of a ligand. The matrix material may be a non-woven material comprising fibres, such as from cellulose. Such fibrous substrate may be based on electrospun polymeric fibres or cellulose fibres, and may e.g. have a cross-sectional diameter of 10- 1000 nm, such as 200-800 nm, 200-400 nm or 300-400 nm. Convection-based chromatography and membrane adsorbers are described in for example US20140296464A1, US20160288089A1, US2019308169A1 and US2019234914A1, hereby incorporated by reference in their entireties.

[0073] A polymer used for a polymeric membrane may be a natural or synthetic polymer, including a derivatized polymer. For example, a hydroxylated polymer may be used, such as to provide a hydroxylated polymeric membrane. A hydroxylated polymer may include a glycopolymer. In addition to cellulose and polyether sulfone as mentioned above, polymers useful in adsorptive membranes include polymers based on polytetrafluoroethylene (PTFE), polypropylene, polyamide, or polyacrylamide.

[0074] The adsorptive membrane could alternatively be a monolithic material, or a conventional membrane made by emulsification. Another alternative is a 3D-printed material. A non-limiting example of a monolith is CIMmultus® (Sartorius, Germany).

[0075] Mean flow pore (MFP) size is an indicator of material flow characteristics, and is measured by capillary flow porometry, based on the displacement of a wetting liquid with a known surface tension from the sample pores by applying a gas at increasing pressure. The higher the MFP size, the larger the flow of liquid through the material at a given pressure. The mean flow pore size is calculated from the point at which 50 % of the flow goes through a sample. Mean flow pore size thus corresponds to the pore size calculated at the pressure where the wet curve and the half-dry curve meet. Cytiva ref. P2023-0219-SE01

[0076] In some embodiments, the support comprises beads having a volume-weighted median diameter (D50v) of from about 20 pm to about 60 pm, such as about 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, or 60 pm, preferably from about 30 pm to about 55 pm, currently more preferably from about 40 pm to 50 pm.

[0077] The chromatography material may comprise a compound which couples the ligand(s) to the support. The terms "linker", "extender", and "surface extender" may be used to describe such a compound, as further described below. In such embodiments, the linking group in Formula (I) is a group of the linker, extender or surface extender.

[0078] In some embodiments, at least a portion of the plurality of ligands attached to the support are directly attached to the support without the presence of a linker. In such examples, in Formula (I), the linking group L is a functional group which is inherently part of the support. For example, the support may be a polysaccharide such as agarose. Agarose is a linear polysaccharide made up of alternating D- galactose and 3,6-anhydro-alpha-L-galactopyranose residues. The repeat unit is shown below. Agarose comprises free hydroxyl groups, including, for example, hydroxyl groups on the C2, C3 and C6 carbon atoms of the galactose ring, as numbered below.

[0079] Attachment of a ligand to agarose may, for example, be achieved as shown below, via an oxygencarbon bond. In this example, the linking group L in Formula (I) is the C6 -CH2O- group of the galactose ring. As will be described in more detail below, such materials can be prepared by the method of the second aspect by reaction of the C6 CH2OH group with a ligand comprising a leaving group.

[0080] Whilst chromatography materials known in the art typically require the addition of linkers as described above in order to achieve successful coupling of the ligand to the support, the inventors have surprisingly found that chromatography materials of the present invention can be prepared through a simple method (i.e. the method of the second aspect) that is suitable for coupling the ligands directly to the support, without the presence of a linker on the support prior to coupling. Cytiva ref. P2023-0219-SE01

[0081] Repeat unit of agarose comprising a ligand of Formula (I) directly attached via the C6 -CH2O- group of agarose.

[0082] In some embodiments, the ligands may be attached to the support via a N, S or O atom of a functional group on the surface of the support. For example, the "free" support (i.e. without ligands) may comprise hydroxyl groups, amine groups or thiol groups, or combinations thereof, and the ligands may be attached to the support via N, S or O atoms of the hydroxyl groups, amine groups or thiol groups, respectively. In other examples, a linker may be attached to the support, wherein the linker comprises hydroxyl groups, amine groups or thiol groups, and the ligands may be attached to the support via N, S or O atoms of the hydroxyl groups, amine groups or thiol groups, respectively, of the linker.

