WO2024068682A1 - Ligand de chromatographie et matériau de chromatographie, et leurs utilisations - Google Patents

Ligand de chromatographie et matériau de chromatographie, et leurs utilisations Download PDF

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WO2024068682A1
WO2024068682A1 PCT/EP2023/076622 EP2023076622W WO2024068682A1 WO 2024068682 A1 WO2024068682 A1 WO 2024068682A1 EP 2023076622 W EP2023076622 W EP 2023076622W WO 2024068682 A1 WO2024068682 A1 WO 2024068682A1
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chromatography
impurities
target molecules
ligand
chromatography material
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English (en)
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Jean-Luc Maloisel
Gunnar Malmquist
Lalita Shekhawat KANWAR
Todd F. MARKLE
Keyhan ESFANDIARFARD
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Cytiva Bioprocess R&D Ab
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28004—Sorbent size or size distribution, e.g. particle size
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3847—Multimodal interactions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28011—Other properties, e.g. density, crush strength
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28016—Particle form
    • B01J20/28019—Spherical, ellipsoidal or cylindrical
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B01J20/289—Phases chemically bonded to a substrate, e.g. to silica or to polymers bonded via a spacer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3291—Characterised by the shape of the carrier, the coating or the obtained coated product
    • B01J20/3293—Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/165—Extraction; Separation; Purification by chromatography mixed-mode chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase
    • B01D15/327—Reversed phase with hydrophobic interaction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/362—Cation-exchange

Definitions

  • the present disclosure is directed to a chromatography ligand and a chromatography material, uses thereof for separating one or more target molecules from impurities, and a method for separating one or more target molecules from impurities.
  • the present disclosure relates to separation of target molecules, such as antibodies or antibody fragments, from impurities comprising aggregates of the one or more target molecules.
  • Bio-macromolecules such as proteins, nucleic acids, and polysaccharides, may often partially occur in the form of aggregates, or multimers, such as dimers, trimers, or higher oligomers.
  • aggregates or multimers, such as dimers, trimers, or higher oligomers.
  • the conditions may favor the formation of such aggregates through intermolecular disulphide linkages or other covalent bonds, or through non-covalent interactions.
  • the presence of such aggregates of a target macromolecule is many times undesired. Protein aggregation is thus a common issue encountered during bioprocess development and manufacturing of biotherapeutics.
  • Aggregated forms of a 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 undesired side-effects. Hence, it is essential for therapeutic safety that a therapeutic protein is in a non-aggregated state and that there are no aggregates of molecules present in the final product.
  • Preparative chromatography remains the primary technique for purification of therapeutic proteins due to its benefits of resolution, scalability, and robustness.
  • Monoclonal antibodies are one of the most powerful therapeutic tools in curing an increasing number of diseases.
  • the continued development in upstream processing has resulted in increased purification complexities with regard to the types and content of impurities, e.g. due to higher titers.
  • the heterogeneity of monoclonal antibodies with differences in their charge distribution and size due to various molecular modifications over its lifespan from cell culture to polishing increases challenges in the polishing steps.
  • the potential molecular modifications include charged acidic and basic variants, Fab fragment, and high molecular weight (HMW) aggregates, as well as glycosylation, deamidation, incomplete disulphide bond formation, oxidation, and isomerization etc. resulting in the formation of additional product related impurities.
  • HMW high molecular weight
  • Multimodal (or mixed mode) chromatography where small molecule ligands provide more than one type of interaction, is an important tool in downstream processing of therapeutic proteins.
  • a commercialized example of a multimodal is CaptoTM MMC ImpRes (Cytiva Sweden AB, Uppsala, Sweden). It is 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.
  • Xi is selected from CO and SO 2 ; each of R1-R5 is independently selected from H, F, Cl, 0, N, S, C1-3 alkyl, and C1-3 a lkyl-X 2 ; any two adjacent moieties selected from R1-R5 together with the atoms to which they are attached may form a 5- or 6-membered heterocyclic or carbocyclic ring; and
  • X 2 is selected from 0, S, NH(CO), (CO)NH, NH(SO 2 ), and (SO 2 )NH; and provided that: i. when each of R1-R5 is H, Xi is S0 2 ; ii. when any two adjacent moieties selected from R1-R5 together with the atoms to which they are attached form a 5-membered heterocyclic containing ring containing two oxygen atoms in the ring, Xi is SO 2 ; and ill. when three of R1-R5 are CH 3 O, Xi is CO.
  • the present disclosure further provides a method for preparing a chromatography material, comprising immobilising a plurality of the above-defined chromatography ligand to a support.
  • chromatography material comprising the above-defined chromatography ligand coupled to a support.
  • the present disclosure also provides 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 chromatography material as disclosed herein; b) eluting the target molecules from the chromatography material; c) optionally eluting the impurities from the chromatography material.
  • Also provided is 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 chromatography material as disclosed herein; b) obtaining the target molecules in a flow-through mode, the target molecules having passed through the chromatography material essentially without binding to the chromatography material; c) optionally eluting the impurities from the chromatography material.
  • Fig. 1 is a flow chart of a first method for separating one or more target molecules from impurities according to the present disclosure.
  • Fig. 2 is a flow chart of a second, alternative method for separating one or more target molecules from impurities according to the present disclosure.
  • Fig. 3 shows a comparison of separation resolution between HMW aggregate and monoclonal antibody (mAb) for different ligand prototypes for the data obtained using Methodi as described in Example 1 herein.
  • mAb monoclonal antibody
  • Fig. 4 shows a comparison of separation resolution between HMW aggregate and mAb for different ligand prototypes for the data obtained using Method2 as described in Example 1 herein.