[0083] Spacers, such as 2-12 carbon alkyls, linear or branched, or 2-12 carbon ethers, can be used between the ligand and a linker, such as vinyl sulfone. The linker may also react with an extender group and link it to the ligand or spacer. The spacer is the length of carbons between the linker and the ligand used to improve the base stability of the linker-ligand bond.

[0084] The ligand may be coupled to the support via an "extender group", or simply "extender". In such embodiments, the linking group L in Formula (I) comprises or consists of an extender. In some embodiments, the extender comprises N, S or O atoms, or a combination thereof, and the ligands are attached to the N, S or O atom of the extender via a covalent bond.

[0085] The extender may be selected from polysaccharide structures and polymeric structures. The extender may be e.g. dextran, acrylamides or polyglycerol. If dextran is used as an extender, it may have a molecular weight in a range of from 5,000 to 2,000,000 Dalton. The extender group can be synthesized from the support through polymerisation (undetermined length) or alternatively coupled to the support surface.

[0086] The second aspect provides a method for preparing a chromatography material according to the first aspect, the method comprising: Cytiva ref. P2023-0219-SE01 providing a support comprising one or more linking groups, L; providing one or more ligands; and mixing the support and the one or more ligands for a period of time; wherein: the one or more linking groups L comprise a nucleophilic group; and the one or more ligands have the structure according to formula (II):

[0087] Formula (II) wherein Ri is as defined in the material of first aspect;

[0088] R2 is H or C1-C5 alkyl; and

[0089] X is a leaving group; optionally wherein X is Cl or Br.

[0090] The C1-C5 alkyl may be linear, branched or cyclic, and may be substituted or unsubstituted. In some embodiments, C1-C5 alkyl is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or isopentyl. In some embodiments, C1-C5 alkyl is methyl or ethyl. In some embodiments, R2 is H, methyl or ethyl. In some embodiments, R2 is ethyl.

[0091] In some embodiments, the nucleophilic group of the linking group L is a hydroxyl group, an amine group or a thiol group. In some embodiments, the linking group L is a hydroxyl group. Some supports may comprise more than one type of linking group L. For example, the support may inherently comprise more than one type of linking group L, or further types of linking groups may be introduced by the attachment of linkers as described hereinabove in relation to the materials of the first aspect.

[0092] In some embodiments, the providing one or more ligands having the structure according to formula (II) may involve providing two or more ligands having the structure according to formula (II). For example, in some embodiments, the method may comprise mixing the support with two ligands each having the same Ri and R2 groups, but a different leaving group X. In some embodiments, the method may comprise mixing the support with two ligands each having the same R2 group and leaving group X, but a different Ri group. This allows a mixed-ligand chromatography material [i.e a chromatography material comprising more than one type of ligand of formula (I)] to be prepared by the method of the second aspect. Cytiva ref. P2023-0219-SE01

[0093] In some embodiments, the method further comprises an additional step of providing one or more ligands. This additional step may be carried out before or after the providing step described hereinabove. This additional step may comprise providing ligands having the structure according formula (II), or it may comprise providing ligands having a different structure, such as other chromatography ligands known in the art. When this additional step comprises providing one or more ligands having the structure according to formula (II), the ligands provided in both steps may be the same or different. For example, the ligands may be the same, and the additional step may be included to increase the ligand density of the chromatography material compared with that which was achieved after a single step. In other examples, the ligands may be different, and the additional step may be included to introduce further ligands to the chromatography material compared with those present after the first step.

[0094] In some embodiments, the method further comprises providing a base. In some embodiments, the base is an alkali metal hydroxide. For example, the base may be KOH or NaOH. In some embodiments, the base is provided along with the support, and the support and base are mixed for a period of time prior to providing the one or more ligands.

[0095] In some embodiments, the mixing is carried out under alkaline conditions, for example at a pH greater than or equal to 7, or greater than or equal to 8, or greater than or equal to 9, or greater than or equal to 10, or greater than or equal to 11, or greater than or equal to 12, or greater than or equal to 13, or greater than or equal to 14. It may be advantageous to carry out the coupling under very basic conditions, such as a pH greater than or equal to 12, or greater than or equal to 13, or greater than or equal to 14.