  • Fig. 5 displays retention data for three of the novel ligand prototypes as described in Example 1 herein.
  • the present disclosure is directed to chromatography ligands for improved separation of one or more target molecules from impurities, wherein the impurities may include aggregates and/or fragments of said target molecules.
  • Capto MMC ImpRes ligand (Cytiva Sweden AB, Uppsala, Sweden) was chosen as a starting point for creating a large, chemically diverse virtual library of 100 Capto MMC analogue ligand structures. Based on these structures, physiochemical properties were predicted in silico, yielding a matrix of numerical descriptors (e.g. pKa, cLogP, etc). A subsequent principal component analysis (PCA) of these ligand descriptors resulted in a large chemical diversity map, which was used to select ligands for synthesis and coupling to an agarose base matrix. High-throughput plate-based screening, as well as analysis of column retention and resolution then generated numerical descriptors of chromatographic separation performance, which were connected to the chemical descriptors, guiding further cycles of synthesis and evaluation of the column separation resolution.
  • PCA principal component analysis
  • the present disclosure describes the selection, synthesis, and chromatographic evaluation of a smaller library of novel multimodal ligands against the reference ligand, i.e., Capto MMC ImpRes. It is shown in the Examples herein that the novel ligands, which are more hydrophobic than the reference ligand, achieved improved separation resolution between a monoclonal antibody and product-related impurities (i.e., aggregates and Fab fragments of said antibody), compared to the separation achieved by the reference ligand, when using linear salt gradient elution. More specifically, the monoclonal antibody was obtained at a higher purity and higher yield by use of the novel ligands.
  • product-related impurities i.e., aggregates and Fab fragments of said antibody
  • the prominent role of the secondary hydrophobic and hydrogen bonding interactions provided by the ligand chemical structure of more hydrophobic ligands in conjunction with electrostatic interactions results in their better performance with regard to removal of Fab fragment and aggregate impurities.
  • the present disclosure provides a chromatography ligand defined by formula I: wherein:
  • Xi is selected from CO and SO 2 ; each of R1-R5 is independently selected from H, F, Cl, 0, N, S, C1.3 alkyl, and C1.3 a lkyl-X 2 ; any two adjacent moieties selected from R1-R5 together with the atoms to which they are attached may form a 5- or 6-membered heterocyclic or carbocyclic ring; and
  • X 2 is selected from 0, S, NH(CO), (CO)NH, NH(SO 2 ), and (SO 2 )NH; and provided that: i. when each of R1-R5 is H, Xi is S0 2 ; ii. when any two adjacent moieties selected from R1-R5 together with the atoms to which they are attached form a 5-membered heterocyclic containing ring containing two oxygen atoms in the ring, Xi is S0 2 ; and ill. when three of R1-R5 are CH 3 O, Xi is CO.
  • 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, all of them are N. When there are a maximum of two heteroatoms, each of them may be independently selected from N, 0, and S.
  • any two adjacent moieties selected from R1-R5, together with the atoms to which they are attached, may form a 5-membered heterocyclic ring containing a maximum of one oxygen atom in the ring.
  • the "support” moiety of formula la represents a support, such as a chromatography bead, to which the ligand can be coupled.
  • the ligand is connected to the support via a covalent thioether bond formed at the thiol.
  • Formula la (a chromatography ligand coupled to a support) may alternatively be depicted as wherein the wavy moiety represents the coupling to said support.
  • support is used in formula la to show where the ligand may bind to the support.
  • chromatography 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 molecules and impurities, which are present in a liquid sample.
  • target molecule is intended to include macromolecules 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.
  • 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.
  • 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 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.
  • the macromolecule may be a nucleic acid sequence, which may for example be used as a vector, such as in a therapeutic application.
  • a macromolecule or a biomacromolecule may for example be a biopharmaceutical, i.e., a biological molecule, including but not limited to a biological macromolecule, 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.
  • 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.
  • 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.
  • 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.
  • 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 bivalent 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, monoclonal antibody or monoclonal antibody fragment covalently linked to other protein.
  • Exemplary antibodies are the immunoglobulin isotypes and different types of fragments, such as Fab, Fab', F (a b') 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).
  • fragments such as Fab, Fab', F (a b') 2, Fv, dAb (single domain antibody), and Fd (fragment obtained by papain hydrolysis of an immunoglobulin molecule followed by reduction of the disulfide bonds
  • scFv fragment obtained by papain hydrolysis of
  • a bispecific monoclonal antibody is an example of a multispecific antibody and is an artificial protein that can simultaneously bind to two different types of antigen or two different epitopes on the same antigen.
  • 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 process manufacturing.
  • vector is herein used to denote 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.
  • 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.
  • 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.
  • 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.
  • impurities is intended to mean any molecule or substance which is present in the liquid sample, and which is not the desired target molecule.
  • the term “impurities” includes aggregates, such as aggregates of the target molecule, such as high molecular weight aggregates of the target molecule.
  • the term “impurities” further includes fragments of the target molecule, for example when the target molecule is an intact antibody and undesired fragments of said antibody are present in the liquid sample.
  • impurities also includes host cell proteins (HCP), and in the case of bispecific antibodies, homodimers.
  • HCP host cell proteins
  • the target molecule according to the present disclosure is typically a non-aggregated macromolecule while the impurities, from which the target molecule is to be separated, typically include aggregates, and/or fragments, of said macromolecule, typically a protein, such as an antibody.
  • 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".
  • the non-aggregated macromolecule 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.
  • a non-aggregated macromolecule essentially does not have hydrophobic moieties or hydrophobic groups exposed on the surface.
  • 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 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.
  • HMW aggregates 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 molecules (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 molecule.
  • the target molecules e.g., a monoclonal antibody having a molecular weight of approx. 150kDa
  • dimers e.g., approx. 300kDa for monoclonal antibody dimers
  • trimers approximatelyx. 450kDa for monoclonal antibody trimers
  • high molecular weight aggregate may refer herein to an aggregate of a target molecule, which aggregate has a molecular weight which is approximately twice the molecular weight of the target molecule, or larger than twice the molecular weight of the target molecule.
  • hydrophobic moiety is intended to mean a hydrophobic part of the macromolecule or a hydrophobic group present in the macromolecule.
  • 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 unionized solutes, and the logarithm of the ratio is thus log P.
  • the log P value is a measure of lipophilicity or hydrophobicity.
  • octanol n-octanol
  • water n-octanol
  • a log P value ⁇ 0 indicates that a higher percentage of the solute is in the hydrophilic phase.
  • a log P value > 0 indicates a higher percentage of the solute in the lipophilic phase, i.e., the hydrophobic phase.
  • 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 molecule also have a larger size than the target molecule as such, and thus have a larger surface area interacting with a chromatography ligand. Consequently, compared to a target molecule's binding to the presently disclosed multimodal ligand, aggregates of the target molecule will exhibit a stronger binding to the ligand. Thereby, in general an aggregate will elute later from a chromatography device than the target molecule.
  • a fragment of a target molecule is smaller in size than the intact target molecule and will therefore have a smaller surface interacting with a ligand.
  • a Fab fragment of a monoclonal antibody will in general mainly interact with a multimodal ligand via electrostatic interactions, while the intact monoclonal antibody generally will interact with the ligand via both hydrophobic and electrostatic interactions. Consequently, an intact monoclonal antibody in general exhibits a stronger binding to the presently disclosed multimodal ligand than a Fab fragment of the monoclonal antibody. Thereby, in general an intact antibody will elute later from a chromatography device than a fragment of the intact antibody.
  • the use of the presently disclosed chromatography ligand is based on utilizing differences in the ligand's binding to non-aggregated macromolecule and aggregates and/or fragments of the macromolecule, respectively.
  • Formula I may be further defined by the following criteria:
  • Xi is selected from CO and SO 2 ; each of R1-R5 is independently selected from H, F, Cl, 0, N, S, C1.3 alkyl, and C1.3 a lkyl-X 2 ; any two adjacent moieties selected from R1-R5 together with the atoms to which they are attached may form a 5- or 6-membered heterocyclic or carbocyclic ring; and
  • X 2 is selected from 0, S, NH(CO), (CO)NH, NH(SO 2 ), and (SO 2 )NH.
  • Formula I may be further defined by the following additional criteria: i. when each of R1-R5 is H, Xi is S0 2 ; ii. when any two adjacent moieties selected from R1-R5 together with the atoms to which they are attached form a 5-membered heterocyclic containing ring containing two oxygen atoms in the ring, Xi is S0 2 ; and ill. when three of R1-R5 are CH 3 O, Xi is CO.
  • the 5- or 6-membered heterocyclic or carbocyclic ring may be unsaturated or saturated.
  • 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, all of them are N. When there are a maximum of two heteroatoms, each of them may be independently selected from N, 0, and S.
  • lysozyme batch binding capacity is intended to mean the binding capacity of a chromatography material to a lysozyme in batch mode, wherein said chromatography material comprises a chromatography ligand coupled to a support as explained elsewhere herein.
  • Lysozyme was one of five model proteins used in a high-throughput plate-based study to test the presently disclosed chromatography ligands when present in said material, as described in detail in Example 1 below. More particularly, the binding capacity was studied by loading 60 pg of lysozyme on 6 pL of chromatography material (i.e., support coupled with the chromatography ligand) at binding conditions pH 7.5 and 480 mM NaCI in batch mode.
  • adsorption isotherm has its conventional meaning in the art. It describes the relationship between the equilibrium concentration of protein in solution and the amount bound on a chromatography ligand at a particular temperature and solution conditions like pH and ionic strength.
  • the protein-ligand selectivity study for screening the pH and ionic strength window is never operated in the saturation part of the isotherm or overloaded conditions, as the selectivity between the protein-ligand is interfered by competitive binding of other impurities (as getting 100% pure monoclonal antibody for the study is not possible) and also protein-protein interactions comes into the picture at overloading conditions which is the characteristic feature of a non-linear isotherm.
  • the maximum limit for equilibrium binding capacity of lysozyme was lOpg/pL of chromatography material (60pg of lysozyme divided by 6pL of chromatography material).
  • the experimentally observed percentage equilibrium binding capacity can be calculated for each resin for each binding condition with respect to the maximum equilibrium binding capacity (lOpg/pL of resin).
  • a selection was made of those ligands which showed at least 50%, such as 60% of the maximum equilibrium binding capacity, i.e., the ligands which achieved an amount of bound lysozyme at equilibrium of at least 50%, such as 60% of the added amount of lysozyme.
  • logS has its conventional meaning in the art.
  • LogS is directly related to the water solubility of a compound and it 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 log S value, the more hydrophobic a chromatography ligand is.
  • the presently disclosed chromatography ligand may also further be defined by having a logS from about -2.5 to about -5.
  • Log S values were calculated for the chromatography ligand structures with the support modeled as a methyl group. In other words, they were calculated for the methyl-S-ligand structures. More particularly, there is provided herein a chromatography ligand, wherein said ligand, when the -SH of formula I has been replaced with a methyl thioether (-S-CH3), has a logS from about -
  • chromatography ligand may be defined by a chemical structure selected from any one of (a)-(h): and (L18).