[0096] The temperature during mixing may be approximately room temperature, or a temperature above room temperature. In some embodiments, the temperature during mixing may be a about 15 to 50 °C; about 20 to 45 °C; about 25 to 40 °C; or about about 30 to 35 °C. For example, the temperature may be about 15 °C, or about 20 °C, or about 25 °C, or about 30 °C, or about 35 °C, or about 40 °C, or about 45 °C, or about 50 °C.

[0097] The method according to the second aspect may be used to prepare a chromatography material having any of the ligand densities described hereinabove with respect to the first aspect. For example, the ratio of the ligand and support may be varied. In some examples, the one or more ligands may be provided in an amount from about 0.1 mmol of ligand per mL of support to about 10 mmol of ligand per m L of support (mmoliigand.m Lsupport-1), or from about 1 mmoliigand.m Lsupport-1to about 5 mmoliigand.mLsupport1. The mixing is typically carried out for a period of time from about 1 hour to about 24 hours or more. However, the skilled person, through routine experimentation, will be able to Cytiva ref. P2023-0219-SE01 determine suitable conditions (e.g. reaction time, ligand:support ratio, number of successive ligand addition steps) to achieve successful coupling and desired ligand densities. Examples of ligands according to formula (II) may be liquids or solids. In the providing step, the ligands may be provided as a neat liquid or solid, other the ligands may be provided as a solution of the ligand in a suitable solvent. Reactions are typically carried out under aqueous conditions i.e. in a solvent consisting or comprising water. Support materials are typically in the form of a gel and so a suitable amount of water may be added in order to achieve adequate stirring of the reaction medium.

[0098] After the mixing steps have been completed, the obtained chromatography material may be washed with further solvents. Suitable washing solvents include alcohols, ketones and water. In some examples, the obtained chromatography material is washed with ethanol, acetone and water.

[0099] In some embodiments, the one or more ligands comprise one or more ligands selected from: Cytiva ref. P2023-0219-SE01

[0100] In some embodiments, the one or more ligands comprise one or more ligands selected from: Cytiva ref. P2023-0219-SE01

[0101] In some embodiments, the one or more ligands comprise one or more ligands selected from:

[0102] 5 In some embodiments, the one or more ligands comprise or consist of: Cytiva ref. P2023-0219-SE01 and preferably

[0103] The third aspect provides a use of the chromatography material according to the first aspect for separating one or more target entities from one or more impurities. The fourth aspect provides a method for separating one or more target entities from one or more impurities, the method comprising adding a liquid sample comprising one or more target entities and one or more impurities to a chromatography material according to the first aspect. The description of the one or more target entities and the one or more impurities applies equally to the uses of the third aspect and the methods of the fourth aspect.

[0104] The one or more target entities may comprise or consist of one or more biomolecules. In some embodiments, the one or more target entities may comprise or consist of one or more polypeptides and / or one or more nucleic acid sequences. In some embodiments, the one or more target entities may comprise or consist of one or more polypeptides. In some embodiments, the one or more target entities may comprise one or more selected from: enzymes, antibodies, parts of antibodies, antibody fragments, carbohydrates and nucleic acid sequences (e.g. RNA and / or DNA). In some embodiments, the one or more target entities may comprise or consist of one or more polypeptides selected from: enzymes, antibodies, parts of antibodies and antibody fragments. In some embodiments, the one or more target entities may comprise or consist of one or more nucleic acid sequences selected from: RNA and DNA.

[0105] In some embodiments, a target entity may be a non-aggregated macromolecule while the impurities, from which the target entity is to be separated, may include aggregates, and / or fragments, of said macromolecule, typically a protein, such as an antibody.