  • chromatography ligands When the above chromatography ligands are coupled to a support, they may be illustrated as follows: a. ) e. . According to presently preferred embodiments, the presently disclosed chromatography ligand may be defined by a chemical structure selected from a group consisting of (a)-(f):
  • Table 1 describes the above-listed ligands (a)-(h) as well as the reference ligand, in terms of their chemical structures, predicted logS values, and lysozyme batch binding capacity.
  • the present disclosure further provides a method for preparing a chromatography material, comprising immobilising a plurality of the above-defined chromatography ligand to a support.
  • 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.
  • linker “extender”, and “surface extender” may be used to describe such a compound, as further described below.
  • surface herein means all external surfaces and includes in the case of a porous support outer surfaces as well as pore surfaces.
  • the separation matrix may be contained in any type of separation device, as further defined elsewhere herein.
  • 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.
  • the herein disclosed chromatography material comprises a support to which the ligand is coupled.
  • support has its conventional meaning in the field of bioprocessing and may alternatively be called a “support material” or a “solid phase”, which are other terms conventionally used in this field.
  • the term "support” at the upper left end of formula la shows where the ligand may be coupled to the support.
  • a linker or extender is used to couple the ligand to the support (as described in detail further below), such a linker or extender may also be included in the term "Support”.
  • the disclosed chromatography material comprises a support to which the ligand is coupled.
  • the support may be made of different types of materials and may have different shapes or forms, as described in more detail below.
  • the support may be made from an organic or inorganic material and may be porous or non-porous.
  • the support is prepared from a native polymer, such as cross-linked carbohydrate material, e.g., agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, pectin, starch, etc.
  • the native polymer supports are easily prepared and optionally crosslinked according to standard methods, such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964).
  • the support is a kind of relatively rigid but porous agarose, which is prepared by a method that enhances its flow properties, see e.g., US 6,602,990 (Berg).
  • the support is prepared from a synthetic polymer or copolymer, such as cross-linked synthetic polymers, e.g. styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters, vinyl amides etc.
  • Such synthetic polymers are easily prepared and optionally cross-linked according to standard methods, see e.g., "Styrene based polymer supports developed by suspension polymerization” (R Arshady: Chimica e L'lndustria 70(9), 70-75 (1988)).
  • Native or synthetic polymer supports are also available from commercial sources, such as Cytiva, Sweden, for example in the form of porous particles.
  • the support is prepared from an inorganic polymer, such as silica. Inorganic porous and non-porous supports are well known in this field and easily prepared according to standard methods.
  • the support of the chromatography material may be in the form of particles, such as substantially spherical, elongated or irregularly formed particles.
  • the Capto ImpRes base chromatography matrix (Cytiva, Uppsala, Sweden) comprises a support in the form of substantially spherical particles or beads, which have a diameter of approx. 40 pm. This is a non-limiting example of a particle suitable for inclusion of the presently disclosed ligands by coupling of the ligand to the support.
  • Suitable particle sizes of the presently disclosed chromatography material may be in a diameter range of 5-500 pm, such as 10-200 pm, e.g., 20-100 pm.
  • the average particle size is in the range of from about 20 pm to about 50 pm, such as about 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, or 50 pm, preferably from about 25 pm to about 40 pm.
  • Suitable average pore sizes of the presently disclosed chromatography material in the form of particles may be of any size larger than the target molecules and impurities to be separated, including but not limited to an average pore diameter of 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.
  • an average pore diameter of 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.
  • the chromatography material may be dried, such as dried particles which upon use are soaked in liquid to retain their original form.
  • a dried chromatography material may comprise dried agarose particles.
  • the support of the chromatography material may alternatively be in the form of magnetic particles.
  • the term "magnetic particle" is defined herein as a particle which is able to be attracted by a magnetic field.
  • magnetic particles for use in the presently disclosed method shall not aggregate in the absence of a magnetic field. In other words, the magnetic particles shall behave like superparamagnetic particles.
  • the particle may have any symmetric shape, such as a sphere or a cube, or any asymmetric shape. Spherical magnetic particles are often called magnetic beads.
  • magnetic particle magnetic bead
  • Magnetic particle Magnetic bead
  • magparticle magparticle
  • magbead magnetic particles having a spherical shape. Magnetic particles suitable for use in the presently disclosed method have been described in WO2018122089, which is hereby incorporated by reference in its entirety.
  • the support of the chromatography material may alternatively take any other shape conventionally used in separation, such as monoliths, filters or membranes, capillaries, chips, nanofibers, surfaces, etc.
  • a suitable average pore diameter in the monolith for the purpose of separating target molecules from impurities ranges 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 pm, such as about 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 pm.
  • such nanofibers may for example comprise electrospun polymer nanofibers.
  • such nanofibers form a stationary phase comprising a plurality of pores through which a mobile phase can permeate.
  • the support of the chromatography material may comprise a membranous structure, such as a single membrane, a pile of membranes or a filter.
  • the membrane may be an adsorptive membrane.
  • a suitable pore diameter in the membranous structure for the purpose of separating target molecules from impurities ranges 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 pm, such as about 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 pm.
  • chromatography material comprises a membranous structure
  • membranous structure may for example comprise a nonwoven web of polymer nanofibers.
  • suitable polymers may be selected from polysulfones, polyamides, nylon, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, and polyethylene oxide, and mixtures thereof.
  • the polymer may be a cellulosic polymer, such as selected from a group consisting of cellulose and a partial derivative of cellulose, particularly cellulose ester, cross-linked cellulose, grafted cellulose, or ligand-coupled cellulose.
  • Cellulose fiber chromatography (known as Fibro chromatography; Cytiva, Sweden) is an ultrafast chromatography purification for short process times and high productivity, which utilizes the high flow rates and high capacities of cellulose fiber.