[0106] In some embodiments, the one or more target entities comprise one or more antibodies and / or parts of antibodies. In some embodiments, the antibodies are monoclonal antibodies. Optionally, the monoclonal antibodies are multispecific monoclonal antibodies, such as bispecific monoclonal antibodies. Alternatively, the one or more target entities may be one or more antibody fragments. Optionally, the one or more antibody fragments may be selected from antigen-binding fragments as described and exemplified in detail elsewhere herein, e.g., Fab, Fab', F(ab')2, scFv, Fv, dAb, or Fd. Cytiva ref. P2023-0219-SE01

[0107] The one or more impurities may comprise aggregates of the one or more target entities, such as high molecular weight aggregates of the target entities. For example, when the one or more target entities comprises an antibody, such as a monoclonal antibody, the one or more impurities may comprise high molecular weight aggregates of said antibody. When the one or more target entities comprise one or more antibody fragments, the one or more impurities may comprise high molecular weight aggregates of the one or more antibody fragments. When the one or more target entities comprise a bispecific antibody, the one or more impurities may comprise product-related impurities like various mismatch entities of said bispecific antibodies, such as homodimers of said bispecific antibodies. Irrespective of the nature of the one or more target entities, the one or more impurities may comprise host cell proteins.

[0108] Figure 1 shows a method 100 for separating one or more target entities from one or more impurities, which is a method according to the fourth aspect. The method 100 comprises: adding 110 a liquid sample comprising one or more target entities and one or more impurities to a chromatography material according to the first aspect; eluting 120 the one or more target entities from the chromatography material according to the first aspect; optionally, eluting 130 the one or more impurities from the chromatography material according to the first aspect.

[0109] In accordance with said method 100, upon adding 110 the liquid sample to the chromatography material and choosing suitable chromatographic conditions, the one or more target entities present in the liquid sample will bind to the ligand(s) of the chromatography material. The one or more impurities may optionally bind to the ligand(s) of the chromatography material, depending on which impurities are present in the liquid sample and on which binding conditions are applied. The pH and the salt concentration are normally kept constant during the binding of the target entities and / or impurities to the ligand(s) of the chromatography material. To do this, the liquid sample in 110 may comprise one or more target entities, one or more impurities, and a binding buffer. The binding buffer may have a pH in the range of about 3-12, or about 4 to 8.

[0110] Fractions that pass through the chromatography material without binding to the ligand(s) of the chromatography material are referred to as "flow-through fractions". These fractions are eluted from the chromatography material prior to eluting 120 the one or more target entities. Impurities which do not bind to the ligand(s) of the chromatography material will be eluted in the flow-through fractions. Cytiva ref. P2023-0219-SE01

[0111] The one or more target entities, if bound to the ligand, may be eluted 120 from the chromatography material by applying an elution buffer comprising a salt. The elution may be performed isocratically, i.e., by keeping the composition of the elution buffer constant throughout the elution process, or by i) a salt gradient, (ii) a pH gradient, or a combination of (i) and (ii). In other words, the elution may 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). The eluate obtained after the eluting 120 of the method 100 comprises the one or more target entities.

[0112] The one or more target impurities, if bound to the ligand, may optionally be eluted 130 from the chromatography material by applying an elution buffer comprising a salt. The elution may be performed isocratically, i.e., by keeping the composition of the elution buffer constant throughout the elution process, or by i) a salt gradient, (ii) a pH gradient, or a combination of (i) and (ii). In other words, the elution may 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). The eluate obtained after the optional eluting 130 of the method 100 comprises the one or more impurities.

[0113] Buffering systems, including the binding buffer and the elution buffer, that are suitable for separation of various types of target entities are well known in the art and can easily be chosen by the skilled person.

[0114] In some embodiments, the eluting 120 of target entities, and optionally eluting 130 of impurities, may be performed within a pH range of about 3-12, or about 4-8, and at a salt concentration of 0.2-2 M NaCI. In some embodiments, the eluting 120 of target entities, and optionally elution 130 of impurities, may be performed at constant pH by applying a salt gradient, where the pH of the binding buffer and the elution buffer is the same and a constant salt gradient from 0 M NaCI to about 1 M NaCI is applied.