  • a suitable pore diameter in the cellulose fiber for the purpose of separating target molecules from impurities ranges from a minimum average pore diameter of about 9-12 nm (e.g., suitable for separating monoclonal antibodies), and up to a maximum pore diameter of about 5 pm, such as about 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 pm.
  • membrane chromatography has its conventional meaning in the field of bioprocessing.
  • membrane chromatography there is binding of components of a fluid, for example individual molecules, associates or particles, to the surface of a solid phase in contact with the fluid.
  • the active surface of the solid phase is accessible for molecules by convective transport.
  • the advantage of membrane adsorbers over packed chromatography columns is their suitability for being run with much higher flow rates. This is also called convection-based chromatography.
  • a convection-based chromatography matrix 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 substance(s) into the matrix or out of the matrix, which is effected very rapidly at a high flow rate.
  • Convection-based chromatography and membrane adsorbers are described in for example US20140296464A1, US20160288089A1, W02018011600A1,
  • the coupling of the ligand to the support of a chromatography material as disclosed herein may be provided by introducing a linker between the support and ligand.
  • the coupling may be carried out following any conventional covalent coupling methodology such as by use of epichlorohydrin; epibromohydrin; allyl-glycidylether; bis-epoxides such as butanedioldiglycidylether; halogensubstituted aliphatic substances such as di-chloro- propanol; and divinyl sulfone.
  • linkers are: polyethylene glycol (PEG) having 2-6 carbon atoms, carbohydrates having 3-6 carbon atoms, and polyalcohols having 3-6 carbon atoms. These methods are all well known in the art and easily carried out by the skilled person.
  • PEG polyethylene glycol
  • the ligand may be coupled to the support via a longer linker molecule, also known as a "surface extender", or simply “extender”.
  • Extenders are well known in this field, and commonly used to sterically increase the distance between ligand and support. Extenders are sometimes denoted tentacles or flexible arms.
  • the extender may be in the form of a polymer such as a homo- or a copolymer.
  • Hydrophilic polymeric extenders may be of synthetic origin, i.e., with a synthetic skeleton, or of biological origin, i.e., a biopolymer with a naturally occurring skeleton.
  • Typical synthetic polymers are polyvinyl alcohols, polyacryl- and polymethacrylamides, polyvinyl ethers etc.
  • Typical biopolymers are polysaccharides, such as starch, cellulose, dextran, agarose.
  • 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.
  • 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.
  • the density of the plurality of ligands immobilised on the support may be from about 15 to about 50 pmol/mL, such as about 15, 20, 25, 30, 35, 40, 45, or 50 pmol/mL, preferably from about 20 to about 35 pmol/mL.
  • chromatography material comprising the above-defined chromatography ligand coupled to a support.
  • the chromatography material and the support are as defined and exemplified above.
  • the support comprises beads having a diameter from about 25 pm to about 50 pm, preferably from about 30 pm to about 45 pm.
  • the herein disclosed chromatography material may have a density of the plurality of ligands, immobilised on the support, of from about 15 to about 50 pmol/mL, such as about 15, 20, 25, 30, 35, 40, 45, or 50 pmol/mL, preferably from about 20 to about 35 pmol/mL.
  • the chromatography material comprising a chromatography ligand as disclosed herein may be further defined by having a lysozyme batch binding capacity in the linear part of an adsorption isotherm at binding conditions pH 7.5 and 480 mM NaCI and further by that the amount of lysozyme bound to the chromatography material at equilibrium is at least 50%, such as 60% of the amount of lysozyme added to the chromatography material.
  • the meaning of the terms "lysozyme batch binding capacity" and "linear part of an adsorption isotherm” is as defined further above.
  • the chromatography ligand may be defined by having a logS from about -2.5 to about -5.
  • the ligand of the chromatography material is defined by Formula I as described in detail further above.
  • the one or more target molecules may be one or more antibodies, as described in detail elsewhere herein.
  • the antibodies are monoclonal antibodies.
  • the monoclonal antibodies are multispecific monoclonal antibodies, such as bispecific monoclonal antibodies.
  • the one or more target molecules may be one or more antibody fragments.
  • the one or more antibody fragments may be selected from antigenbinding fragments as described and exemplified in detail elsewhere herein, e.g., Fab, Fab', F(a b') 2 , scFv, Fv, dAb, or Fd.
  • the impurities may comprise aggregates of the one or more target molecules, such as high molecular weight aggregates of the target molecules, as described in detail elsewhere herein.
  • the impurities may comprise aggregates of said antibody, such as high molecular weight aggregates of the one or more antibody fragments.
  • the impurities may comprise aggregates of the one or more antibody fragments, such as high molecular weight aggregates of the one or more antibody fragments.
  • the present disclosure also solves or at least mitigates the problems associated with existing methods for separating one or more target molecules from impurities by providing, as illustrated in Fig. 1, 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 chromatography material as disclosed herein; b) eluting the target molecules from the chromatography material; c) optionally eluting the impurities from the chromatography material.
  • the target molecules and optionally the impurities may be 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).
  • Elution buffers suitable for separation of various types of target molecules, e.g., monoclonal antibodies, are well known in the art and can easily be chosen by the skilled person.
  • 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.
  • the present disclosure further provides an alternative method for separating one or more target molecules from impurities, as illustrated in Fig. 2, which method comprises: a) adding a liquid sample comprising one or more target molecules and impurities to a chromatography material as disclosed herein; b) obtaining the target molecules in a flow-through mode, the target molecules having passed through the chromatography material essentially without binding to the chromatography material; c) optionally eluting the impurities from the chromatography material.
  • the above-described flow-through method may be especially suitable when using a chromatography material comprising a support in the form of cellulosic fiber (e.g., Fibro) or nanofibers, as described in detail further above.
  • a chromatography material comprising a support in the form of cellulosic fiber (e.g., Fibro) or nanofibers, as described in detail further above.
  • Figs. 1 and 2 The above-described methods for separating target molecules from impurities, as illustrated by Figs. 1 and 2, are based on multimodal interactions between the chromatography ligand and the molecules present in the liquid sample, i.e., electrostatic interactions, hydrophobic interactions, hydrogen bonding etc.
  • the one or more target molecules may be one or more antibodies, as described in detail elsewhere herein.
  • the antibodies are monoclonal antibodies.
  • the monoclonal antibodies are multispecific monoclonal antibodies, such as bispecific monoclonal antibodies.
  • the one or more target molecules may be one or more antibody fragments.
  • the one or more antibody fragments may be selected from antigenbinding fragments as described and exemplified in detail elsewhere herein, e.g., Fab, Fab', F(a b') 2 , scFv, Fv, dAb, or Fd.
  • the impurities may comprise aggregates of the one or more target molecules, such as high molecular weight aggregates of the target molecules, as described in detail elsewhere herein.
  • the impurities may comprise aggregates of said antibody, such as high molecular weight aggregates of the one or more antibody fragments.
  • the impurities may comprise aggregates of the one or more antibody fragments, such as high molecular weight aggregates of the one or more antibody fragments.
  • step (al) comprises pre-treating the liquid sample.
  • said pre-treating may comprise subjecting a target molecule-containing cell culture harvest to cell lysis, clarification, and/or filtration.
  • step (a2) preceding step (a), wherein step (a2) comprises pre-purifying the one or more target molecules by separating target molecules from a target molecule-containing cell culture harvest, thereby obtaining a pre-purified liquid sample comprising target molecules, before adding said prepurified liquid sample comprising target molecules to the herein disclosed chromatography material.
  • said pre-purifying may comprise subjecting the target molecule-containing cell culture harvest to chromatography, or to clarification followed by chromatography.
  • This Example shows experiments demonstrating a successful separation of a monoclonal antibody from Fab fragments and high molecular weight aggregates by use of the herein disclosed novel ligands.
  • the load contained a monomeric monoclonal antibody (mAb) of a purity of 92%, and in addition 6% of Fab fragments and 2% of high molecular weight (HMW) aggregates.
  • mAb monomeric monoclonal antibody
  • HMW high molecular weight
  • the mAbl has a pl value of 8.6, a molecular weight of 150 kDa and an extension coefficient of 1.58.
  • the concentration of buffer exchanged mAbl sample (pH 7 and conductivity 2.94 mS/cm) used for isocratic retention study was ⁇ 20 mg/mL and a monomeric purity of 98.5% and contained 1.5% HMW aggregates, and for linear salt gradient elution study was ⁇ 18 mg/mL and a monomeric purity of 92% and contained 6% of a Fab fragment and 2 % of HMW aggregates.
  • the Fab fragment was produced from mAbl using Papain digestion method and spiked into the mAbl sample.
  • five model proteins i.e., Cytochrome C, a-Lactalbumin, Lysozyme, Ovalbumin, and Human serum albumin (HSA) (Sigma- Aldrich, St. Louis, MO, USA) and the monoclonal antibody (mAbl) were used.
  • HSA Human serum albumin
  • Capto MMC ImpRes multimodal resin and the Capto ImpRes base matrix were obtained from Cytiva (Uppsala, Sweden), Chemicals including L-Homocysteine thiolactone hydrochloride was obtained from Acros Organics and acyl and sulfonyl chlorides, bromine, dichloromethane, and ethyl acetate were obtained from Sigma Aldrich. All other chemicals including sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, sodium acetate, sodium acetate trihydrate, sodium hydroxide, glacial acetic acid, blue dextran 2000 used were of analytical grade and were purchased from Merck (Darmstadt, Germany).
  • UV 280 nm obtained by SEC-HPLC was performed using the 'Agilent ChemStation' software and by Akta chromatography system using Unicorn 5.31. Titrations were performed using a Metrohm auto-titrator 905 Titrando system from Metrohm AG (Herisau, Switzerland) and analysed with the Tiamo software.
  • the multimodal Capto MMC thioether ligand is derived from a thiolactone proligand.
  • a Capto MMC ligand prototype library was synthesized via acylation or sulfonylation of the commercially available D, L-homocysteine thiolactone hydrochloride with an equimolar amount of the appropriate acyl, or sulfonyl, chloride in dichloromethane (DCM) in the presence of diisopropyl ethylamine (DIPEA).
  • 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 seporatory funnel with aqueous solution of citric acid (10% w/v), aqueous solution of K 2 CO 3 10% (w/w), water, and brine.
  • EtOAc ethyl acetate
  • the organic phase is dried over MgSO , filtered, and evoparated under the reduced pressure to afford the product which is generally a white or pale yellow solid.
  • N-toluoyl-D, L-homocysteine thiolactone (L01) - D,L-Homocyteine thiolactone hydrochloride (18.75 mmol, 2.87 g) and diisopropylethylamine (37.5 mmol, 6.53 mL) are dissolved in DCM (30 mL) and the mixture is cooled in an ice bath.
  • Toluoyl chloride (18.75 mmol) is added slowly to the stirring solution as described in the general procedure to afford the product as white solid.
  • N-pentafluorobenzoyl-D, L-homocysteine thiolactone (L02) - D,L-Homocyteine thiolactone hydrochloride (18 mmol, 2.787 g) and diisopropylethylamine (36.9 mmol, 6.492 mL) are dissolved in DCM (40 mL) and the mixture is cooled in an ice bath.