[0115] Figure 2 shows a method 200 for separating one or more target entities from one or more impurities, which is another method according to the fourth aspect. The method 200 comprises: adding 210 a liquid sample comprising one or more target entities and one or more impurities to a chromatography material according to the first aspect; obtaining 220 the one or more target entities in a flow-through mode, the one or more target entities having passed through the chromatography material essentially without binding to the chromatography material; optionally eluting 230 the one or more impurities from the chromatography material. Cytiva ref. P2023-0219-SE01

[0116] In the method 200, the one or more target entities essentially do not bind to the chromatography material while at least one or more of the impurities essentially bind to the chromatographic material. In this example, the one or more target entities are obtained in the flow-through fractions. As such, the method 200 may be described as a "flow-through method" and may be especially suitable when using a chromatography material comprising a support in the form of a membrane (e.g., Fibro).

[0117] The above-disclosed methods 100 and 200 for separating one or more target entities from one or more impurities may comprise a step 102 preceding step 110, or a step 202 preceding step 210, respectively, wherein step 102 / 202 comprises pre-treating the liquid sample. Optionally, said pre-treating may comprise subjecting a target entity-containing cell culture harvest to cell lysis, clarification, and / or filtration. Methods of cell culture clarification are known in the art and may include one or more processes such as (but not limited to) centrifugation, tangential flow filtration, depth filtration, and sterile filtration.

[0118] The methods 100 / 200 for separating one or more target entities from one or more impurities may comprise a step 104 preceding step 110, or a step 204 preceding step 210, respectively, wherein step 104 / 204 comprises pre-purifying the one or more target entities by separating target entities from a target entity-containing cell culture harvest, thereby obtaining a pre-purified liquid sample comprising target entities, before adding said pre-purified liquid sample comprising target entities to the herein disclosed chromatography material. Optionally, said pre-purifying may comprise subjecting the target entity-containing cell culture harvest to chromatography. In some examples, the pre-purifying step 104 / 204 may comprise applying affinity chromatography. For example, a Protein A based affinity resin may be used for purification of monoclonal antibodies. Alternatively, the pre-purifying step may comprise using magnetic beads, monoliths, or membranes. The affinity chromatography may be performed by means of periodic counter current (PCC) chromatography. In some examples, the methods 100 / 200 may comprise both the pre-treating 102 / 202 (e.g. clarification) and the pre-purifying 104 / 204.

[0119] The methods 100 / 200 for separating one or more target entities from one or more impurities may comprise one or more purification steps 140 being performed after the step of eluting 120 the one or more target entities, or one or more purification steps 240 being performed after the step of obtaining 220 the one or more target entities, respectively, from the presently disclosed multimodal chromatography material. Non-limiting examples of such one or more purification steps 140 may include hydrophobic interaction chromatography, chromatography using Capto Core beads (Cytiva, Sweden), an additional orthogonal ion exchange step, such as an anionic exchange chromatography step, and / or multimodal anionic exchange chromatography. Cytiva ref. P2023-0219-SE01

[0120] The fifth aspect provides a chromatography device, comprising a chromatography material according to the first aspect.

[0121] In some embodiments, the chromatography device may be a chromatography column. The chromatography column may comprise chromatography material according to the first aspect, wherein the support comprises porous particles, a monolith, expanded bed media, or non-porous particles, such as polystyrene beads.

[0122] In some embodiments, the chromatography device may be a membrane adsorber unit, such as a capsule or a cassette. The membrane adsorber unit may comprise chromatography material according to the first aspect, wherein the support comprises a convective flow material, such as a porous adsorptive membrane or a fibrous material.

[0123] In some embodiments, the chromatography device may be a container comprising non-porous particles, such as solid glass beads, or porous particles, such as magnetic beads.

[0124] The present disclosure further provides a use of the above-described chromatography device for separating one or more target entities from one or more impurities.

[0125] As is well-known in the art, a chromatography cycle normally includes the following steps: equilibration of the chromatography material, loading of sample comprising target entities and impurities, washing of the chromatography material, elution of target entities (unless obtained in the flow-through during the loading step), cleaning-in-place (CIP) of the chromatography material, and re-equilibration of the chromatography material. It is to be understood that the chromatography material is normally present in a chromatography device throughout all steps of a chromatography cycle.

[0126] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art related to this invention. Also, the singular forms "a", "an", and "the" are meant to include plural reference unless it is stated otherwise.