  • Pentafluorobenzoyl chloride (18 mmol, 2.62 mL) is added slowly to the stirring solution as described in the general procedure to afford the product as white solid.
  • N-benzenesulfonyl-D,L-homocysteine thiolactone (L05) - D,L-Homocyteine thiolactone hydrochloride (16 mmol, 2.478 g) and diisopropylethylamine (32.8 mmol, 5.771 mL) are dissolved in DCM (40 mL) and the mixture is cooled in an ice bath.
  • Benzenesulfonyl chloride (16 mmol, 2.063 mL) is added slowly to the stirring solution as described in the general procedure to afford the product as white solid.
  • N-(4-ethylbenzoyl)-D,L-homocysteine thiolactone (L08) - D,L-Homocyteine thiolactone hydrochloride (18.75 mmol, 2.87 g) and diisopropylethylamine (37.5 mmol, 6.53 mL) are dissolved in DCM and the mixture is cooled in an ice bath.
  • 4-Ethylbenzoyl chloride (18.75 mmol) is added slowly to the stirring solution as described in the general procedure to afford the product as white solid (> 90%).
  • N-(2-naphthoyl)-D,L-homocysteine thiolactone (Lil) - D,L-Homocyteine thiolactone hydrochloride (18.75 mmol, 2.87 g) and diisopropylethylamine (37.5 mmol, 6.53 mL) are dissolved in DCM (30 mL) and the mixture is cooled in an ice bath.
  • 2-Naphthoyl chloride (18.75 mmol) is added slowly to the stirring solution as described in the general procedure to afford the product as white solid (92%, 4.67 g).
  • N-pentafluorobenezensulfonoyl homocysteine thiolactone (L17) - D,L-homocysteine thiolactone HCI (10.02 mmol, 1.02 eq, 1.567 g), dichloromethane (20 mL), and diisopropylethylamine (2.04 eq, 20.04 mmol, 3.553 mL), are added to a Schlenk flask and flushed with N2 and stirred until dissolved. The solution is cooled in an ice bath and pentafluorobenzenesulfonyl chloride (1 eq, 10 mmol, 2.666 g, 1.484 mL) is added dropwise.
  • the reaction mixture is allowed to warm to rt while stirring overnight under N2.
  • the reaction is complete by TLC (DCM with drops of EtOH, or 1:1 EtOAc/cyclohexane).
  • the reaction mixture is concentrated on the rotovap, then diluted with EtOAc (100 mL). This is 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 is concentrated on the rotovap, and the brown solid residue (1.7 g, theoretical yield is 3.75g) was dried on the house vac.
  • the crude product was recrystallized from boiling EtOH/H2O (ca.
  • N-(3,5-diethoxy-benzoyl) homocysteine thiolactone (L18) - D,L-homocysteine thiolactone HCI (10.2 mmol, 1.02 eq, 1.567 g), dichloromethane (20 mL), and diisopropylethylamine (2.04 eq, 20.4 mmol, 2.636 g, 3.55 mL), are added to a Schlenk flask and flushed with N2 and stirred until dissolved. The solution is cooled in an ice bath and the acid chloride (1 eq, 10 mmol, 2.287 g) in DCM (5 mL) is added dropwise.
  • the resulting A/-acylated, or sulfonylated, thiolactone is hydrolyzed in aqueous NaOH providing the carboxylate cation exchange group, and a thiol which is then used to n ucleoph ilica lly couple to the functionalized agarose base matrix (Capto ImpRes, Cytiva).
  • the agarose base matrix is first functionalized with terminal allyl groups using allyl glydicyl ether (AGE), which are subsequently brominated with elemental bromine.
  • AGE allyl glydicyl ether
  • the brominated gels form epoxy termini under basic conditions which are good electrophiles to react with MMC ligand prototypes via S N 2 nucleophilic substitution.
  • Multimodal ligand loadings were 25-30 pmol/mL ReS in as determined by titration.
  • BC binding capacity
  • BC data at low protein loading of 10 pg/pL ReS m was determined in 6pL PreDictorTM 96-well filter plates for four different binding pH (4.5, 5.5, 6.5, 7.5) and eight different NaCI salt concentrations (0, 55, 133, 257, 480, 750, 1250, 1750 mM) using an automated liquid handling system Tecan robotic workstation procured from Tecan Group Ltd. (Mannedorf, Switzerland). Thus, the ligand prototypes were tested for 32 different binding pH and salt conditions.
  • the binding pH 4.5 and 5.5 were based on a 25 mM acetate buffer and pH 6.5 and 7.5 based on a 50 mM phosphate buffer and the stock volumes for binding pH were calculated using a proprietary Excel application (Cytiva, Uppsala, Sweden). All protein solutions were prepared using 5 mM pH 7 phosphate buffer whereas the mAbl load was buffer exchanged in 5 mM pH 7 phosphate in order to avoid the influence of buffer on targeted pH and salt concentration. Several experiments were repeated in duplicates.
  • the external dead volume of the Akta system was estimated by the pulse injection of 200 pL IM NaCI in pH 6.5 10 mM phosphate buffer in the absence of column i.e. a zero dead volume connector was used in place of the column.
  • the column porosity, e c of manually packed columns with resin Capto MMC ImpRes coupled with different synthesized ligand prototypes was estimated by the pulse injection of 200 pL of 3 mg/mL of blue dextran 2000 (Mol. Wt. 2000 kDa) in pH 6.5 10 mM phosphate buffer into column.
  • the column porosity, e c was calculated using the equation: is the column retention volume of blue dextran, V S ystemdead ' s the Akta system dead volume, and V c is the geometric column volume.
  • e T is the total column porosity which was estimated from the pulse injection of 200 pL protein solution (of concentration ⁇ 8.23 mg/mL) under non-binding condition of pH 12 100 mM Na 3 PO into column.
  • the total column porosity, e T was calculated using the equation:
  • t 0 is the retention time of protein under non-binding condition
  • Q is flow rate
  • V c column volume
  • Step 1 Equilibration (5 CV; pH 7 25 mM phosphate buffer + X (200 mM to 1800 mM) NaCI); Step 2: Sample loading ( ⁇ 2 mg of protein per mL of resin; 200 pL sample load volume); Step 3: Isocratic elution (until UV is less than 2 mAU); Step 4: Strip (5 CV; pH 7.5 50 mM phosphate buffer + 1000 mM NaCI ); Step 5: Clean in place (CIP, 3CV) step with IN NaOH for column regeneration and sanitization. The flow rate was set at 0.5 mL/min. The fluid effluent was monitored at 280 nm for peak detection. The retention factor, k, was calculated using the equation: is the system dead volume corrected retention volume of the protein under isocratic condition and V o is the column void volume and was calculated using the equation:
  • V o V C £ C (5)
  • the isocratic retention factor data was fitted to the simplified form of Eq. (6) given by:
  • the model parameters and y are related to the (Av + +Av_) and (Avj) by the following expressions:
  • R s 1.18 Where t R is the retention time of peak and w 0 , 5h is the full width at the half maximum peak height.
  • the retention time of mAbl and Fab fragment peak is denoted by t R2 and t R1 , respectively, and the full width at the half maximum peak height of Fab and mAbl is denoted by IV O 5?li and W o _ 5h , respectively.
  • the prototypes displayed a "U"- shaped dependence for the protein retention as a function of salt concentration, with a retention minimum at ca. 800 mM NaCI.
  • the "U"-shape behaviour is a characteristic of multimodal resins, and a consequence of the contributions from ion-exchange, and other secondary interactions, such as hydrophobic interactions.
  • protein retention decreases with increasing salt concentration.
  • buffer cations compete with the positively charged mAb for the negatively charged sites on the multimodal.
  • protein retention increases with salt concentration, behaving like a HIC resin.
  • ca. 800 to 1000 mM NaCI there is a transition taking place in mAbl adsorption mechanism from primarily electrostatic interactions to secondary hydrophobic interactions.
  • Fig. 3 displays the comparison of isocratic mAbl retention factor, ln(k), vs. NaCI concentration varied from 200 mM to 1800 mM at pH 7 for L09, L02, and LOO. More particularly, Fig. 3 shows a plot of In (fc) vs. In (C s ) for mAbl adsorption onto ( ⁇ ) LOO, (x) L09, ( ⁇ ) L02 at pH 7. The solid lines ( - ) are overlapping fits to Eq. (7) and Eq. (10).
  • Table 2 shows high throughput binding capacity data in terms of % bound of specific protein at a given binding condition.
  • Table 3 shows the R s values for the separation resolution between Fab fragment and the monoclonal antibody (mAbl). All new ligand prototypes showed better separation resolution than the reference ligand (LOO), with L09 having the highest separation resolution, followed by L02, L05, L01 and L08.
  • LEO reference ligand
  • Fig. 4 shows a comparison of separation resolution between HMW aggregate and monoclonal antibody (mAb) for different ligand prototypes for the data obtained using Methodi as described further above.
  • the load contained ⁇ 2% of HMW aggregate.
  • LEO reference ligand
  • Fig. 5 shows a comparison of separation resolution between HMW aggregate and mAb for different ligand prototypes for the data obtained using Method2 as described further above.
  • the load contained ⁇ 1.95% of HMW aggregates.
  • Example 1 An experimental design is performed as in Example 1 with other target molecules than in Example 1, for example a bispecific antibody (approx. 200kDa), or a protein not being an antibody, e.g., a protein of 50-100 kDa.
  • a bispecific antibody approximately 200kDa
  • a protein not being an antibody e.g., a protein of 50-100 kDa.

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Abstract

La présente divulgation concerne un ligand de chromatographie défini par la formule I : X1 est choisi parmi CO et SO2; chacun de R1-R5 est indépendamment choisi parmi H, F, Cl, O, N, S, alkyle en C1-3, et un alkyle en C1- 3-X2; deux fractions adjacentes quelconques choisies parmi R1-R5 conjointement avec les atomes auxquels elles sont attachées peuvent former un cycle hétérocyclique ou carbocyclique à 5 ou 6 chaînons; et X2 est choisi parmi O, S, NH (CO), (CO) NH, NH NH(SO2), et (SO2)NH; et à condition que : i lorsque chacun de R1-R5 est H, X1 est SO2; ii. lorsque deux fractions adjacentes quelconques choisies parmi R1-R5 conjointement avec les atomes auxquels elles sont attachées forment un cycle hétérocyclique à 5 chaînons contenant deux atomes d'oxygène dans le cycle, X1 est SO2; et iii. lorsque trois de R1-R5 sont CH3O, X1 est CO. La présente divulgation concerne également un matériau de chromatographie comprenant un support et le ligand de chromatographie divulgué dans la présente divulgation accouplé au support. Est en outre divulguée une utilisation dudit matériau de chromatographie pour séparer une ou plusieurs molécules cibles d'impuretés, ainsi qu'un procédé pour séparer une ou plusieurs molécules cibles à partir d'impuretés.
PCT/EP2023/076622 2022-09-29 2023-09-27 Ligand de chromatographie et matériau de chromatographie, et leurs utilisations WO2024068682A1 (fr)

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