[0127] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0128] EXPERIMENTAL SECTION

[0129] The experimental data disclosed herein demonstrate a successful separation of several different target entities using a chromatography material according to the first aspect. The target entities were as follows: Ribonuclease A (RNAse), Cytochrome C (Cyt C), Lysozyme (Lys) and a-Chymotrypsinogen. Cytiva ref. P2023-0219-SE01

[0130] Materials and methods

[0131] The Capto MMC ImpRes multimodal resin and the MMC ImpRes base matrix were obtained from Cytiva (Uppsala, Sweden). The target entities (RNAse, Cyt C, Lys and a-Chymotrypsinogen) were obtained from Sigma Aldrich. Sodium chloride and sodium dihydrogen phosphate were obtained from VWR (Randor, USA). Citric acid monohydrate, sodium citrate dihydrate and sodium chloride were of analytical grade and were purchased from Merck (Darmstadt, Germany).

[0132] Synthesis of the Chromatography Materials

[0133] The chromatography materials used in this example consisted of the same support and ligand. Several chromatography materials were tested, each having a different density of the ligand. The support was an agarose base matrix (MMC ImpRes, Cytiva). The chromatography materials were synthesised as follows:

[0134] The agarose base matrix MMC Impres (8 mL) was washed with distilled water 5 times. The gel was drained and added to a 50 mL glass reactor with hanging magnetic stirrer bar. Distilled water (1 mL) was added to the drained gel after which NaOH 50% w / w (3.12 mL) was added. The slurry was stirred for 30 min in a 33 °C water bath. The ligand ethyl a-bromophenylacetate was added slowly (almost dropwise) to the gel slurry over a 10 min period. The reaction was stirred at 33 °C for a period of time. In some examples, a further ligand addition step was included using the same ligand ethyl a- bromophenylacetate. The gel was then washed 3 times with EtOH, 2 times with acetone, and 6 times with distilled water. The drained gel was then stored in 20% EtOH / O.2 M NaOAc solution in a fridge. The reaction of the ligand with a surface hydroxyl group of the base matrix is shown below in Scheme 1.

[0135] Agarose base matrix

[0136] MMC ImpRes

[0137] Chromatography Material

[0138] Scheme 1 Cytiva ref. P2023-0219-SE01

[0139] Five materials were synthesized. The conditions are summarized in Table 1 and the materials are labelled A-E. The reaction conditions were varied to achieve different ligand densities, including: varying the supportdigand ratio; varying the reaction time; and including a second addition of the ligand ethyl a-bromophenylacetate (referred to as reaction number 2 in Table 1). The resulting materials had ligand densities from 27 to 116 pmol mL1(as determined by titration).

[0140] Table 1 Chromatography materials A-E tested in this example.

[0141] The ligand densities given in Table 1 were determined by titration as follows:

[0142] The chromatography material was washed with distilled water, then KCI 1 M (3 x 1 gel volume). The gel is drained and packed in a 1 mL cubing device. The drained gel cubes (1 mL) together with 19 mL of 1 M KCI solution are transferred to a 50 mL titration cup. The pH of the mixture is adjusted to 9-10 and titration with 0.1 M HCI gives the ligand density of the chromatography material in pmol mL1. A blank sample of 20 mL of 1 M KCI is also titrated with 0.1 M HCI for background subtraction.

[0143] A reference chromatography material, Capto ImpRes MMC, was used for comparison with the materials A-E. The ion capacity of this reference material was around 34-42 pmol mL1. This reference material comprises the same agarose base matrix as the chromatography materials A-E, but a different ligand. The structure of Capto ImpRes MMC is shown below. To prepare this reference material, the base matrix needs to be activated first by allylation using allyl glycidyl ether (AGE), followed by bromination using Br2. The ligand N-phenyl-D,L-homocysteine thiolactone is synthesized by coupling D,L-homocysteine thiolactone hydrochloride with benzoyl chloride, then activated with NaOH prior to reaction with the allylated base matrix to form the Capto ImpRes MMC. The method used to prepare Cytiva ref. P2023-0219-SE01 the chromatography materials A-E of the present invention is comparably much simpler. In addition to hydrophobic interactions due to the aromatic ring and cation exchange by the carboxylate group, this material is also capable of hydrogen bonding due to the amine group and thiophilic interactions due to the sulphur atom in the backbone.

[0144] Structure of the reference chromatography material Capto ImpRes MMC.

[0145] Linear salt gradient elution study

[0146] Sample Preparation

[0147] Target entity samples were prepared in two different buffers to test their performance at two different pH values: (1) pH 4.5 and (2) pH 7.6. The samples each comprised all four target entities at a concentration of approximately 5 mg mL1of each of the proteins.

[0148] For pH 4.5, the target entity samples were prepared in an "Elution A" buffer of 20 mM sodium citrate at pH 4.5. To apply a salt gradient, an "Elution B" buffer of 20 mM sodium citrate at pH 4.5 and 1 M sodium chloride was used.

[0149] For pH 7.6, the target entity samples were prepared in an "Elution A" buffer of 35 mM sodium phosphate at pH 7.6. To apply a salt gradient, an "Elution B" buffer of 35 mM sodium phosphate at pH 7.6 and 1 M sodium chloride was used.

[0150] Column preparation

[0151] HiTrap 1 mL chromatography columns (Cytiva) were packed with one of the chromatography materials A-E, or the reference material Capto MMC ImpRes.

[0152] Chromatography Experiments

[0153] The tests were conducted on an AKTA Explorer 10XT liquid chromatography system. Each chromatography experiment was performed in 5 different steps including equilibration, sample loading, column wash, elution, cleaning-in-place (CIP), and re-equilibration. After equilibration (7 column volumes), the target entity sample (20 pL) was loaded onto the column and the column was washed with Elution A buffer (1 column volume). The elution was conducted in a salt gradient of 20 Cytiva ref. P2023-0219-SE01 column volumes from 0 to 100% "Elution B buffer" (i.e. starting at 100% Elution A buffer / 0% Elution B buffer and ending at 0% Elution A buffer / 100% Elution B buffer). CIP was carried out with 1 M NaOH (5 column volumes), followed by re-equilibration (8 column volumes).

[0154] Fractions from elution were collected and UV chromatography was used to determine the presence of the target entities in the fractions. A wavelength of 405 nm was used for identification of Cytochrome C (Cyt C) in the UV chromatogram, and 215 nm or 280nm nm was used for identification of Ribonuclease A (RNAse), Lysozyme (Lys) and a-Chymotrypsinogen. Conductivity measurements were used to quantify the amount of each target entity in the fractions.

[0155] Results and discussion The results at pH 4.5 and pH 7.6 are shown in Figures 3 and 4, respectively. Despite having different ligand structures, the chromatography materials A-E performed comparably to the reference material Capto ImpRes MMC. In particular, chromatography materials C and D showed very similar absolute conductivity to the reference material, as well as similar selectivities for the four proteins. Material E showed much higher overall conductivity, which may have been due to the much higher ligand density of this material compared with the reference material.

[0156] This example shows that chromatography materials of the present invention possess a much simpler ligand structure than a known chromatography material, yet still perform comparably in chromatography tests.

Claims

Cvtiva ref. P2023-0219-SE01CLAIMS1. A chromatography material comprising a support and a plurality of ligands attached to the support, wherein at least a portion of the plurality of ligands have the structure given by Formula (I):Formula (I) wherein:L is a linking group covalently attached to the support; andRi is aryl, substituted aryl, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

2. The chromatography material according to claim 1, wherein the ligand is attached to a N, S or O atom of the linking group via a covalent bond.

3. The chromatography material according to claim 1 or claim 2, wherein Ri is:wherein:Rs is H, halo, or C1-C5 alkyl; and* indicates the point of attachment of Ri in Formula (I).

4. The chromatography material according to claim 3, wherein R3 is H, Cl or Br.

5. The chromatography material according to claim 1 or claim 2, wherein Ri is C?-Cg alkyl or C?-Cg halo-substituted alkyl.

6. The chromatography material according to any of the preceding claims, wherein the ligand having the structure given by Formula (I) is present at a concentration of about 15 to about 150 pmol per mL of the chromatography material.Cytiva ref. P2023-0219-SE017. A method of preparing a chromatography material according to any of the preceding claims, the method comprising: providing a support comprising one or more linking groups, L; providing one or more ligands; and mixing the support and the one or more ligands for a period of time; wherein: the one or more linking groups L comprise a nucleophilic group; and the one or more ligands have the structure according to formula (II):Formula (II) wherein Ri is as defined in any of the preceding claims;R2 is H or C1-C5 alkyl; andX is a leaving group; optionally wherein X is Cl or Br.

8. The method according to claim 7, wherein the one or more ligands comprise one or more ligands selected from:Cytiva ref. P2023-0219-SE019. The method according to claim 8, wherein the one or more ligands comprise or consist of:

10. The method according to any of claims 7 to 9, wherein the nucleophilic group of the linking group L is a hydroxyl group, an amine group or a thiol group.

11. The method according to any of claims ? to 10, wherein the mixing is carried out at a pH greater than or equal to 10.

12. Use of the chromatography material according to any of claims 1 to 6 to separate one or more target entities from one or more impurities.

13. The use according to claim 12, wherein the one or more target entities comprises or consists of one or more biomolecules, optionally wherein the biomolecules are polypeptides or nucleic acid sequences.

14. The use according to claim 12 or 13, wherein the one or more target entities comprise one or more antibodies and / or parts of antibodies, preferably wherein the antibodies are monoclonalCytiva ref. P2023-0219-SE01 antibodies, optionally wherein the monoclonal antibodies are multispecific monoclonal antibodies, optionally wherein the multispecific antibodies are bispecific antibodies and the one or more impurities comprise mismatch entities of said bispecific antibodies, such as homodimers of said bispecific antibodies.

15. The use according to claim 12 or 13, 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.

16. A method for separating one or more target entities from one or more impurities, the method comprising: adding a liquid sample comprising one or more target entities and one or more impurities to a chromatography material according to any of claims 1 to 6; eluting the one or more target entities from the chromatography material; optionally eluting the one or more impurities from the chromatography material.

17. The method according to claim 16, wherein the one or more target entities, and optionally the one or more impurities, are eluted from the chromatography material by using an elution buffer comprising a 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.

18. A method for separating one or more target entities from one or more impurities, comprising: adding a liquid sample comprising one or more target entities and one or more impurities to a chromatography material according to any of claims 1 to 6; obtaining the one or more target entities in a flow-through mode, the target entities having passed through the chromatography material essentially without binding to the chromatography material; optionally eluting the one or more impurities from the chromatography material.

19. The method according to any of claims 16-18, wherein the one or more target entities comprises or consists of one or more biomolecules, optionally wherein the biomolecules are polypeptides or nucleic acid sequences.

20. The method according to any of claims 16-18, wherein the one or more target entities comprise one or more antibodies and / or parts of antibodies, preferably wherein the antibodies are monoclonal antibodies, optionally wherein the monoclonal antibodies are multispecific monoclonal antibodies, optionally wherein the multispecific antibodies are bispecific antibodies and the one or more impurities comprise mismatch entities of said bispecific antibodies, such as homodimers of said bispecific antibodies.

21. The use according to any of claim 14, or the method according to claim 20, wherein the one or more impurities comprise aggregates of the one or more antibodies.Cytiva ref. P2023-0219-SE0122. The method according to any one of claims 16-18, wherein the one or more target entities comprise 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')j, scFv, Fv, dAb, or Fd.

23. The use according to any of claim 15, or the method according to claim 22, wherein the one or more impurities comprise aggregates of the one or more antibody fragments.

24. The use or method according to any of claims 12 to 23, wherein the one or more impurities comprise host cell proteins.

25. A chromatography device, comprising a chromatography material according to any of claims 1 to 6; optionally wherein the chromatography device is disposable, optionally wherein the chromatography device is selected from: a. a chromatography column, optionally comprising chromatography material having a support comprising porous particles, a monolith, expanded bed media, or non-porous particles; b. a membrane adsorber unit, optionally comprising chromatography material having a support comprising a convective flow material, such as a porous adsorptive membrane or a fibrous material; and c. a container comprising non-porous particles, such as solid glass beads, or porous particles, such as magnetic beads.

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